INTERNATIONAL
REVIEW OF CYTOLOGY VOLUME87
ADVISORY EDITORS H. W. BEAMS HOWARD A. BERN GARY G. BORISY PIET BORST BHARAT B. CHATTOO STANLEY COHEN RENE COUTEAUX MARIE A. DIBERARDINO CHARLES J. FLICKINGER OLUF GAMBORG M. NELLY GOLARZ DE BOURNE YUKIO HIRAMOTO YUKINORI HIROTA K. KUROSUMI GIUSEPPE MILLONIG ARNOLD MITTELMAN AUDREY MUGGLETON-HARRIS ALEXANDER
DONALD G. MURPHY ROBERT G. E. MURRAY RICHARD NOVICK ANDREAS OKSCHE MURIEL J. ORD VLADIMIR R. PANTIC W. J. PEACOCK DARRYL C. REANNEY LIONEL I. REBHUN JEAN-PAUL REVEL JOAN SMITH-SONNEBORN WILFRED STEIN HEWSON SWIFT K. TANAKA DENNIS L. TAYLOR TADASHI UTAKOJI ROY WIDDUS YUDIN
INTERNATIONAL
Review of Cytology EDITED BY
G . H. BOURNE
J. F. DANIELLI
S t . George's University School of Medicine St. George's, Grenada West Indies
Danielli Associates Worcester. Massachusetts
ASSISTANT EDITOR K. W. JEON Department of Zoology University of Tennessee Knoxville, Tennessee
VOLUME
87
1984
ACADEMIC PRESS, Inc. (Harcourt Brace Jovanovich, Publishers)
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LIBRARY OF CONGRESS CATALOG CARDNUMBER: 52-5203 ISBN 0-12-364487-9 PRINTED IN THE UNITED STATES OF AMERICA 84 85 86 8 1
9 8 1 6 5 4 3 2 1
Contents
CONTRIBUTORS
...........................................
ix
The Modeling Approach BARBARAE. WRIGHT I. The Citric Acid Cycle in Dicryosrelium discoideum: A Steady-State Model. . . . . . 11. Glycogen Metabolism in Rat Liver and Morris Hepatoma: Transition Models. . . . 111. Carbohydrate Metabolism in Candida albicuns: A System to Model . . . . . . . . . . . IV. Information Required for a Cundidu Steady-State Model . . . . . . . . . . . . . . . . . . . . . V. Information Required for a Candidu Transition Model . . . . . . . . . . . . . . . . . . . . . . . VI.
Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1
4 10
12 13 14 I5
Protein Diffusion in Cell Membranes: Some Biological Implications MICHAEL MCCLOSKEYA N D MU-MINGPO0 1.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11. Protein Diffusion: Known and Unknown.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Implications in Membrane Biology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
IV. Closing Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
19 20 34 71 72
ATPases in Mitotic Spindles
I.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11. Energy Requirements for Mitosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. ATPase in the Mitotic Spindle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
IV. Calmodulin Regulation of Spindle ATPase . . . . . . . . . . . . . . . . . . . . . . . . . . V. Spindle ATPase and Models of Mitosis.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Concluding Remarks ...................... References . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
V
86
96 101 101
vi
CONTENTS
Nucleolar Structure GUYGOESSENS
I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1 .
...........
111. IV .
V . General Conclusion . . .
.........................
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
i07 108 126 148 151 152
Membrane Heterogeneity in the Mammalian Spermatozoon W . V . HOLT
.......... I . Introduction . . . . . . . . . . .................... I1 . Summary of Sperm Struc 111. Structural Differentiations in Sperm Membranes ............................ IV . Developmental Aspects of Mammalian Sperm Membranes V . Epididymal Contribution to Sperm Surface Heterogeneity .................... VI . Functional Aspects of Membrane Heterogeneity in Spermatozoa . . . . . . . . . . . . . . . VII . Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References .............................
159 160 161 171 179 182 188 189
Capping and the Cytoskeleton LILLYY . W . BOURGUIGNON A N D GERARD 1. BOURGUIGNON
I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1 . General Capping Phenomena . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Microfilaments ......................
IV . V. VI . VII . V111.
Microtubules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Intermediate Filaments .................. Capping-Related Regul .................. Concluding Remarks ........................ References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
195 196 198 203 207 208 213 217 220
The Muscle Satellite Cell: A Review DENNISR . CAMPION
.............................................. .................... 111. Gross Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ................................... IV . Fine Structure . . V . Situations Affecting Satellite Cell Content .................... I. I1 .
225 226 231 233 238
CONTENTS VI . Activation Stimulus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
vii 245 246 247
Cytology of the Secretion in Mammalian Sweat Glands KAZUMASA KUROSUMI. SUSUMU SHIBASAKI. AND TOSHIOITO I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1 . Historical Survey of Light Microscopic Studies of Sweat Gland Morphology . . . . 111. Ultrastructures and Their Functional Significance of the Human Sweat Glands . . . IV . Ultrastructural Cytology of the Secretory Activity in the Sweat Glands of Nonhuman Mammals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CONTENTS OF RECENTVOLUMES AND SUPPLEMENTS ..............................
253 254 263 309 325 326 331 335
This Page Intentionally Left Blank
Contributors
Numbers in parentheses indicate the pages on which the authors’ contributions begin
GERARDJ. BOURGUIGNON ( 1 9 9 , Department of Anatomy and Cell Biology, School of Medicine, University of Miami, Miami, Florida 33101 LILLYY. W. BOURGUIGNON (195), Department of Anatomy and Cell Biology, School of Medicine, University of Miami, Miami, Florida 33101
DENNISR. CAMPION(225), USDA-SE-ARS, Animal Physiology Unit, Richard B. Russell Agricultural Research Center, Athens, Georgia 30613, and Department of Foods and Nutrition, University of Georgia, Athens, Georgia 30602 GUYGOESSENS(107),lnstitut d’Histologie, Universite‘de 1’Etat a Liage, Likge, Belgium W. V . HOLT (159), Gamete Biology Unit, Department of Reproduction, Institute of Zoology, Zoological Society of London, London h W I 4RY, England
TOSHIOITO (253), Department of Anatomy, Teikyo University School of Medicine, Tokyo, Japan KAZUMASAKUROSUMI(253), Department of Morphology, Institute of Endocrinology, Gunma University, Maebashi, Japan MICHAELMCCLOSKEY ( 19), Department of Physiology and Biophysics, University of California, Iwine, California 9271 7 MU-MINGPo0 ( 1 9), Department of Physiology and Biophysics, University of California, Irvine, California 92717 M. M. PRATT(83), Department of Anatomy and Cell Biology, University of Miami, School of Medicine, Miami, Florida 33101 ix
X
CONTRIBUTORS
SUSUMUSHIBASAKI (253), Department of Anatomy, Gunma University School of Medicine, Maebashi, Japan
BARBARA E. WRIGHT(I), Microbiology Department, University of Montana, Missoula, Montana 59812
INTERNATIONAL REVIEW OF CYTOLOGY, VOL. 87
The Modeling Approach BARBARA E. WRIGHT Microbiology Department, Universiq of Montana, Missoula, Montana I. The Citric Acid Cycle in Dicryosrelium discoideum: A Steady-State Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. 111.
IV. V. VI.
Glycogen Metabolism in Rat Liver and Morris Hepatoma: Transition Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Carbohydrate Metabolism in Cundidu ulbicans: A System to Model Information Required for a Candida Steady-State Model . . . . . . . . . . Information Required for a Candida Transition Model.. . . . . . . . . . . Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I 4 10 12 13 14 15
“Infatuation with over-simple answers to complex problems is one of the earmarks of intellectual mediocrity.” (Theodosius Dobzhansky)
If one is interested in understanding complex metabolic changes in living cells, the use of kinetic models can be very rewarding. Relatively simple (steadystate) models can aid in the interpretation of radioactive tracer data, provide information on reaction rates in vivo, and make predictions about intracellular and intercellular compartmentation. More complex (transition) models can be used to integrate, over time, data on enzyme mechanisms, metabolite concentrations, and flux. These models can help define those events which are most critical to a particular metabolic transformation, for example, in development, aging, malignancy, dimorphic fungi, or a fermentation process. The modeling approach is also useful in the analysis of specific areas of metabolism in higher animals, e.g., glycogen metabolism in liver, or glycolysis in the perfused heart. The purpose of this article is twofold. First, to summarize briefly the kind of data required for, and the kind of information obtained from, a steady state as compared to a transition model; second, to describe a system which has not yet been modeled, but is highly amenable to such an analysis. A more technical treatment of various modeling approaches has appeared recently (Wright and Kelly, 1981).
I. The Citric Acid Cycle in Dictyostelium discoideum: A Steady-State Model In order to better understand energy metabolism in this simple differentiating system, intermediates of the citric acid cycle were labeled with tracer levels of 1 Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-364487-9
2
BARBARA E. WRIGHT
amino acids, isolated, and their specific radioactivities determined. It was necessary to construct kinetic models to interpret these data. These models simulate metabolism under steady-state conditions, i.e., during a brief period of time, such that metabolite concentrations, as well as metabolite flux into and out of each metabolite “pool,” remain constant. As will become apparent, these models successfully integrated a mass of data within a dynamic framework, and resulted in many predictions about compartmentation and exchange rates between like pools inside and outside of the mitochondria. The model used is shown in Fig. 1 (Kelly et al., 1979a). Metabolites labeled 1 are intramitochondrial and the others are extramitochondrial. Cellular protein and hence amino acids serve as the energy source during development in this system; the five major points of entry of carbon into the cycle are indicated in Fig. 1. The rates of these reactions were based upon the known rate of net protein degradation and upon the amino acid composition of typical cell proteins (Kelly et al., 1979b). The flow of carbon or flux through the cycle was based on oxygen consumption; at one stage of differentiation in this system (culmination), this amounted to 0.4 pmol/minute/ml packed cell volume. Thus, all fluxes into the cycle must total 0.4 mmol/minute. The total cellular levels of each metabolite shown in Fig. 1 were known, but the relative concentrations inside and outside the mitochondria were unknown. The distribution of one metabolite in and out of the mitochondria, as well as the rates of exchange between like pools inside and outside the mitochondria,
Citrate 2 * - - - - +Citrate I’‘.--
..
Succinalc I + - - - - -Protein
\ ..,‘
Acctyl-CoA
Fumarate I
&---+ Fumarate 2
i Malale 2
FIG. 1 . Model used in initial simulations of the [14C]glutamatelabeling data. The numbers I and 2 or 3 indicate intra- and extramitochondrial pools, respectively (see Kelly et al., 1979a).
3
THE MODELING APPROACH
were deduced in attempting to simulate tracer experiments such as that shown in Fig. 2. On cell rupture, the intra- and extramitochondrialpools will mix, and the specific radioactivities obtained will be the average for the two pools of each metabolite. We assumed that the initial rapid labeling of cycle intermediates was a result of labeling the mitochondria1 pools, as the whole cycle only functions within the mitochondria. Mitochondria1 malate, fumarate, and citrate then exchange with their counterpart cytoplasmic pools, accounting for the slow rise in their (average) specific radioactivities. Since 2-ketoglutarate and succinate remain constant in specific radioactivity, there is (1) only one pool of each, or (2) their cytoplasmic pools are labelled immediately, or (3) no exchange occurs between the pools in the cytoplasm and mitochondria. The relative sizes of the intra- and extramitochondrial pools and the fluxes between them cannot be measured, but can be predicted by constructing a model and using a computer program called TFLUX. Relative pool sizes and fluxes are considered as unknowns, and their values are varied until the computer specific radioactivity curves match the data. To use the TFLUX program (Sherwood et al., 1979), the following input information is required: (1) a metabolic map describing the reactions (Fig. 1); (2) the specific radioactivity of [14C]glutamateat t=O; (3) flux through the cycle; (4) the relative sizes of each pool; and ( 5 ) the fluxes between them. The program calculates the specific radioactivities over time of all metabolites shown in Fig. 1, as well as the averaged values for the two pools of a given metabolite. For example, the latter value for malate at any point in time will be
2 -0XOGWTARATE
0
5
10
15
20
25
30
Time in Minutes FIG. 2. Simulation of the [“Tlglutamate data. The solid lines represent computer output super2-oxoglutarate (O), succinate fumarate (@), imposed on the experimental data for malate (A), and citrate (A)(see Kelly e r a / . . 1979a).
(o),
4
BARBARA E. WRIGHT
+
total counts in mitochondrial malate total counts in cytoplasmic malate/total pmoles in mitochondria1 malate total pmoles in cytoplasmic malate. Output from the model attempting to match the specific radioactivity data (symbols) is actually represented by the solid lines in Fig. 2. The fit is quite good. Amino acids other than [14C]glutamatewere also used to label the cycle intermediates. Having arrived at a model giving output consistent with all the experimental data, variations were explored, to examine how unique or constrained it was (Kelly et al., 1979a). These analyses allowed us to estimate compartmentation ratios between cycle and noncycle pools for seven metabolites (oxaloacetate and aspartate pools not shown), as well as the exchange rates between them. A number of other predictions and insights into this area of metabolism were formulated as a result of this investigation, and are discussed elsewhere (Kelly et al., 1979a; Wright and Emyanitoff, 1982). A steady-state model of the citric acid cycle in rat liver was constructed by Sauer et af. (1970). Other models of energy metabolism have been analyzed by Heath and Threlfall (1968), Reich et al. (1968), and Randle et al. (1970). Katz, Wood, and collaborators have developed over the years a series of very sophisticated steady-state models of carbohydrate metabolism (Katz and Wood, 1960; Katz et al., 1966, 1974). A steady-state model has also been used recently in the analysis of intercellular compartmentation between the two cell types in the Diczyostelium system (Wright et al., 1982).
+
11. Glycogen Metabolism in Rat Liver and Morris Hepatoma:
Transition Models The purpose of this investigation was (1) to construct three models compatible with data obtained for normal liver, a hepatoma (tumor of the liver), and host liver (the liver of the tumor-bearing rat), and (2) to determine which parameters in the model of normal liver could be altered to produce the models of host liver and of the hepatoma (Anderson and Wright, 1980; Anderson et af., 1980). These are transition models, because they simulate changes in metabolism during feeding, or, more specifically, during the conversion of glucose to glucose-6-P (G6P) and glucose- 1-P (G 1P), uridine diphosphoglucose (UDPG), and glycogen. Thus, pool sizes and reaction rates vary, in contrast to steady state conditions. The rats were fasted, and the experiments performed over a 700-minute period after feeding, while glycogen accumulated at a linear rate. During this period, the concentrations of glycogen, G6P, and UDPG were determined in normal liver (Fig. 3). Comparative values for host liver and hepatoma 7787 at one time point are given in Table I. The rate of glycogen synthesis was determined at a time point 360 minutes after the onset of feeding. These values were based upon the concentration of glycogen and the specific radioactivities of glycogen and its
5
THE MODELING APPROACH HEPATOMA7787 AT 6 HOURSAFTER FEEDING
T I
Y
1
6PM
1
12 PM
I
I
I
6AM
FIG. 3. Levels of glycogen (H)(mM glucose equivalents), G6P liver. Bars show ? SEM (see Anderson et al.. 1980).
(A), and UDPG (0)in normal
precursor, UDPG. Striking differences were observed, as can be seen in Table 11. Given these data and information from the literature regarding the enzymes of glycogen metabolism, a transition model was constructed (Fig. 4). A specialized program called METASIM has been developed as a general purpose metabolic simulator of in vivo, multienzyme systems undergoing longterm changes in metabolite concentrations and enzyme activities (for a review, see Wright and Kelly, 1981). These models required the following input information: (1) a metabolic map such as that shown in Fig. 4; (2) initial metabolite concentrations determined experimentally (Fig. 3); (3) enzyme kinetic expressions and constants taken from the literature; (4) time functions giving “inTABLE I TISSUE CONTENT OF GLYCOGEN, G6P, AND U D E , IN NORMALLIVER, HOST LIVER, HEPATOMA7787 AT 6 HOURSAFTER FEEDINGO Tissue
Glycogen
G6P
Normal liver Host liver Hepatoma 7787
219 +. 14 36 +. 4 7.3 t 0.8
0.53 0.02 0.74 ? 0.05 0.43 t 0.02
*
AND
UDPG 0.61 0.92 0.50
? ? ?
0.06 0.07 0.04
“All values are given in mM (or mM glucose equivalent in the case of glycogen) and are means of number of rats shown in parentheses t SEM (see Anderson er al., 1980).
6
BARBARA E. WRIGHT TABLE I1 RATE OF GLYCOGENSYNTHESIS, EXPRESSED AS MILLIMOLES GLUCOSE EQUIVALENTS INCORPORATED PER MINUTEa
Tissue
Rate
Normal liver Host liver Hepatoma
0.27 0.033 0.006
Percentage normal 100
12 2
OSee Anderson et al. (1980).
dependent metabolite’’ concentrations, which conform to experimental values. These metabolites impinge upon, but are not part of the model, e.g., ATP and Pi of Fig. 4; ( 5 ) time functions giving external flux rates, e.g., perturbation of this system by external glucose (see below); and (6) enzyme “activation functions. *’ The activity of an enzyme is called V,, and is adjusted such that output from the model is consistent with all data on metabolite concentrations and reaction rates (v) determined experimentally, and with other parameters assumed to be valid, e.g. (2) and (3). Treating enzyme activity as an unknown is unique to this modelling approach (see Wright and Kelly, 1981). The use of an enzyme activaGlycogen
pi H20 PPi Pi R2
UDPG ATP
ADP
R4
FIG.4. The model of glycogen metabolism. The reactions are catalized by the following enzymes or pathway: R1, G6P phosphohydrolase; R2, UDPG pyrophosphorylase; R3, glucokinase; R4, G6P catabolism; R5, glycogen synthase; R6, glycogen phosphorylase. For further details, see text and Anderson and Wright (1980).
THE MODELING APPROACH
7
tion function [Vv(t)], to increase or decrease activity as a function of time, implies nothing with respect to the mechanism(s) responsible for the change in enzyme activity (e.g., new synthesis, inhibition etc.). Enzyme activities are represented as V , when a known kinetic mechanism is used, and K, when a simple rate constant is invoked (Wright and Gustafson, 1972). Reaction rates, v , are expressed in terms of cell water (mmol/min). A model was constructed which gave output consistent with the data regarding metabolite concentrations and the rate of glycogen synthesis in normal liver. In this model all enzyme activities are constant except R4, representing G6P catabolism (glycolysis pentose cycle), which becomes active after 100 minutes. The Nordlie mechanism (Nordlie, 1972) for G6Pase (Rl) was chosen, together with glucokinase (R3), to depict the relationship between glucose and G6P. The reaction catalyzed by glucokinase (R3) is expressed as an ordered mechanism using kinetic constants similar to those of rabbit liver (Salas et al., 1965). The G l P and G6P pools are integrated and held at a constant ratio of 1:20 (Fruton and Simmonds, 1960). Glycogen synthetase (R5) and glycogen phosphorylase (R6) are shown as simple rate constants. UDPG pyrophosphorylase(R2) was modeled after the mechanism of Turnquist and Hansen (1973), and Munch-Peterson (1955). The METASIM program contains 22 different enzyme mechanisms, and the user need only indicate the appropriate one for each reaction and supply the kinetic constants. For example, in the case of UDPG pyrophosphorylase, the following information was specified:
+
R2: UDP-glucose pyrophosphorylase (EC 2.7.7.9) UTP + glucose-1-P G PP, + UDP-glucose Ordered Bi Bi K (UTP) = 0.05 mM, K (glucose-1-P) = 0.1 mM, K (PPi) = 0.2 mM, K (UDP-glucose) = 0.05, Keq = 0.3, Ki(UTP) = 0.05 mM, Ki(GlP) = 0.1 mM, Ki (PPi) = 0.2 mM, Ki(UDP-glucose) = 0.05 mM. V,l and Vv2 = 150 mmol/min. An external flux of glucose was used to perturb the system and produce the metabolite accumulation patterns shown in Fig. 5 . Note the similarity of these patterns to those in Fig. 3. The rate of glycogen synthesis (not shown) was also similar to that determined experimentally (Table 11). Having achieved a model of normal liver which simulated the rate of glycogen synthesis and the accumulation patterns observed experimentally, parameters were varied in an attempt to determine how unique or constrained the model was. In spite of its simplicity, the model parameters were quite constrained by the data. For example, lowering glucokinase (R3) to two-thirds of its activity gives unacceptably low values for all fluxes and metabolite concentrations; raising its activity gives unacceptably high values. The activities of the enzymes catalyzing R4, R3, and R1 are constrained by the levels of G6P and G l P required to produce a rate of UDPG
8
BARBARA E. WRIGHT MM
1
..................................................................................................
-
GLUCOSE 6- PHOSPHATE
I
8 ;
!
......._..
II
* .........,.........
:.
........,.............................,.........,.........,.........,........
UDP- GLUCOSE
~
2 ; ... I
*
I
1
(1.1
*I*
*
II
I..*
*t
I: .
11.11 1.11.
;
I'
.I*.
.I.,..
*
*
**.*** *..****
I.. t i l t .*..l..*
tI
I
....I.*.
.*.***.
......
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . 1 . . . . . . . . . , . . . . . . . . . , . . 1 ._ _._ . . . . . 1
2oo
;
**.**.**l**lt
GLYCOGEN
***in.* I**
I I
100
t.
1* . * . . *
*.** *..*
****..*. *.**. .***** I**.*
t***...*
I.. .I.
;
** *.* It.
I I
1.
1. i
I
****.*
I..
ft.
MM I1 ......... 0
.I . ........:
.....
. I
.......
250
. _ I
.,
........ ...
.,........ SO0
TIME IN MINUTES FIG.5. Metabolic accumulation patterns resulting from a computer simulation using the model shown in Fig. 4. (From Anderson and Wright, 1980.)
synthesis compatible with the observed rate of glycogen synthesis. Attempts were then made to vary parameters to produce a model of host liver and hepatoma 7787. Changing the activities of glucokinase (R3),glycolysis (R4), UDPG pyrophosphorylase (R2), glycogen phosphorylase (R6),or combinations thereof, were not effective in simulating the data for host liver or hepatoma 7787. The critical enzyme which can, alone, be perturbed in the model of normal liver and produce a model of host liver and of hepatoma 7787 is glycogen synthetase (R5).Table 111 demonstrates that decreases of 12-fold and of 62-fold, respectively, in the activity of this enzyme, give output simulating the data for host liver and for hepatoma 7787. Thus a decrease in glycogen synthetase activity alone can induce all four of the changes observed in comparing normal glycogen metabolism to that of the host liver and hepatoma: a lower rate of
9
THE MODELING APPROACH
TABLE I11 OUTPUTAT 350 MINUTES FROM VARIATIONS IN THE ACTIVITY OF GLYCOGEN SYNTHETASE (K,of R ~ ) o Normal
Host
(K, = 0.5
(K, = 0.04
mmol/minute)
mmol/minute)
Hepatoma 7787 (K, = 0.008 mmol/minute)
Parameter
Exp.
Model
Exp.
Model
Exp.
Model
Flux R2 (mmol/min) Glycogen (mM) UDPG (mM) G6P (mM)
0.27 219 0.61 0.53
0.27 209 0.54 1.66
0.033 36 0.92 0.74
0.027 45 0.66 I .93
0.006 7.3 0.50 0.43
0.005 7.4 0.67 1.96
aSee Anderson and Wright (1980).
glycogen synthesis, a lower level of glycogen, and, in the case of host liver, higher steady-state levels of UDPG and G6P. The agreement between the data and the models is good, except for G6P. This apparent discrepancy may be due to compartmentation of G6P (Anderson and Wright, 1980). The decreased activity of glycogen synthetase in the model for the hepatoma is consistent with the literature. A number of investigators have found that either the active form of glycogen synthetase and/or the total amount of active + inactive forms is reduced in tumor compared to normal liver (Sato et al., 1973). However, a number of other changes have also been observed in this and similar slowly growing tumors: ( 1) glucokinase activity decreases or is undetectable, while hexokinase increases in activity (Manjeshwar et al., 1965); (2) G6P dehydrogenase, and hence presumably catabolism, increases somewhat in activity (Weber and Lea, 1966); and (3) glycogen phosphorylase activity decreases (Sato et al., 1973). These alterations were therefore explored in the model of tumor metabolism, to determine whether or not they were also compatible with the data. Incorporation of the first two changes (1 and 2) in the hepatoma model made remarkably little difference in the output. However, to be consistant with the data, the rate of glycogen degradation, catalyzed by glycogen phosphorylase, must remain unchanged or increase in the hepatoma. This is an excellent example of the use of kinetic models for testing the relevance of in vitro data to metabolism in vivo, i.e., the lower enzyme activity in extracts of the hepatoma cannot indicate or reflect a lower reaction rate in vivo. This conclusion gives rise to some testable predictions: Either this enzyme is not rate limiting in vivo (i.e., it could decrease in activity and not affect the reaction rate), or an in virro artifact is involved (e.g., enzyme instability, loss of an activator, etc.). To summarize the two sections presented, one steady-state and one transition
10
BARBARA E. WRIGHT
model have been examined. Both models served to integrate the available data within a dynamic framework, in order to simulate metabolism in vivo. Both models provided information which could not be obtained through direct experimentation. From the steady-state model, many specific predictions were made regarding intracellular metabolic compartments and flux relationships. From the transition model, information was obtained concerning which changes in the enzyme activities of normal liver could, and which could not, be responsible for producing the metabolism characteristic of the hepatoma and host liver.
111. Carbohydrate Metabolism in CandidQ albicans: A System to Model C . albicans is a dimorphic fungus which exhibits both yeast and hyphal morphologies. Although the yeast phase is predominant in those individuals harboring C . albicans as part of the normal flora, the hyphal phase is generally associated with the pathological state (Hill and Gebhardt, 1965; Mackenzie, 1974; Tashdjian and Kozinn, 1970). It is thought that the dimorphic transition is involved in the invasive capacity of the organism (Young, 1958; Odds, 1979). This dimorphic and metabolic transition would be ideal to model for a number of reasons. The process has medical significance, and the literature is rich with in vitro data on the system, although no attempt has been made to integrate these data under in vivo conditions. With relatively little effort, flux through the system under steady-state conditions could be determined. This information, together with available and additional data on the enzymes involved, could form the basis for a transition model to aid in the analysis of rate-limiting events controlling the yeast to hypha transformation. Information available: the development of the hyphal form is thought to be a consequence of an alteration of the process of cell growth and division reflected primarily in changes in cell wall composition (Nickerson, 1963). Analysis of the cell wall of yeast and hyphal forms reveals quantitative, rather than qualitative differences (Chattaway et al., 1968), particularly in terms of glucosamine, glucose, mannose, and protein content. Chitin, the cell wall glucosamine polymer, is four times more abundant in the hyphal cell walls than in the yeast cell walls. The total amounts of mannose and glucose in yeast wall are roughly equivalent to those in hyphal wall, but their distribution between alkali soluble and insoluble fractions varies. The major metabolic pathways in this system are indicated in Fig. 6. There have been numerous investigations of isolated metabolic components in either yeast phase or hyphal phase cells. The biosynthesis of chitin has been examined in both yeast and hyphal cells. In vitro activities of enzymes in the biosynthetic pathway have been reported, including ~glutamine-~F6P aminotransferase (Chattaway et al., 1973), N-acetylglucosamine kinase (Bhattacharya et al., 1974), chitin synthase, (Braun and Calderone, 1979) and
THE MODELING APPROACH
11
GLUCOSE
I
/--
P
HMP-G6
F l , 6 P-F6
1
P-
WALL PROTEIN GLUCANS
--
TREHALOSE GLYCOGEN MANNAN
FIG.6. A model depicting major metabolic pathways in Cundidu ulbicuns. G6P, Glucose-6-P; F6P, fructose-6-P; HMP, hexose monophosphate pathway; F1, 6P, fructose-l,6-diphosphate;TCA, tricarboxylic acid cycle; GlcNAc6P, N-acetylglucosamine-6-phosphate;UDP-GlcNAc, UDP-Nacetylglucosamine.
glucosamine-6-phosphate deaminase (Singh and Datta, 1979). Chiew et al. ( 1980) have also determined kinetic constants for ~-glutamine-~-F6P aminotransferase and chitin synthase. Enzymes of the glycolytic and hexose monophosphate pathway have been demonstrated (Ton and Karunairatnam, 1976; Rao et al., 1960), and phosphofructokinase has been characterized (Chattaway et al., 1973). Tricarboxylic acid cycle enzymes have been demonstrated in extracts from whole cells (Rao et al., 1962; Kot et al., 1976) and from isolated mitochondria1 fractions (Nozu et al., 1958; Yamaguchi et al., 1971). Shepherd et al. (1978) reported both a classical and an alternate electron transport system in C. albicans. Some of the data published by Chattaway et al. (1973) were obtained from both yeast and mycelial cells, as well as from cells undergoing transition from yeast to mycelium. These data include measurements of in v i m activity of phosphoglucose isomerase, phosphofructokinase, glucosed-phosphate dehydrogenase, and the total pathways for chitin and mannan synthesis. They also determined the concentrations of glucose 6-phosphate, fructose 6-phosphate (F6P), ATP, ADP, and AMP. Changes in the levels of the storage carbohydrates trehalose and glycogen have been measured by Arnold and McLellan (1975). Although there are in v i m data on isolated metabolic parameters in either yeast or hyphal cells, there is relatively little information on metabolic activity in vivo. Chattaway et al. (1973) measured the flux of [14C]glucose through the hexose monophosphate pathway. Rates of substrate utilization and respiration in both morphological types have been determined (Ward and Nickerson, 1958; Shepherd and Sullivan, 1976), and data are available on the rate of incorporation of N-acetylglucosamine into chitin (Braun and Calderone, 1979). Limitations of in vitro data: There has been no attempt to integrate the data
12
BARBARA E. WRIGHT
available in order to examine the metabolic network for cell wall biosynthesis as it functions in vivo. In fact, much of the available data would not be suitable to such an analysis. Most previous work on carbohydrate metabolism relevant to cell wall biosynthesis in this and similar systems has focused on in vitro enzyme activities. These values rarely reflect in vivo activities since (1) enzymes frequently are not. rate limiting in vivo, and (2) cell rupture imposes drastic changes upon the organization of cell components and on the chemical environment in which the enzyme functions. Subtle changes in enzyme activity in vivo, of the kind expected to occur during the yeast to hyphal transition, may be masked or destroyed when measuring in vitro activities. Alternatively, seemingly significant changes may be artifacts induced by in vitro conditions. For these reasons, we culculute enzyme activities in a transition model, as discussed earlier in connection with the analysis of glycogen metabolism in rat liver. Importance of in vivo flux analyses: The most appropriate analysis of in vivo metabolism is a determination of the overall (net) flux through a system under steady-state conditions, in conjunction with the steady-state concentrations of the relevant metabolites (Hess and Brand, 1965). Such data provide the most information about the overall operation of a metabolic network. Proper analysis of these parameters, which are the experimentally obtainable data most applicable to the intact organism, may reveal the existence of some form of intracellular compartmentation, and indicate control points or rate-limiting steps in metabolism. As demonstrated earlier in the Dictyostelium model, the analysis of such data at any level of complexity beyond the most simple linear metabolic scheme requires the use of computer software designed to integrate flux patterns within complex, interrelated metabolic pathways.
IV. Information Required for a Cundidu Steady-State Model Ideally, the data to be analyzed should be obtained from the two morphological forms of the same strain. Manning and Mitchell (1980) have recently described a strain which grows as pure yeast at 24°C or pure hyphae at 37°C in a defined medium. The concentration, based on cell volume, of the following intermediates and end products should be determined in yeast, hyphae, and in a population in transition: G6P, F6P, mannose 6-phosphate (M6P), GDP-mannose (GDP-Mann), N-acetylglucosamine-6-phosphate (GlcNAc6P), UDP-N-acetylglucosamine (UDP-GlcNAc), UDPG, trehalose, mannan, glycogen, glucan, and chitin. The concentrations of these metabolites at one point in time, together with their specific radioactivities following tracer experiments, constitute the necessary input for a steady state model. (Their changes in concentration during the yeast to hypha transformation will be necessary for a transition model.) Overall flux and rates of end product synthesis: By measuring the rate of
THE MODELING APPROACH
13
glucose utilization and I4CO, production from [1-14C]-, [6-14C]-, and [U-14C]glucose, the total amount of glucose metabolized and the fraction metabolized via the hexose monophosphate pathway may be determined (Wang, 1972; Chattaway et al., 1973). Carbon flux through the citric acid cycle may be determined by oxygen uptake (Q,,). Following tracer labeling with [U-'4C]glucose, flux into most of the end products may be calculated by dividing the increase in total radioactivity in a product by the specific radioactivity of its precursor, and expressing the rate of synthesis as micromoles incorporated per unit time. UDPG is the immediate precursor of both trehalose and glycogen. For flux calculations, UDPG, GDP-Mann, and UDP-GlcNAc may be used as the precursors of glucan, mannan, and chitin, respectively. These calculations of flux can be used only if the system is in metabolic steady state, that is, over a time period during which the concentration of precursor is constant. Furthermore, the incorporation of precursor into end product must increase linearly, while the precursor specific radioactivity is fairly constant. If no such labeling pattern can be found in cells at steady state, flux into end products can be calculated using a computer program such as TFLUX described earlier. Fluxes between phosphorylated intermediates: Since these pools are likely to have rapid turnover rates and dissimilar patterns of labeling, TFLUX must be used to calculate fluxes by curve fitting the specific radioactivity patterns for all the measured metabolites simultaneously. As in the case of the steady-state model described earlier, the initial input information required is (1) a metabolic map (Fig. 6); (2) the specific radioactivity of [U-14C]glucoseat t =O; (3) the size of each pool; and (4)the known or estimated fluxes between them. This analysis will reveal whether the various independently determined flux relationships are compatible and consistent with each other. If they are not, critical analyses of the computer output may suggest alterations in the initial assumptions made. Such alterations as assumptions about compartmentation, rate limiting steps, or the rate of equilibration of various pools can be tested, using TFLUX, to see if they provide a pattern more consistent with the experimental data. Analysis of the patterns of in vivo steady-state metabolism in yeast, hyphal, and transition cells should provide a rational approach to understanding the dynamics of metabolism in each type of cell population. This knowledge of flux patterns is also essential input to a transition model simulating the yeast to hypha transformation.
V. Information Required for a Candida Transition Model This model will be similar in kind to that described earlier in which glycogen metabolism in rat liver was simulated. The purpose of the Candidu transition model will be to analyze those events most critical in the change from yeast to
14
BARBARA E. WRIGHT
hypha metabolism. In connection with the steady-state models, information on flux, metabolite, and end product accumulation patterns would be obtained. The additional kind of data required for a transition model is the characterization of critical enzymes in the Candida system. Fortunately, ~-glutamine-D-F6Paminotransferase, chitin synthase, and phosphofructokinasehave already been characterized in this system (Chiew er d . , 1980; Chattaway er al., 1973). The kinetic mechanism and constants for a given enzyme, regardless of the source from which it was purified, are frequently similar. Therefore, until information is available for the other key enzymes in Candida, literature data for these enzymes isolated from similar sources (e.g., yeast) could be used in a preliminary model. Trehalose and glycogen synthase would fall into this category. These enzymes and perhaps ~mannose-6-phosphateketol isomerase (Gracy and Noltmann, 1968) would be among the most critical to purify and characterize in the Candida system. In summary, the following information would be required as input for a transition model: (1) a metabolic map (Fig. 6); (2) initial metabolite concentrations (i.e., in the yeast); (3) enzyme kinetic expressions and constants for the most critical reactions, and mass action expressions for the others; (4) enzyme activation functions and rate constants (see earlier discussion); and (5) time functions for external flux rates and for “independent metabolites.” The enzyme activation functions and rate constants would be adjusted such that the metabolite concentrations and flux relationships at r = 0 correspond to the data and to the steady state model of yeast metabolism. Model parameters would then be altered individually and in combination, attempting to simulate a transition, within a realistic time frame, from the yeast to the hyphae flux patterns, metabolite, and end product accumulation patterns (e.g., a fourfold higher concentration of chitin). Hopefully, as in the case of the Dictyostelium transition models (Wright and Kelly, 1981), the Candida models would provide testable predictions and define critical variables controlling the transformation from the yeast to the hypha metabolism.
VI. Discussion Although many kinetic models have been constructed and appear in the literature, most are primarily theoretical in nature. Of the more strongly data-based models, relatively few have been tested, i.e., there has been no persistent experimental follow-up to refute or substantiate assumptions and predictions inherent in the models. Most models tend to be a summary, or the end of an investigation, rather than an integral part of a continuing investigation. Thus far, in the analysis of metabolism in organisms such as Candida albicans, flux studies are relatively rare, perhaps because they are difficult to
THE MODELING APPROACH
15
interpret. However, these studies are essential to the construction of kinetic models, which in turn are essential for a realistic understanding of metabolism in living cells (Wright and D avison, 1980; Wright and Kelly, 1981). The Cundidu problem is only one of many which would benefit from a strongly data-based, dynamic systems analysis of the kind described in this article. Such transition models are currently being used to understand carbohydrate and energy metabolism in Dictyostelium (Wright and Emyanitoff, 1982) and purine metabolism in human fibroblasts (Holland and Kelleher, 1980). Many other systems are sufficiently described to make them amenable to the modeling approach. These include, for example, glycolysis during sporulation in Succhuromyces (Fonzi et al., 1979) and in Bacillus (Donohue and Bernlohr, 1978); citric acid cycle metabolism in suspension cultures of Paul’s Scarlet Rose (Fletcher and Beevers, 1970; Hunt and Fletcher, 1976); cellulose synthesis in Acanthamoebu (Potter and Weisman, 1976); chitin synthesis in Blustocladiellu (Selitrennikoff et ul., 1976); ornithine metabolism in Neurosporu (Davis et ul., 1978; Legerton and Weiss, 1979); and industrial processes such as citric acid production in Aspergillus (Kubicek and Rohr, 1978; Smith and Ng, 1972).
ACKNOWLEDGMENTS This work was supported by Public Health Service Grants AGO0260 and AGO0433 from the National Institutes of Health.
REFERENCES Anderson, P. J., and Wright, B. E. (1980). Inr. J. Biochem. 12, 361-369. Anderson, P. J., Rotenberg, S. A,, Moms, H. P.,and Wright, B. E. (1980). Int. J . Biochem. 12, 37 1-378. Arnold, W. N., and McLellan, M. N. (1975). fhysiol. Chem. fhys. 1, 369-380. Bhattacharya, A., Puri, M., and Datta, A. (1974). Biochem. J . 141, 593-595. Braun, P. C., and Calderone, R. A. (1979). J. Bacreriol. 140, 666-670. Chattaway, F. W., Holmes, M. R., and Barlow, J. E. (1968). J. Gen. Microbiol. 51, 367-376. Chattaway, F. W., Bishop, R., Holmes, M. R., and Odds, F. C. (1973). J. Gen. Microbiol. 75, 97-109. Chiew, Y. Y., Shepherd, M. G., and Sullivan, P. A. (1980). Arch. Microbiol. 125, 97-104. Davis, R. H., Bowman, B. J., and Weiss, R. L. (1978). J. Supramol. Srruct. 9, 473-488. Donohue, T. J., and Bernlohr, R. W. (1978). J . Bacreriol. 135, 363-372. Fletcher, J. S . , and Beevers, H. (1970). flanrfhysiol. 45, 765-772. Fonzi, W. A., Shanley, M., and Opheim, D. J. (1979). J. Bacreriol. 137, 285-294. Fruton, J. S . , and Simmons, S. (1960). In “General Biochemistry,” p. 461. Wiley, New York. Gracy, R. W., and Noltmann, E. A. (1968). J. Biol. Chem. 243, 3161-3168. Heath, D. F., and Threlfall, C. J. (1968). Biochem. J. 110, 337-361.
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Hess, B., and Brand, K. (1965). In “Control of Energy Metabolism” (B. Chance, R. Estabrook, and S. Williamson, eds.), pp. 11 1-222. Academic Press, New York. Hill, D. W., and Gebhardt, L. P. (1965). Proc. SOC.Exp. B i d . Med. 92, 640-644. Holland, M. J. C., and Kelleher, J. (1980). J. Cell Biol. 87 (Abstr.). Hunt, L., and Fletcher, J. S. (1976). Plant Physiol. 57, 304-307. Katz, J., and Wood, H. G. (1960). J. Biol. Chem. 235, 2165-2177. Katz, J . , Landau, B. R., and Bartsch, G. E. (1966). J . Biol. Chem. 241, 727-740. Katz, J . , Wals, P. A,, and Van de Velde, R. (1974). J. Biol. Chem. 249, 7348-7357. Kelly, P. J . , Kelleher, J., and Wright, B. E. (1979a). Biochem. J . 184, 589-597. Kelly, P. J . , Kelleher, J., and Wright, B. E. (1979b). Biochem. J. 184, 581-588. Kot, E. J., Olson, V. L., Rolewis, L. J., and McClary, D. 0. (1976). Antonie van Leeuwenhoek 42, 33-48. Kubicek, C. P., and Rohr, M. (1978). Eur. J . Appl. Microbiol. Biotechnol. 5 , 263-271. Legerton, T. L., and Weiss, R. L. (1979). J . Bacteriol. 138, 909-914. Mackenzie, D. W. R. (1974). Sabouraudia 3, 225-232. Manjeshwar, R., Sharma, C., Moms, H. P., Donnelly, A. J., and Weinhouse, S. (1965).Adv. Enz. Regul. 3, 317-324. Manning, M., and Mitchell, T. G. (1980). J . Bacteriol. 142, 714-719. Munch-Peterson, A. (1955). Acta Chem. Scand. 9, 1523-1535. Nickerson, W. J. (1963). Bacreriol. Rev. 27, 305-324. Nordlie, R. C. (1972). In “The Enzymes” (P. Boyer, ed.), 3rd ed. Vol. 4, pp. 543-610. Academic Press, New York. Nordlie, R. C., and Anon, W. J. (1964). J . Biol. Chem. 239, 1680-1685. Nozu, K., Takagi, S., Kaniki, T., and Kashiwara, M. (1958). Eikens J. 1, 35-44. Odds, F. C. (1979). “Candida and Candidosis.” Univ. Park Press, Baltimore, Maryland. Potter, J. L., and Weisman, R. A. (1976). Biochim. Biophys. Acta 428, 240-252. Randle, P. J . , England, P. J . , and Denton, R. M. (1970). Biochem. J. 117, 677-695. Rao, G. R., Ramakrishnan, T., and Sirsi, M. (1960). J. Bacteriol. 80, 654-658. Rao, G. R., Sirsi, M., and Ramakrishnan, T. (1962). J. Bacteriol. 84, 778-783. Reich, J. G., Till, U.,Gunther, J., Zahn, D., Tschisgale, M., and Frunder, H. (1968). Eur. J. Biochem. 6, 384-394. Salas, J., Salas, M., Vinuela, E., and Sols, A. (1965). J. Biol Chem. 240, 1014-1018. Sato, K., Weinhouse, S., and Morris, H. P. (1973). Adv. Enz. Regul. 11, 343-358. Sauer, F., Ertle, J. D., and Binns, M. R. (1970). Eur. J . Biochem. 17, 350-363. Selitrennikoff, C. P., Allin, D., and Sonneborn, D. R. (1976). Proc. Natl. Acad. Sci. U.S.A. 73, 534-538. Shepherd, M. G., and Sullivan, P. A. (1976). J . Gen. Microbiol. 93, 361-370. Shepherd, M. G . , Chin, C. M., and Sullivan, P. A. (1978). Arch. Microbiol. 116, 61-67. Sherwood, P., Kelly, P., Kelleher, J. K., and Wright, B. E. (1979). Comp. Prog. Biomed. 10, 66-74. Singh, B., and Datta, A. (1979). Biochim. Biophys. Acta 583, 28-35. Smith, J . E., and Ng, W. S . (1972). Can. J . Microbiol. 18, 1657-1664. Tashdjian, C. L., and Kozinn, P. J. (1970). Ann. N . Y . Acad. Sci. 174, 606-622. Ton, S. H . , and Karunairatnam, M. C. (1976). Sourheast Asian J . Trop. Med. Publ. Health 7, 359-362. Tumquist, R. L., and Hansen, R. B. (1973). I n “The Enzymes’’ (P. D. Boyer, ed.), Vol. 8, pp. 51-69. Academic Press, New York. Wang, C. H. (1972). In “Methods in Microbiology” (J. R. Norris and D. W. Ribbons, eds.), Vol. 6B, Ch. 7. Academic Press, New York. Ward, J. M., and Nickerson, W. J. (1958). J. Gen. Physiol. 41, 703-721.
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Weber, G . , and Lea, M. A. (1966). Adv. Enzyme Regul. 4, 115-145. Wright, B. E., and Davison, P. F. (1980). Mech. Aging Dev. 12, 213-219. Wright, B. E . , and Emyanitoff, R . G. (1983). “Fungal Differentiation” (J. Smith, ed.), Ch. 2, pp. 19-41. Dekker, New York. Wright, B . E . , and Gustafson, G. L. (1972). J . Biol. Chem. 247, 7875-7884. Wright, B. E., and Kelly, P. J . (1981). Curr. Top. Cell. Regul. 19, 103-158. Wright, B. E., Thomas, D. A., and Ingalls, D. J . (1982). J . B i d . Chem. 257, 7587-7594. Yamaguchi, H . , Kanda, Y . , and Iwata, K . (1971). Sabouraudia 9, 221-230. Young, G . (1958). J . Infect. Dis. 102, 114-120.
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INTERNATIONAL REVIEW OF CYTOLOGY. VOL. 87
Protein Diffusion in Cell Membranes: Some Biological Implications MICHAEL MCCLOSKEY AND MU-MINGPo0 Department of Physiology and Biophysics, Universiry of California, Irvine, California Introduction ........ Protein Diffu ....................... A. Experimental Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Biological Scope of Measurements........................ C. Temperature Dependency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Short- versus Long-Range Diffusion E. Diffusional Constraints . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Implications in Membrane Biology. ........................... A. Pattern Creation and Maintenance ........................ B. Reactions in Two Dimensions.. .......................... C. Biological Signaling at the Membrane D. Self-Assembly and Sorting .............................. IV. Closing Remarks ..... ......................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.
11.
19 20 20 21 22 24 25 34 34 45 59 66
I1 12
I. Introduction In the overall economy of a living cell, what difference does it make whether membrane proteins are capable of rapid diffusion or immutably immovable? How significant is the observation that a given protein has a translational diffusion coefficient of, say, and not lop9 cm*/second? Current efforts to capture and quantitate the motion of proteins in membranes are predicated on the assumption that this phenomenon has important biological implications. Our purpose here is to review some of the bases of that assumption, to speculate further on how passive diffusion of membrane proteins contributes directly to certain biological functions, and to consider how cells might deal with some of the potentially negative consequences of protein lateral motion. The intent is to provide an interpretive overview rather than a comprehensive summary of all published work in the field. Interested readers may find other recent reviews useful (Cherry, 1979; McConnell, 1979; Shinitzky and Henkart, 1979; Jacobson, 1980; Edidin, 1981; Jacobson and Wojcieszyn, 1981; Peters, 1981; Schlessinger and Elson, 1981; Webb et al., 1982; Axelrod, 1983). In 1968-1969 the seminal observation that hydrophobic regions of nerve and 19 Copyright 0 1984 by Academic Press. Inc. All rights of reproduction in any form reserved. ISBN 0-12-364487-9
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MICHAEL MCCLOSKEY AND MU-MINGPO0
muscle membranes are “low-viscosity liquid-like’’ bilayers sparked a revolution in cell biology which has profoundly influenced present concepts of membrane structure (Hubbell and McConnell 1968, 1969). During the intervening 15 years there has been an explosion of empirical and theoretical research dealing with molecular motion and structural dynamics in both model and biological membranes. As a result, our knowledge of characteristic kinetic and thermodynamic properties of simplified lipid and lipid-protein systems has advanced rapidly. On the other hand, our ability to explain the workings of biological membranes in terms of those pure physical-chemical properties has not kept pace. Consider the following as illustrations of this point: (1) On the basis of a number of observations one must infer that bilayer fluidity or some closely related property is essential for normal life processes. This has been apparent for some time, and while qualitative rationale abounds an unequivocal molecular explanation does not exist. (2) Except for the erythrocyte we do not know what structural constraints govern the slower than theoretical diffusion rates of many integral membrane proteins. (3) Where there is striking long-range order in the lateral distributions of different lipid and protein species, we do not know what generates and maintains this topography. (4)On a smaller distance scale, say I0.1 pm, the homogeneity of the separate classes of lipids and proteins in cell membranes is largely unknown. ( 5 ) Although cholesterol is a ubiquitous and prominent constituent of animal cell plasma membranes, and though there exists an impressive catalogue of peculiar effects attributable to cholesterol in model systems, we do not know the primary function(s) of cholesterol in membranes. These questions provide an inkling of the true structural complexity of biomembranes. It is our belief, however, that persistent mechanistic dissection of the physical-chemical aspects of membrane-mediated events will eventually yield major clues about how biological membranes work.
11. Protein Diffusion: Known and Unknown A. EXPERIMENTAL TECHNIQUES
Since the initial finding in 1970 that surface histocompatibility antigens on mouse-human heterokaryons rapidly intermix after cell-cell fusion (Frye and Edidin, 19701, our understanding of protein mobility in cell membranes has taken a circuitous path. In 1973-1974 the first reported measurements of the rate of lateral diffusion led to the belief that membrane proteins are capable of “rapid” diffusion, with lateral diffusion coefficients in the range of 1 to 5 X IOW9 cm2/second (Edidin and Fambrough, 1973; Po0 and Cone, 1974; Liebman and Entine, 1974). Subsequent results using the method of fluorescence recovery after photobleaching (FRAP) have until recently yielded a 10 to lo3 times slower
PROTEIN DIFFUSION IN CELL MEMBRANES
21
lateral diffusion coefficient for membrane proteins. Moreover, in nearly all measurements with this method, a substantial fraction of the protein has appeared immobile for the duration of an experiment, usually ca. 30 minutes. Besides FRAP, a variety of other techniques has been employed to monitor protein lateral diffusion: intermixing of membrane components after chemically or virally induced cell-cell fusion (Frye and Edidin, 1970; Fowler and Branton, 1977; Schindler et al., 1980b); lateral spreading of locally applied labels (Edidin and Fambrough, 1973); redistribution of membrane proteins after electric field induced migration (Poo, 1981); passive accumulation of specific receptors at a ligand template (Michl et al., 1979); recovery of channel activity in locally inactivated zones (Poo, 1982). Most of the quantitative results, however, have come from FRAP. In this method, a fluorescent molecule like fluorescein or rhodamine is covalently attached to a probe, e.g., an antibody, lectin, or hormone, and the cell is treated with this specific fluorescent probe to label a particular membrane protein. Fluorescence on the cell surface is then irreversibly bleached in one or more regions by brief exposure to a high-intensity laser beam tuned to the excitation maximum of the fluorophore. The rate of recovery of fluorescence at the bleached region(s) is used to calculate a diffusion coefficient (Dvalue), assuming that recovery is due to lateral diffusion of proteins from the nearby unbleached regions of the membrane. The extent of recovery for proteins in biological membranes is, as a rule, less than 100%. Some recent experiments excluded, typical D values fall in the range of to lo-'* cm*/second, about lo3 to lo5 times slower than an average soluble protein in aqueous solution. On the other hand, isolated integral proteins diffuse at close to the theoretical limit (ca. lo-* cm*/second) and with nearly complete recovery when incorporated into artificial bilayers composed of fluid phase phospholipids. Fluorescent lipid analogs are generally rapidly mobile (ca. l o p 8 cm2/second)in both synthetic and biological membranes. Recovery is usually high for lipid diffusion in both systems.
SCOPEOF MEASUREMENTS B. BIOLOGICAL With few exceptions (Stuhmer and Almers, 1982; Young and Poo, 1983) protein mobility has not been studied in intact tissues; isolated single cells of four principal classes have been the preferred objects of study. These include various cells from the blood and lymph, primary animal cell cultures, established cell lines, and vertebrate photoreceptors. Consequently, little is known about the diffusibility of membrane proteins in intact multicellular animal tissues. As will be discussed in a later section, there is now evidence that intercellular interaction can provide a passive localization mechanism for proteins in the animal cell plasma membrane. Metcalf et al. (1982) have published the only direct measurement of protein lateral diffusion in plant cell membranes of which we are aware.
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MICHAEL MCCLOSKEY AND MU-MING PO0
Although the number of membrane proteins whose mobility has been examined continues to grow, there are some conspicuous gaps. As a class, peripheral proteins have not received adequate attention. Cytochrome c mobility in mitochondrial membranes has been directly assessed for the first time only in the last year, although its capacity to serve as a freely diffusing lateral shuttle of electrons has been discussed for some time (see Section III,B,2). Other peripheral proteins whose biological function(s) may depend upon lateral diffusion are plastocyanin, ferredoxin, and ferredoxin-NADP reductase in chloroplast thylakoids, phosphodiesterase and GTPase complex in rod outer segments, and perhaps the G-unit of hormone-activated adenylate cyclase in a host of cell types. Some cell surface proteoglycans, e.g., heparan sulfate (HS), are believed to be anchored to the membrane by a hydrophobic polypeptide (Kjelltn et al., 1981) as well as by specific interaction of the carbohydrate moieties with other membrane associated components, e.g., fibronectin; recent reports on the colocalization of heparan sulfate and ACh receptors in membranes of developing muscle (Anderson and Fambrough, 1982; Bayne et al., 1982) have heightened interest in the lateral mobility of surface bound HS, and it is likely that HS-ACh receptor coaggregates are assembled by a diffusion-dependent process. Changes in the type and amount of surface exposed proteoglycans accompany a variety of biological events such as mitosis (Kraemer and Tobey, 1972; Kojima and Koizumi, 1974), phagocytosis (Cappelletti et al., 1980), lymphocyte blastogenesis (Hart, 1982), and malignant transformation (Sampaio et al., 1977). Purified glycosaminoglycans can exert both inhibitory and stimulatory effects on cell division and growth (Chiarugi and Vannucchi, 1976; Takeuchi et al., 1976), and the aberrent adhesive properties of cancerous cells may arise in part from altered expression of cell surface proteoglycans. Aside from the potential significance of lateral diffusion in specific regulatory and recognition/ adhesion functions suggested for proteoglycans, from a purely structural standpoint some knowledge of the motional properties of glycocalyx constituents seems desirable, simply because the cell coat is such an intimate part of most animal cell plasma membranes (Rambourg, 1971; Luft, 1976). C. TEMPERATURE DEPENDENCY In view of the widespread interest in phase behavior of lipid-protein systems and the dramatic effects that lateral phase separations can have on protein diffusion in model bilayers (Wu et al., 1978; Smith et al., 1980; Vaz et al., 1981; Peters and Cherry, 1982), it is surprising that few systematic studies on the temperature dependence of protein diffusion in biomembranes have been done. In light of the current emphasis on cytoskeletal control of protein diffusibility and the lability of microtubules at low temperatures (Olmsted and Borisy, 1973), the common practice of holding freshly isolated cells on ice prior to FRAP measure-
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ments is also somewhat suspect. In fact, cell deformability-and presumably the degree of polymerization and/or cross-linking of cytoskeletal polymers-can change appreciably between 37 and 4°C (Lichtman, 1970; Petersen et al., 1982). Because cytoplasmic levels of metabolites like ATP (Schindler et al., 1980a; Edidin and Wei, 1982), membrane potential (Edidin and Wei, 1982), or other conceivably temperature-sensitive factors may influence protein mobilities, the issue of temperature assumes even further significance. This seems especially pertinent for those cells removed from a native environment where temperature is usually closely regulated about values near 37”C, i.e., warm-blooded animals and incubators. In isolated membranes, several enzymes and transport proteins show breaks in their Arrhenius plots at characteristic temperatures beneath the host organism’s normal growth temperature (Kimelberg, 1977; Quinn, 1981; Ohyashiki et al., 1982; Brasitus, 1983; Rimle et al., 1983). As an isolated fact this could be explained by processes unrelated to thermotropic transitions in the bilayer. For example, a fairly temperature-dependent pK of an ionizable group in the active site or an altered K, due to a directly induced shift in an equilibrium between two conformational states of the protein might explain it. However, there are correlated changes in partitioning of small lipid-soluble molecules, tumbling of fluorescent probes, EPR and NMR order parameters, intensities of characteristic Raman and infrared bands, etc., which many of us take as evidence for the onset or completion of lipid phase separations. There is thus a widely shared belief that abrupt changes in the apparent activation energies for some enzymatic reactions and transport processes of membrane proteins are caused by thermally driven lipid phase changes; it is logical to hypothesize that part of the reason for “unexpectedly” low D values of cell membrane proteins is that surrounding lipids are partially solidified or otherwise segregated at the usual experimental (room) temperature. Available evidence on the temperature dependence of protein diffusion in cell membranes provides little support for this hypothesis. Using biotin depletion and fatty acid supplementation to enrich the membranes of chick myotubes with either high melting (elaidic) or low melting (oleic) fatty acids, Axelrod et al. (1978) found no more than a threefold change in the lateral diffusion coefficient of ACh receptors upon shifting the temperature from 12 to 3 1°C-for elaidate enriched, oleate enriched, or control cells. Similarly, over the interval 5 to 37”C, Hillman and Schlessinger (1982) found a modest threefold decrease in the lateral diffusion rate of ligated EGF receptors on a human epidermal cell line. Petit and Edidin ( 1974) witnessed a curious biphasic temperature dependence for intermixing of cell surface antigens on mouse-human heterokaryons which they attributed to lipid phase separations. As the temperature was lowered from 42 to 5°C mixing rates first dropped, then peaked at 15”C, then fell monotonically. It was suggested that reduction of the total diffusion space by channeling of pro-
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MICHAEL MCCLOSKEY AND MU-MING PO0
teins within fluid paths of a solid-fluid mosaic enhanced the macroscopic diffusion rates and resulted in faster mixing at 15”C, but subsequent theoretical calculations have cast some doubt on this interpretation (Saxton, 1982). If one can extrapolate from this limited sample to other proteins and cells, how is he to rationalize the apparently small temperature dependency of protein diffusion with the above mentioned indications of thermotropic transitions? It is possible that extrinsic constraints are so great that lipid phase changes produce little extra effect. A more intriguing possibility which has found recent experimental support is that living organisms may suppress solid to fluid transitions observed in isolated membranes by promoting lipid fluidity through currently unknown processes (Cameron et al., 1983). D. SHORT-VERSUS LONG-RANGE DIFFUSION The possibility that biomembranes are laterally inhomogeneous over short distances accentuates one shortcoming of the FRAP method, namely, that it is only suited for measuring “long-range” (11 pm) lateral diffusion. The best one can do is obtain an average over all the short-range or “microscopic” D values which may exist. This limited spatial resolution is significant for a few reasons. First, it would be useful to be able to directly measure lateral diffusion of proteins and lipids in small cells like bacteria and sperm, and also in organelles such as the Golgi complex, endoplasmic reticulum, and thylakoid or mitochondrial membranes. FRAP is in general not applicable to these relatively small systems (for exceptions see Schindler et al., 1980b; and Hochman et al., 1983). Second, chemical reactions and other collision-dependent protein-protein interactions (e.g., self-assembly of multisubunit enzymes or channels) are probably more directly related to local than to long-range diffusion. Thus, if a 6receptor appears completely immobile using FRAP, one cannot say that it is incapable of reacting with cyclase by collisional encounters, because the local, or FRAP invisible, mobility may be significant. Of course, evidence of rotational immobility and/or visible aggregates is a separate matter. Third, heterogeneous microscopic D values would provide an indication that proteins are diffusing through locally inhomogeneous environments, and this might be a useful complement to biochemical studies aimed at characterizing short-range membrane structure. Short-range lateral diffusion of lipid analogs has been estimated with magnetic resonance techniques (Popp and Hyde, 1982, and references therein), triplet-triplet annihilation (Razi Naqvi et al., 1974), and pyrene excimer fluorescence (Galla and Sackmann, 1974); however, it is unlikely that any of these collision-dependentmethods will be extended to include proteins. On the basis of a recent study using spin labeled lipids, Laggner (1981) suggests that sar-
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coplasmic reticulum membrane contains local diffusion barriers holding about 50 lipid molecules. Several techniques for measuring the rotational motion of membrane proteins are now available (see Cherry, 1979; Johnson and Garland, 1981), and these offer two distinct advantages over FRAP methods. First, the immediate environment of a protein is reflected more directly by the rate of its rotational diffusion than that of its translational diffusion. Second, rotational diffusion is more strongly dependent on molecular diameters than is translational diffusion (Saffman and Delbruck, 1975; Cherry, 1979; Hughes et a l . , 1982). This latter makes rotation rates a valuable probe of small-scale protein-protein association (e.g., dimerization, oligomerization) .
E. DIFFUSIONAL CONSTRAINTS I . Slow Mobility and the “Immobile Fraction”-What Do They Mean? Much current interest in the field centers on interpretation of the slow mobility and apparent immobility of proteins in cell membranes. As noted earlier, diffusion coefficients of proteins in cell membranes as measured by FRAP methods have in general been much smaller than those expected for proteins in a fluid lipid bilayer. Furthermore, in most photobleaching studies fluorescence recovery is incomplete, often ranging from 20 to 70% of prebleached levels. Partial recovery presumably results from immobility of the remainder of the labeled protein, or “immobile fraction. Consistent with this interpretation is the finding that roughly 100%recovery is obtained after a few successive bleaches of the same region. In model phospholipid bilayers in the fluid state, intrinsic proteins usually have a very large mobile fraction and they diffuse nearly as fast as fluorescent lipid analogues, certainly within a factor of 5 (see references in Section 11,E,6). This finding is more or less consistent with the theoretical predictions of Saffman and Delbruck (1975) for diffusion in bilayer membranes, namely, that lateral diffusion coefficients are weakly dependent on molecular diameters and inversely proportional (roughly) to the length of the diffusing molecule within and normal to the bilayer. Hence, for a protein with bilayer-spanning segments the predicted D value is about half that for a typical lipid molecule, which spans only half the membrane. Yet in membranes of intact cells the difference in diffusion rates of lipid and protein is often apparently one to three orders of magnitude, a result clearly at odds with Saffman-Delbruck theory. Two limiting assumptions of the original Saffman-Delbruck formulation are that intrinsic bilayer viscosity is much greater than that of the bathing aqueous phase and that diffusing molecules do not project beyond the hydrophobic bilayer core. Typical estimates of viscosity for the hydrocarbon region of fluid lipid ”
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MICHAEL MCCLOSKEY AND MU-MING PO0
bilayers range from 1 to 10 poise (P), while that of water or physiological buffers is about 0.01 P. For synthetic lipid membranes the first assumption is reasonable and the second is therefore of little consequence; but just the opposite situation may exist in biomembranes. Hughes et al. (1982) have extended the Saffman-Delbruck theory to include lateral and rotational diffusion rates for all relative values of the bathing and bilayer viscosities. When applied to proteins and lipids for which both rotational and translational D values are available, the new calculations are more consistent with a high bathing viscosity (ca. 1 P) and a relatively low bilayer viscosity (ca. 0 . 2 P). One gathers that hydrophilic protein segments protruding into the aqueous phase may experience much more viscous drag than those within the bilayer, a notion to which we will return later in a discussion about the role of cell surface glycocalyces in restricting protein mobility. It is generally felt that constraints imposed by extramembranous macromolecules account for the slower than expected protein mobility, and the prevailing emphasis is on the role of cytoplasmic constraints. This and other possible mechanisms will be discussed in the following sections. It is worth noting, however, that even if we can pinpoint the cause(s) of slow diffusion, the existence of an immobile fraction also demands clarification. The puzzling fact is that any protein, whether it is a homogeneous or heterogeneous molecular population, always appears to contain an “immobile” subpopulation when examined with FRAP. Is there an anchoring mechanism (say cytoskeleton connection) that always works on a fraction of all the proteins in the membrane? Or, is there perhaps a wide spectrum of diffusion rates for any given protein, with the D value measured by FRAP representing an average rate of detectable diffusion, and the “immobile” fraction representing that subpopulation whose diffusion is too slow to measure? Whatever the explanation it will make a great difference in terms of our ideas about protein diffusion in cell membranes.
2. The Cytoskeleton There is clear evidence that cytoskeletal structures can somehow impede the motion and alter the distribution of integral proteins. Studies purporting to document the effect are far too numerous to cite here. Discussed below are results from three systems where FRAP has been used to demonstrate a direct correlation between cytoskeletal alterations and changes in lateral diffusion rates. a. The Erythrocyte. For the erythrocyte there is compelling evidence that lateral motion of band 3 and other intrinsic proteins is severely retarded by the spectrin-actin-ankyrin matrix which subtends the membrane. In normal mouse erythrocytes band 3 has a lateral D value of 4.5 X lo-” cm*/second at 24”C, while in erythrocytes from mice with a hereditary deficiency of cytoskeltal components the corresponding value is 55-fold greater (Sheetz et al., 1982). In human erythrocyte ghosts a reversible 50-fold increase in the lateral D value and
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a jump in the mobile fraction (10 to 90%) of band 3 results from manipulations of temperature (21 to 37°C) and buffer concentration which are believed to destabilize the cytoskeleton and specifically disrupt band 3-ankyrin interactions (Golan and Veatch, 1980, 1981). Polyphosphate compounds like ATP and 2,3-diphosphoglyceric acid which partially depolymerize the erythrocyte cytoskeletal network cause a moderate (ca. 5 X ) increase in the lateral D value of band 3 (Schindler et al., 1980a; Sheetz et al., 1982). Based upon their work with the erythrocyte Koppel et al. (1981) worked out a detailed mathematical model of integral protein diffusion which treats the cytoskeleton as a matrix with labile cross-links whose rate of formation and breakage sets the observed lateral diffusion rate of intrinsic proteins. Using this conceptual framework they obtain a result consistent with the erythrocyte viscoelastic mechanical properties measured by Evans and Hochmuth (1978). Hypotheses on the nature of band 3-cytoskeleton interactions have come full cycle in the last decade. Original electron microscopic studies led to the conclusion that most of band 3 is directly bound to cytoskeletal polymers (Pinto da Silva and Nicolson, 1974; Elgsaeter e t a l . , 1976; Yu and Branton, 1976). Cherry et al. (1976) then observed that essentially complete elution of spectrin from the erythrocyte membrane did not alter the rotational diffusion rate of band 3, and concluded that no direct linkage could exist between band 3 and the cytoskeleton. In subsequent work the same group discovered that proteolytic cleavage of a cytoplasmic portion of band 3 significantly enhanced its rotational diffusion, and that elution of ankyrin with salt treatments also released constraints on the rotational motion of band 3 (Nigg and Cherry, 1980). They concluded that as much as 40% of band 3 is directly bound to ankyrin, a component of the erythrocyte cytoskeleton which links spectrin to the membrane (Bennett and Stenbuck, 1979; Luna et al., 1979; Yu and Goodman, 1979). Consistent with this notion, Golan and Veatch (1981) found that selective proteolytic cleavage of ankyrin totally releases diffusive constraints on band 3, and that addition of the released ankyrin fragment to untreated ghosts causes a modest rise in lateral mobility of band 3. Bennett and Stenbuck (1979) also showed that solubilized band 3 binds to purified ankyrin. Hence, current evidence does not favor the hypothesis that purely spatial barriers limit band 3 lateral motion, although as Koppel et al. (1981) point out, steric hindrance may well be the major impediment to long-range diffusion of the “mobile” fraction of band 3. b. Anchorage Modulation. ‘‘Anchorage modulation” refers to the inhibitory effect of Con A binding on the patching of several antibody cross-linked membrane proteins in various cell types (Edelman, 1976). The effect is global in that binding of Con A to local regions will inhibit patching over the entire cell surface. Because patching is a passive diffusion-mediated process is has been assumed that Con A inhibits patching by reducing receptor diffusion rates. Anchorage of these potentially patchable proteins is presumably controlled by
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cytoskeletal constituents assembled into a hypothetical “surface modulating assembly” (Edelman, 1976). Recently it was shown directly that localized binding of Con A to mouse lymphocytes reduces the mobility of cell surface IgG (Henis and Elson, 1981). In the presence of Con A the D value and mobile fraction determined by FRAP are about sixfold less than in untreated cells. As with patching inhibition, this effect is global and occurs at a threshold value of about 10% coverage. Above 10% the response is coverage independent and beneath it no effect is observed. The strength of this experiment lies mainly in the finding that colchicine and cytochalasin B act synergistically to return both the D value and mobile fraction of surface IgG on Con A-treated lymphocytes to control values. Here it departs significantly from many previous experiments which had failed to document enhanced diffusion upon treatment with chemicals known to depolymerize microfilaments and microtubules. In accordance with prior studies these pharmacological agents did not raise D values or mobile fractions above control values on cells not treated with Con A. One may surmise from this work that cytoskeletal elements are somehow participating in Con A induced immobilization of surface IgG; nevertheless, the experiment reveals nothing whatsoever about why IgG diffusion is restricted to 5.3 X 10- l o cm2/second on untreated cells-and that seems to be a major question. c. Blebs and Ballooned Cells. A recent intriguing result is that lateral diffusion coefficients of several integral proteins are remarkably increased in plamalemma “blebs” on various cell types and in “bulbous” lymphocytes;both structures are induced to form by chemical treatment with formaldehyde/ mercaptans, phallacidin, or Con A (Tank et al., 1982; Wu et al., 1982). From cytochemical staining it appears that the plasma membrane in these structures is lifted off the underlying cytoskeleton; coincident with this structural alteration is a change in lateral D values of ACh, Con A and LDL receptors from less than 10- l o to about 3 X 10- cm2/second. Fluorescence recovery after photobleaching is also complete. Why is diffusion much faster in “blebs” than in untreated plasma membranes? Webb and associates favor the interpretation that loss of cytoskeletal connections to the plasmalemma releases diffusive constraints in “blebbed” membrane, but recognize that other factors could be involved. Until a rigorous biochemical analysis of lipid, protein, and carbohydrate constituents of “blebbed” membrane and plasmalemma fractions of bulbous cells is performed, other interpretations remain tenable. For example, externally disposed, highmolecular-weight components of the cell coat could be excluded from “blebbed” regions or lost from bulbous lymphocytes. In fact, Sugrue and Hay (1981) have demonstrated that corneal epithelial cells generate membranous blebs when stripped of their extracellular matrix, and that readdition of certain extracellular matrix constituents causes the blebs to retract. We have found (McCloskey et al., unpublished) that when rat basophilic leukemia or Xenopus cells are treated with
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glycosaminoglycan degrading enzymes or trypsin, large protrusions, or “blebs,” sometimes appear on the cell surface. One might speculate that they form where the cell coat has been thinned the most by enzymatic attack. Interestingly, in these protrusions the FRAP-determined lateral diffusion coefficients of IgE Fc and Con A receptors are rapid and recovery is nearly complete. While the above-mentioned ‘‘blebs” are artificially induced structures, blebs do arise spontaneously on cells from many types of animals, both in culture and in vivo. These are transparent, hemispherical protrusions about 2-5 p,m in diameter which form within seconds and then slowly recede (Albrecht-Buehler, 1981). The bleb interior contains ribosomes and other small inclusions but lacks filamentous structure; it is thought to be much more fluid than the remaining cytoplasm. In culture, blebs are most prevalent as cells flatten out after division or after being plated; they form a dynamic ferris-wheel array on cells in some developing amphibian embryos. It would be of interest to measure protein diffusion rates in naturally occurring blebs to see if constraints are relaxed as in the induced “blebs.” If so, it would imply that proteins are capable of sudden, large-scale changes in mobility during their residence on the cell surface.
3 . Intrinsic Diffusion Barriers Although extramembranous constraints are undeniably important, the possibility that lateral motion of integral membrane components is also hindered by structural heterogeneity of the bilayer or by direct interactions with other integral components has not been rigorously excluded. In bilayers consisting of a binary mixture of cholesterol and dimyristoyl phosphatidylcholine the coexistence of solid and fluid regions can dramatically influence long-range diffusion of fluorescent lipids (Rubenstein et a l . , 1980; Owicki and McConnell, 1980). Measured D values for the solid-fluid mixture differ by more than an order of magnitude from theoretical microscopic D values in either phase. Klausner and Wolf (1980) find that certain fluorescent lipid analogs are largely immobile (mobile fraction 25%) in a solid-fluid mixture of di-C,,- and di-(=,,-lecithin while other analogs are almost 100% mobile, and they interpret this as due to selective partitioning of the different probes into either solid or fluid domains. Although biomembranes probably do not often contain the percentage of solid phase lipids present in either of these model systems, solid-fluid phase separation is only one mechanism whereby intrinsic lipid organization could influence protein mobilities. Several cases of fluid-fluid immiscibility involving phospholipids are now known (for references, see Berclaz and McConnell, 1981). Given the selective lipid dependence and preferential lipid-protein association constants exhibited by proteins like cytochrome oxidase (Marsh et a l . , 1982) it is possible that some proteins would partition unequally between two fluid lipid phases. In this case the protein would diffuse rapidly within both phases but conceivably more slowly across the phase boundary. If the protein enriched phase happened to be dis-
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MICHAEL MCCLOSKEY AND MU-MING PO0
persed then the protein would be partially trapped, and its long-range diffusion somewhat impeded. Another potential intrinsic constraint in some systems is binding between the pendant oligosaccharide groups of integral proteins and glycolipids. The rotational motion of spin-labeled ganglioside head groups is restricted by interaction with glycophorin in reconstituted membranes (Sharom and Grant, 1978). Protein lateral diffusion might also be retarded if the interaction were multivalent for both protein and l i p i d d u e to multiple sites per molecule or to glycolipid clusters like those suggested by Sharom and Grant (1977, 1978). It is unlikely that the above constraints will fully explain the slow mobility or immobility, but they do merit consideration as potential contributions. Intrinsic diffusion limits of a different nature are mentioned in Section II,E,6. 4. Difluusion of Ligand-Free and Ligand-Bound Proteins
One serious reservation we have regarding FRAP-determined diffusion coefficients stems from a problem inherent to the experimental protocol. As generally practiced, photobleaching measures the diffusion not of native proteins but of extrinsically labeled species. Binding of specific ligands to cell surface receptors can have large effects on their mobility and lateral distribution. Examples of ligand-induced clustering or dispersal include the opioid peptide receptors (Hazum et a l . , 1980), thyrotropin receptors (Avivi et a l . , 1981), p-adrenoreceptors (Henis et a l . , 1982), chemotactic peptide receptor (Niedel et al., 1979), and receptors for hormones like EGF and insulin (Schlessinger et al., 1978). Oliver and Berlin (1982) discuss several cases of ligand-induced receptor migration in leukocytes and propose an “entrainment” of the ligand-receptor complexes in actively propagated membrane ‘‘waves. ” There is also the distinct possibility that lectin receptors may globally modulate their own mobility by signaling cytoplasmic attachments upon binding (Henis and Elson, 198I ) . Aside from these specific and sometimes biologically important effects, we are concerned with a possible nonspecific effect due to ligand binding. In particular, does the attachment of a large probe like an antibody Fab fragment impede diffusion of integral proteins due to its interaction with the thick, carbohydraterich cell coat (glycocalyx) which appears to surround all animal cells? Several experimental results bear directly on this issue. First we note that in most FRAP studies fluorescently labeled antibodies, antibody fragments, lectins, or other macromolecular ligands have been used to label the membrane proteins. As previously stated, the D values and percentage recovery are consistently lower than for integral proteins in reconstituted systems or for rhodopsin in the disk membrane. When a small (MW 441) fluorescent antagonist was used to label padrenoreceptors in Chang human liver cells the mobile fraction had a high D value of 1.4 X lop9 cm*/second at 23°C (Henis et al., 1982). Diffusion coefficients of free, or nonliganded proteins can be determined by back diffusion after
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in situ electromigration (Po0 er al., 1979) and postinactivation recovery of functional activity (Poo, 1982). Using these methods the diffusion coefficients for Con A and acetylcholine (ACh) receptors in embryonic Xenopus muscle cells were estimated to be in the range of 1-4 X cm2/second. Using postelectric field relaxation D. W. Tank et al. (personal communication) have estimated that unliganded LDL receptors on an internalization defective human fibroblast cell line diffuse at a rate of 1.1 X cm2/second (22°C). Using FRAP, this same group finds a D value of 1.4 X 10- cm2/second for the LDL-receptor complex at 21°C (Barak and Webb, 1982). They found no evidence for cytoskeletal disruption by the electric fields, and suggested that LDL particles may interact with extracellular matrix components and thus impede lateral motion of ligated LDL receptors. There is also more indirect evidence that ligand-free proteins may diffuse quite rapidly. For example, when murine macrophages are plated on an immunoglobulin G (1gG)-coated cover slip the IgG Fc receptors disappear from noncontacting portions of the plasma membrane. The process is not blocked by metabolic poisons but is prevented by soluble IgG. Assuming it is due to passive diffusion of receptors to the IgG “trap,” a high D value of about 2.5 X l o p 9 cm2/second was calculated for the free receptor (Michl et al., 1979, 1983). Although the effect of ligand binding on protein diffusion remains a debatable issue, its final resolution will have important implications not only in the interpretation of experimental results, but also for a number of biological processes, e.g., hormonal and immune responses.
5 . The Glycocalyx and Extracellular Influences In lipid-protein model systems, an artificial cell coat can have remarkable effects on both the binding and diffusion of macromolecular “ligands.” The affinity of wheat germ agglutinin (WGA) for lipid vesicles containing the integral protein glycophorin is increased tremendously by the presence of a mere monolayer of high-molecular-weight dextran (Ketis et al., 1980); WGA binding also appears to become cooperative when dextran is present. These effects are not unique to dextran: other high polymers and bovine serum albumin work just as well. Ketis and Grant (1982) also find that by butressing the relatively sparse cell coat of extensively washed human erythrocytes with a layer of adsorbed BSA they can substantially increase the number of high affinity WGA binding sites. Restriction of oligosaccharide rotational and translational mobility may be involved in this phenomenon. Indeed, spin labels on the terminal sugars of liposome-bound glycophorin report a 20% reduction in rotational diffusion rate when the liposomes are coated with dextran and immunoglobulin or BSA (Lee and Grant, 1979). When specific anti-nitroxide antibodies are bound to bilayers containing phospholipids with a nitroxide head group, the antibody-hapten complex diffuses at
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MICHAEL MCCLOSKEY AND MU-MING PO0
the same rate the lipid hapten does (Smith et al., 1979b). This is in the absence of an added “cell coat.” When a monomolecular layer of BSA is added to a similar system the lateral diffusion rate of the antibody-hapten complex is an order of magnitude smaller than the lipid hapten itself (Hafeman et al., 1981). One cannot help but conclude that the more firmly attached layer of proteoglycans, the covalently bound oligosaccharides of integral proteins, the carbohydrate chains of glycosphingolipids and accumulated detritus at the surface of real cells would provide a more effective diffusion barrier than a monolayer of BSA. We have tested this possibility by using FRAP to quantitate mobility of plasma membrane proteins in living cells after partially removing the cell coat. Subsequent to mild treatment with hyaluronidase, chondroitin lyase ABC, and Flavobacterium heparinase, there is as much as a 20% increase in the mobile fraction of soybean agglutinin bound to cultured Xenopus muscle cells, although the D value is apparently unchanged (Liu et al., unpublished observation). Whether more complete removal of the same glycosaminoglycans or other glycocalyx constituents would further increase the mobile fraction remains to be determined. Rapid diffusion of integral proteins in spectrin-depleted erythrocytes appears inconsistent with the glycocalyx being a significant diffusional constraint; however, of cells which have been stained for cell coat material the erythrocyte seems to have the thinnest and least dense coat of all (Luft, 1976). Furthermore, directly bound, small-molecular-weightfluorophores rather than antibodies have usually been used to label the erythrocyte proteins for diffusion studies. For further discussion of the glycocalyx, see Section 111,A,6. 6 . What Determines the “Viscous Limit” ? Why is lateral diffusion of integral proteins often appreciably faster in artificial lipid bilayers than in any of the natural membrane systems where extrinsic constraints are presumed to be absent? Diffusion is now generally referred to as “rapid” (D 2 cm2/second) for rhodopsin in disk membranes, band 3 in spherocytic erythrocytes, and several different proteins in blebbed membranes. It is commonly accepted that diffusion in this regime is limited soley by lipid “viscosity. ” However, dimyristoylphosphatidylcholine (DMPC) at 25°C is bound to be more viscous than disk membrane phospholipids, which contain on average about six cis double bonds per molecule. Yet purified bacteriorhodopsin diffuses about six times faster in fluid DMPC than does rhodopsin in the disk membrane (Peters and Cherry, 1982). Similarly, purified band 3 protein diffuses about sixfold faster in DMPC than it does in spherocytic erythrocytes (Chang et al., 1981; Koppel et al., 1981).’ ‘Other integral membrane proteins found to diffuse at 5 cm2/second when reconstituted into phospholipid bilayers include murine histocompatibility (H-2K) and vesicular stomatitis G proteins
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Part of this disparity could easily derive from an increased bilayer viscosity due to the high protein content (ca. 50 wt%) of biological membranes relative to that of most reconstituted systems previously studied with FRAP. Cherry et al. (1977) found a 50-fold decrease in the rotational diffusion coefficient of bacteriorhodopsin in DMPC bilayers as the protein content was increased from 25 to 63 wt%. In analogy with the marked effects of dissolved soluble proteins on water viscosity (Fahey and Green, 1938; Treffers, 1940), they ascribed the drop in bacteriorhodopsin rotational rates to an increase in lipid viscosity due to the added protein. The observation of Jacobson et al. (1981) that lateral diffusion of lipid analogs is significantly faster in multibilayers constructed from fibroblast plasma membrane lipids than in the plasma membrane itself is also consistent with an effect of the protein on lipid viscosity, although the possibility of probe-protein binding also exists. The same argument applies to the finding that di-I diffusion in inner mitochondria1membrane preparations can be enhanced ca. three- to fourfold by dilution of the proteins with exogenously added phospholipid (Chazotte et al., 1983). Should integral proteins in reconstituted systems not be faithfully integrated into the bilayer then some enhancement of diffusion might ensue. As mentioned in Section II,E,3, lipid phase boundaries could restrict protein diffusion, and some NMR work on disk membrane lipids is consistent with the existence of two lipid domains, fluid and less fluid, with rhodopsin concentrated in the former (Brown et al., 1977). Adsorbed proteins like the GTP binding protein and phosphodiesterase in photoreceptor disk membrane, serum proteins on cultured cells and erythrocytes, or general nonfilamentous cytoplasmic proteins may also limit translational diffusion of integral proteins which protrude significantly into the aqueous phase. The effect of adsorbed BSA on diffusion coefficients cited in the previous section is in line with this idea. Finally, the intrinsically discrete (molecular) structure of membranes may help account for the discrepancy between the “viscous limit” in natural and model membranes. Saffman-Delbruck theory is a hydrodynamic model which treats the membrane as a featureless continuum characterized by a bulk viscosity. Yet we know that the third dimension of “two-dimensional” biomembranes is often asymmetric, with a unique lipid composition in each monolayer (see Smith ef ul., 1977, for evidence on photoreceptor disk membrane). Moreover, separate monolayers in these (Tanaka and Ohnichi, 1976; Schroeder, 1980) and artificial membranes (Seul et al., 1983) can apparently undergo independent phase changes. Diffusion in isotropic fluids may depend upon density fluctuations which open up local voids of free volume (Cohen and Turnbull, 1959). Is it (Cartwright et al., 1982); glycophorin (Vaz et al., 1981; Wu et al., 1981); the coat protein of M-13 phage (Smith ef 01.. 1979~);cytochrorne P-450 (Wu and Yang, 1980); and lipophilin (Derzko and Jacobson, 1978).
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possible that in anisotropic bilayers the diffusion of large transmembrane proteins is limited by the independent, and unequally probable occurrence of critically sized voids in opposing monolayers? It might be of interest to construct artificial bilayers both with and without pronounced transbilayer compositional differences and compare the diffusion rates of purified proteins therein. Related to the discontinuous structure of bilayers, we note that proteins can influence local lipid order, and that in principle this can lead to either attractive or repulsive interactions between the proteins (see references in Section III,A,2). Since these effects are most important at high protein concentrations, they must be considered when comparing diffusion rates in biological and model membranes. Such treatment is beyond our present scope however.
111. Implications in Membrane Biology A. PATTERNCREATION AND MAINTENANCE Living organisms are highly ordered and improbable arrangements of molecules, and only by constantly expending metabolic energy can they preserve this order. This is true for fluid “two-dimensional” membrane assemblies just as it is for three-dimensional structures like cells and tissues. Two striking examples where long-range membrane order is a prerequisite for biological function are the localization of ACh receptors at neuromuscular junctions in skeletal muscle and sodium channels at nodes of Ranvier in myelinated nerve fibers. Other examples include gap junctions, tight junctions, and epithelial cells with polarized lateral distributions of enzyme and transport activities. Short-range ordering of membrane components is important in the formation of nuclear pores, budding of membrane-enveloped viruses and adsorptive pinocytosis of polypeptide hormones through coated pits. In thylakoid membranes, sequestration of photosynthetic pigments in light harvesting and reaction center polypeptide complexes reflects an even finer pattern of order, probably crucial for efficient energy transfer. Diffusion is a direct manifestation of the Second Law, and superficially it is expected to counter the existence of organized features like those above. Certainly a cell must consume metabolic energy to contain potentially mobile proteins within functionally specialized regions of the membrane. The central question is how this is done. While many hypotheses have been advanced to explain immobilization and localization of membrane proteins, their success rate and predictive value have been unremarkable. In the following sections we briefly highlight some major concepts which have been entertained. Before doing so, it is worth noting that pattern creation and pattern stabilization are not necessarily inextricably coupled to one another. For instance, proteins could be actively
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driven to their destination by linkage to a submembranous contractile apparatus, while they might be kept there by passive protein-protein interactions leading to the formation of immobile aggregates. While the two processes are intertwined in what follows, it will be useful to keep the distinction in mind. 1. Contractile Macromolecules The most popular models of pattern creation and maintenance invoke direct attachment of dispersed or patched proteins to the cytoskeleton or cytoskeletonassociated molecules. A previous section has covered some evidence for cytoskeletal participation in maintaining the localization of membrane proteins. ATP-driven, directed motion of the attached species to specialized zones on the cell surface, analogous to the sliding filament model of muscle contraction, has often been proposed to account for localizing movement. Because such a disproportionate amount of attention has already been paid to this subject, we defer to previous articles by de Petris (1977), Weatherbee (1981), Oliver and Berlin (1982), Levine and Willard (1983), and Heath (1983). 2. Protein Solubility and Aggregation Several theoretical papers on so-called “lipid mediated protein-protein interaction” have appeared (Gruler, 1975; Marcelja, 1976; Schroeder, 1977; Owicki and McConnell, 1979), one treating explicitly the creation of long-range order (Gershon, 1978). Experimental verification that lipid structural alterations can mediate membrane functions by promoting the association and dissociation of specific proteins was recently obtained by Siege1 et al. (1981). They found that in spinach chloroplast thylakoids the stoichiometric interaction of lightharvesting pigment-protein complexes with photosystem I1 (PS 11) reaction center complexes was progressively disrupted by successive addition of soybean lipids to the membrane, and that energy transfer to the PS I1 trap dropped off in parallel. Disruption of the laterally dispersed supramolecular structures was accompanied by formation of extensive lattices of the light-harvesting complex. Lipid-mediated protein-protein interactions, although potentially selective in nature, are really one manifestation of the overall problem of protein solubility within the bilayer. Two widely known structures to which protein insolubility may contribute are gap junctions and the purple membrane of Halobacterium. In both cases there is long-range membrane order due to laterally separated arrays to densely packed integral proteins, and in neither case are cytoskeletal components implicated as a stabilizing factor. For the gap junction, gross localization of individual connexons is a direct consequence of the intercellular interaction of complementary proteins in two contacting membranes. Whatever their mobility, once formed they cannot diffuse out of the cell-cell contact zone without first breaking apart. But that does not explain why connexons are often assembled in discrete, organized plaques within the contact zone. Perhaps the formation of
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these plaques from individual connexons reflects a reduction in solubility over the separate connexon precursors, due for instance to proteolytic cleavage or other covalent modification. Or perhaps locally high concentrations of connexon proteins resulting from diffusion-mediated trapping exceed the solubility limit for that species and initiate precipitation. Crystallization of bacteriorhodpsin in vivo may be promoted by high bacteriorhodopsinconcentrationscoupled with the unique neutral lipid composition of Halobacterium membranes, which contain significant amounts of squalene. Pertinent to this speculation is the observation that increasing concentrations of cholesterol in fluid phoshatidylcholine bilayers cause progressive lateral segregation of purified bacteriorhodopsin (Cherry et al., 1980). Integral membrane proteins may be thought of as colloidal particles dispersed in a thin layer of fluid. Rules that govern the behavior of colloidal dispersions and the alteration of the behavior that results from specific molecular modification might well be applied to certain membrane situations. A balance of electrostatic repulsion between charged protein moieties in the aqueous phase and van der Waals attraction within the lipid matrix is likely to be an important factor for molecular interactions in the cell membrane, in particular, the formation of protein aggregates. Gingell (1976) and Gingell and Ginsberg (1978) have discussed interesting implications of colloidal theory in membrane biology. Rubin et al. (1981) used Smoluchowski colloidal aggregation theory to model the redistribution of integral membrane components which accompanies thylakoid membrane stacking and destacking, and derived a lateral diffusion coefficient for the PS I1 complex. 3. Bulk Membrane Flow Bulk membrane flow is an active process that could impart a motive force to membrane proteins (Bretscher, 1976; Harris, 1976). It was originally invoked to explain whole-cell locomotion and ligand-induced capping, but in principle is applicable to contact-induced redistribution. One theory (Bretscher, 1976) has it that flow is sustained by insertion of membrane lipid at one or more sources and simultaneous removal at a spatial remote sink. A molecular filter at the sink was originally postulated which would selectively retain proteins on the surface while passing the lipids through; the coated pit was later assigned this role (Bretscher et al., 1980). Rapidly mobile proteins will resist the flow by randomization against the concentrating effect of flow, but relatively immobile species like protein aggregates or “patches” will be swept up and concentrated at the sink, as observed during capping or surface receptors. There are some interesting experimental findings at least partially consistent with this hypothesis. For example, on different mammalian cell types several monovalent lipid “receptors” will patch upon treatment with multivalent ligands, and the patches migrate into a cap (Revesz and Greaves, 1975; Sedlacek
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et al., 1976; Stem and Bretscher, 1979; Schroit and Pagano, 1981). As with capping of integral proteins, lipid capping is in some cases energy dependent and blocked by cytochalasin B. Using FRAP, Schroit and Pagano (1981) measured a cm*/second for a cappable lipid on lateral diffusion coefficient of 8.5 X murine lymphoma cells. Because the lipids in question cannot span the bilayer and since this rapid diffusion is not indicative of an interaction with transmembrane proteins, it is argued that direct cytoskeletal mediation of lipid capping is impossible. Some idea of why a monovalent lipid should even patch may help elucidate the mechanism of lipid capping. The classic observations of Ingram (1969) and Abercrombie et al. (1970) on the directed rearward motion of particles attached to the dorsal surfaces of advancing fibroblasts formed the primary experimental basis for the hypothesis of membrane flow. More recent kinetic experiments on the surface distribution of specific Golgi-processed proteins in locomoting fibroblasts are consonant with the earlier particle experiments, and have been interpreted as local insertion of Golgi-derived vesicles at the leading edge coupled with removal at several perinuclear sites (Bergmann et al., 1983). A few arguments could be raised against the efficiency of membrane flow as a mechanism of protein redistribution and localization. First, by itself it is not a very selective method for localizing proteins. Second, in cases where localization of a protein is undesirable, the protein must be capable of quite rapid translational diffusion (D > l o p 9 cm*/second) to remain dispersed under the influence of membrane flow (Bretscher, 1976). If one can believe the results from FRAP experiments very few plasma membrane proteins have such high mobility. Third, even if they do, some extra supposition must be added to the basic hypothesis to explain how specific proteins are diffusionally immobilized yet still susceptible to the flow. In summary, flow would provide a general impetus for directed migration of many membrane components. Given some selective molecular interaction at the sink this might offer a rate enhancement over other potential collection processes.
4. Localization by Electric Fields Exogenously applied electric fields can induce migration and segregation of cell surface components in many cultured cells (see review, Poo, 1981). There is a distinct possibility that endogeneous electric fields within tissue may serve to localize or segregate components in differentiated cells. There are two likely regions where this might occur. First, a steady potential across an organized layer of cells could segregate different components to two sides of the cell layer. In epithelial tissue, steady potential drops on the order of millivolts across the cell layer have been detected. This is within the same order of magnitude of steady potentials that were found to cause migration of cell surface components
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in cultured cells. Second, high-frequency focal current occurring at localized regions in nervous systems, e.g., synaptic junctions, nodes of Ranvier of myelinated axons, and axonal branch points, may cause migration and localization of membrane receptors and ionic channels (see discussion in Fraser and Poo, 1982). The second possibility is of particular interest for neurobiology, since it suggests a possible cellular mechanism for use-dependent modulation of signal transmission, a key problem in understanding the plasticity of nervous systems. While direct in vivo experimental evidence is lacking, theoretical modeling of electromigration of membrane components in the presence of pulsed, focal fields suggests that under favorable conditions of high electrophoretic mobility to diffusion coefficient ratio (e.g., for protein aggregates), membrane components could be moved laterally by repetitive focal potentials (S. H. Young, personal communication). Lateral electrophoretic displacement of integral membrane components could also be induced by increasing their net electrical charge, for example by phosphorylation. In fact, a light activated protein kinase catalyzes phosphorylation of the light harvesting chlorophyll alb protein complex (LHC)in chloroplast thylakoids, and the phosphorylated LHC migrates out of appressed grana membranes and into the stroma exposed thylakoid regions. This event is believed to regulate the distribution of excitation energy between the two laterally separated photosystems, thus permitting a maximal quantum efficiency of photosynthesis under different light regimes (see Section III,D,2). Whether or not one accepts the specific models advanced to explain this phosporylation-induced redistribution, it is not hard to imagine that the high negative charge density within closely spaced grana stacks repels the negatively charged LHC. 5 . Tight Junctions in Segregation
Epithelial cell monolayers which carpet the lumen of organs like intestine, renal tubes, and pancreas perform a crucial homeostatic function in mediating the directional transport of nutritive substrates, ions, water, and digestive enzymes between the internal environment and the external world. Interstitial passage of these materials between apposed aqueous chambers is blocked to one degree or another by tight junctions, or zona occludens-specialized intercellular connections which seal the individual epithelial cells together into continuous sheets. Associated with this vectorial transport is a distinctly nonuniform lateral distribution of membrane components, which in the intestinal epithelial cell (IEC)is quite striking (Michell et al., 1976). Digestive enzymes and various sodiumsolute symport activities are concentrated in the lumenal membrane but nearly absent from the basolateral membrane. Transcellular sodium concentration gradients that drive nutrient absorption by these symport systems are created by Na-K-ATPase activity found almost exclusively in the basolateral membrane. Adenylate cyclase and receptors for various blood-borne stimuli like hormones
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and cholinergic agonists are similarly restricted to the basolateral membrane. Some lipids are also asymmetrically distributed, with the cholesterol/phospholipid ratio of the lumenal membrane being twice that of the basolateral membrane. To the extent that plasma membrane proteins are capable of lateral motion, what prevents mixing of these various activities from bringing directional transport to a halt? Several groups have postulated that tight junctions preserve lateral asymmetry by blocking the translational diffusion of membrane components between lumenal and basolateral surfaces (Galli et a l . , 1976; Pisam and Ripoche, 1976; Parr and Kirby, 1979; Sang et a l . , 1979; Evans, 1980; Matsuura et a l . , 1982). Tight junctions disassemble in the absence of calcium, and isolation of epithelial cells dissociated by calcium chelators leads to randomization of polarized membrane topographies. For example, generally labeled glycoproteins and proteoglycans restricted to the lumenal face of frog urinary bladder epithelial cells become uniformly dispersed within 80 minutes of tight junction disruption (Pisam and Ripoche, 1976). Histocompatibility antigens confined to the basolateral membrane of mouse IEC also undergo complete redistribution after cell dissociation (Parr and Kirby, 1979). In pancreas and kidney epithelia the intramembranous particle (IMP) density observed with freeze-fracture electron microscopy is appreciably higher in the lateral than in the lumenal membranes, and in both cell types this polarity is lost concomitantly with cell isolation and tight junction disruption (Galli et a l . , 1976; Sang et al., 1979). Using freezefracture to follow the reappearance of IMP polarity in reassociating kidney cells Sang et al. (1979) observed that as soon as a single continuous string of junctional components had formed, differential IMP densities began to appear on opposite sides of the junction. Ziomeck et al. (1980) estimated the translational diffusion coefficients of leucine aminopeptidase (LAP) and alkaline phosphatase (AP) by measuring relaxation kinetics of the initially polarized distribution of these enzymes on freshly isolated mouse IEC. Although the relaxation rate was boosted by drugs which alter membrane potential and ATP levels, redistribution was apparently passive, with a half time of 20-30 minutes at 22°C. Because “though somewhat unraveled, substantial amounts of tight junctions are found on single cells and in cell pairs as late as 20 minutes after the IEC is isolated,” Ziomeck and associates deemed it unlikely that AP and LAP diffusion is restricted by tight junctions. Dragsten et al. (1981) showed that various fluorescent lectins bound to either the apical or basolateral surface of cultured kidney epithelial cell monolayers did not migrate to the opposite surface. However, using photobleaching they found the lectins to be essentially immobile (D < 10- l 2 cm2/second) along the apical membrane itself, and concluded that a barrier function for the tight junction need not be invoked to explain the polarized lectin distributions (see next section for alternative view). On the other hand, fluorescent antibody fragments bound to
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the Na-K-ATPase in one of the same kidney cell lines are only 50% immobilized in either confluent or subconfluent cultures (Jesaitis and Yguerabide, 1981). In subconfluent cultures (junctions absent) the pump is uniformly distributed around the cells, while in confluent cultures (junctions present) it is restricted almost exclusively to the basolateral surface. Independent of the presence or absence of tight junctions, the mobile fraction diffuses at 5 X 10-lo cm2/ second. At this rate the mobile Na-K-ATPase molecules should be able to redistribute uniformly around the cell within an hour at most, provided they had a free path. To summarize, available evidence does not permit a definitive rejection or embracement of the hypothesis that tight junctions block lateral motion of integral membrane proteins; the possibility certainly remains viable for at least some proteins. In any case, diffusion barriers will only help in the maintainence of patterned topographies; localized insertion, direct cytoskeleton-mediated segregation, or some other mechanism must work in concert with the tight junction to form an initial pattern. Evans (1980) discusses evidence for some of these processes in his review on plasmalemma polarity in the liver epithelial cell. 6 . Entrapment in Extracellular Matrices In vivo, intestinal epithelial cells bear perhaps the thickest glycocalyx of any mammalian cell type. Smithson et al. (1981) have presented evidence that it impedes the diffusion of small molecules like sucrose, lactose, and oligopeptides to the plasmalemma enzymes which hydrolyze them. Thinner, but appreciable “fuzzy” coats also adorn the apical surfaces of other kinds of epithelial cells. In many instances this carbohydrate-rich layer is greatly depleted on the basolateral relative to the lumenal surface (Rambourg, 1971), and randomization of this pattern has been observed following tight junction disassembly and cell dissociation (Pisam and Ripoche, 1976). Lectins (MW lo4) are somewhat larger than sucrose, and one might expect their diffusion through the glycocalyx matrix to be significantly impaired; indeed, even unlabeled integral proteins which protrude into the external aqueous phase may suffer the same fate. Perhaps this helps explain the observations mentioned above that lectins bound to apical surfaces of epithelial cells are immobile, while Fab fragments bound to the Na-K-ATPase on basolureral surfaces have considerably greater lateral mobility. If this hypothesis is correct then the tight junction may block the lateral motion of lectin receptors in an indirect manner, by serving as a “dam” to retain certain glycocalyx constituents on the apical surfaces of epithelial cells.2 Leucine aminopeptidase and alkaline phosphatase are confined to the apical Qermane is the recent report by Rizki and Rizki (1983) that wheat germ agglutinin binding sites in the plasmalemma of Drosophila larval fat body cells are directly held in an asymmetric pericellular distribution by the adjacent basement membrane.
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(lumenal) surface of IEC, yet Ziomeck et al. (1980) report fairly rapid diffusion cm2/ coefficients for these two proteins ( D values 0.5 X l o p 9 and 0.9 x second) upon cell dissociation. Perhaps this reflects glycocalyx thinning (redistribution) of the kind demonstrated by Pisam and Ripoche (1976). In addition, hyaluronidase treatment used in the isolation protocol may have trimmed the cell coat sufficiently to release severe diffusive constraints. 7. DifSusion-Mediated Trapping a. Evidence and Background. Diffusion-mediated trapping (DMT) is perhaps the simplest concept that embodies mechanisms for both development and maintenance of ordered biomembrane topographies. As a direct consequence of their Brownian motion, molecules will willy-nilly blunder into a trap region to which they chemically bind and simultaneously disappear from the surrounding membrane. In principle, the trap can be furnished by a neighboring cell or the host, and it may reside outside or inside the cytoplasm. If contributed by a touching cell it may consist of uniformly dispersed, mobile molecules having affinity for complementary species on the host cell. Upon comparison with other mechanisms its efficiency is fairly apparent; in the simplest case metabolic energy is spent only on the synthesis of mutually sticky proteins. DMT will clearly not explain all cases of pattern creation in biomembranes; for example, capping and various receptor redistributions which occur in chemotaxing, dividing, or phagocytosing blood cells occur too rapidly to be accounted for by diffusion alone (de Petris, 1977; Koppel et al., 1982; Oliver and Berlin, 1982). Other internally coordinated topographical rearrangements like those which occur during adsorptive endocytosis and virus budding may well follow the DMT route. We anticipate that molecular redistribution induced by membrane- membrane contact will prove to be a major biological application of DMT. Edwards and Frisch (1976) were the first to consider DMT in the control of cell surface topography. They attempted to account for ACh receptor turnover in neuromuscular synapses by postulating random insertion of newly synthesized ACh receptors in the extrajunctional region of the muscle followed by diffusion to the junction. In discussing the general problem of cell-cell adhesion Bell ( 1979) proposed that intercellular linkage of complementary receptors would result in the diffusion of more and more free receptors into the contact zone, thus rapidly increasing the strength of binding. Under experimental circumstances several contact-induced redistributions have been observed. When ferritin-conjugated Con A was used to agglutinate a mixture of pigeon erythrocytes and peripheral lymphocytes, the ferritin-Con A was found concentrated on the contacting regions of membrane and depleted elsewhere (Singer, 1976). Because both cell types bind Con A it was assumed that the tetravalent lectin acted as an intercellular bridging ligand, thus facilitating passive accumulation. Asialoglycoprotein receptors in the plasmalemma of rat hepatocytes undergo a strik-
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ing accumulation in the zone of contact with surfaces of a synthetic galactosidecontaining polymer (Weigel, 1980). Galactose is the terminal hexose on asialoglycoproteins recognized by this receptor; replacement of galactose in the polymer with other sugar residues fails to induce receptor accumulation. According to Kampe and Peterson (1979), gene products of the major histocompatibility complex become localized in the contacting membranes of cytotoxic T lymphocyte-target cell conjugates. These are all suggestive pieces of evidence, but until recently neither the kinetics nor the mechanism of such processes had been investigated. Work in our laboratory has now provided several concrete examples of pure DMT, including the passive contact-induced accumulation of FcR-IgE complexes in the membrane of rat basophilic leukemia cells touching haptenated polyacrylamide beads (McCloskey and Poo, unpublished) as well as the concentration of both soybean and wheat germ agglutinin receptors in the contacting membranes of Xenopus embryonic muscle cell pairs in culture (Chow and Poo, 1982). Chao et al. (1981) have developed a quantitative model for molecular trapping by perfect sinks which relates the average lifetime of untrapped species to their diffusion coefficient and the area of membrane-membrane contact. Weaver (1983) has recently extended this theory to include imperfectly absorbing traps. b. ACh Receptor Localization. During synaptogenesis of the skeletal neuromuscular junction, ACh receptors over the surface of the embryonic muscle become highly concentrated at the subsynaptic muscle membrane. In Xenopus embryonic nerve and muscle culture, it was clearly demonstrated that contact by appropriate nerve processes can induce clustering of preexisting ACh receptors at the site of contact (Anderson and Cohen, 1977; Cohen and Weldon, 1980). The mechanism by which a nerve induces such a modulation of ACh receptor topography is unknown. Of various possibilities, the simplest explanation is that the ACh receptors, freely diffusing within the plane of the muscle membrane, are trapped at the site of nerve contact by binding to specific molecules associated with the nerve membrane or the intercellular matrix between the nerve terminal and the muscle. This diffusion-mediated trapping mechanism is plausible only if the diffusion rate of ACh receptors is rapid enough to account for the time-course of ACh receptor clustering both in vitro and in vivo, given the dimension of the developing muscle cells that diffusion must cover and the size of nerve-muscle contact. A rough estimate based on results obtained in culture (Cohen et al., 1979) and in intact tadpole (Chow, 1980) suggests that ACh receptors must diffuse with a D value of at least l o p 9cm2/second for the diffusion trap model to be plausible (Poo, 1982; Young and Poo, 1983). For the turnover rate of ACh receptors in mature muscle endplate to be explained by diffusion trapping a similar D value is also required (Edwards and Frisch, 1976). Early FRAP measurements of ACh receptor mobility in rat myotubes yielded a much smaller D value of 5 X 10- * cm2/second (Axelrod et a l . , 1976). Due to
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the concern that FRAP is measuring diffusion of the complex of ACh receptor and its labeling ligand, a-bungarotoxin, rather than the native free receptor, an entirely different method, postinactivation recovery (PIR) was used to measure the diffusion of functional free ACh receptors in the muscle membrane. In this method, ACh receptors on muscle surface were locally inactivated by a pulse of a-bungarotoxin, a snake toxin that binds irreversibly to the ACh receptor. The recovery of ACh sensitivity at the site of inactivation was mapped by an iontophoretic method to reveal the lateral diffusion of the functional, toxin-free ACh receptors in the membrane. The D values obtained for both Xenopus culture preparations and intact developing muscle fibers of Xenopus tadpoles were found to be within the range of 1-4 X lop9cm2/second (Poo, 1982; Young and Poo, 1983). This diffusion rate is rapid enough to account for nerve-induced clustering of ACh receptors by a diffusion trap mechanism. What edge could the diffusion trap mechanism have over other conceivable mechanisms such as local insertion of ACh receptors at the nerve-muscle contact site together with selective removal of receptors from extrajunctional regions? Besides a possibly lower energy cost, diffusion-mediated trapping provides the simplest signaling mechanism for receptor localization. Teleologically speaking, the cell does not need to know where on the surface the nerve has made contact. All it has to do is to disperse ACh receptors on the surface; the receptor will be trapped wherever the contact happens to be made. This perhaps is one of the reasons why embryonic muscle puts a large quantity of ACh receptors into its plasma membrane before the nerve arrives (Blackshaw and Warner, 1976). c. Receptor-Mediated Endocytosis. Coated pits are small (50-200 nm) specializations of the eukaryotic plasmalemma which mediate the acquisition from extracellular fluids of a variety of nutritional, hormonal, immunological, enzymatic, and other macromolecular substances (Goldstein et d . , 1979). The prevailing conception is that these clathrin-coated depressions capture laterally diffusible ligand-receptor complexes from adjacent regions of the membrane and then pinch off intracellularly to form coated vesicles, which deliver the cargo to cytoplasmic compartments. The detailed dynamic events which occur between binding of ligands to their specific receptors and the appearance of clustered ligand-receptor complexes in coated pits have yet to be worked out; whether or not cells employ a simple strategy involving trapping of monomeric ligandreceptor complexes by preformed coated pits is currently unknown. Thus, some propose that ligand-induced receptor clustering provides a cue (e.g., membrane warping) that initiates clathrin assembly underneath the clusters (Roth and Woods, 1982). Notwithstanding the gaps in our knowledge regarding receptor-mediated endocytosis, it is possible to place certain restrictions on the lateral diffusion rates demanded by simple diffusion-mediated trapping of monomeric ligand-receptor complexes in preexisting coated pits. Assuming the coated pits to be perfect
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sinks, Goldstein et al. (198 1) used published data on receptor densities, internalization rates, coated pit dimensions, copies per cell, and steady-state concentration of trapped vs free receptors to calculate the apparent two-dimensional rate constant for trapping of LDL receptors on a human fibroblast cell line. Upon comparison with the theoretical rate constant calculated from a previously measured D value of 2-3 x lo-" cm2/second (Barak and Webb, 1981) they concluded that if the foregoing mechanism does operate, it must do so at very close to the diffusion-limited rate. One can rationalize why a relatively small diffusion coefficient would suffice for the harvesting of LDL by considering the short distance involved. For human fibroblasts at 4°C the surface density of coated pits is about 0.58/pm2 (Orci et al., 1978), which gives an average interpit spacing of roughly 1.3 microns. In contrast, ACh receptors being trapped at the neuromuscular synapse must traverse the entire developing muscle, which may be hundreds of microns long. Because diffusion times depend on the square of the distances covered, the vastly different time frames of endocytosis and ACh receptor accumulation can be accounted for by the observed diffusion coefficients. Most indications are that insulin and epidermal growth factor receptors exist in a highly dispersed state prior to ligand binding. Ligand-induced clustering and internalization of EGF receptors is blocked at 4"C, and this could be due to a reduced lateral mobility of the receptors at 4°C if trapping of EGF were a diffusion limited process. Hillman and Schlessinger (1982) find only a threefold drop in the lateral D value of EGF-EGF receptor complexes over the span of 37 to 5°C and from this they estimate roughly the expected drop in encounter frequencies of bound receptors with coated pits. While they believe that harvesting of EGF proceeds by diffusion-mediated trapping, they conclude that the rates of visible patching and endocytosis of EGF receptor-EGF complexes are not determined by the lateral diffusion rates of EGF receptor-EGF complexes. A more conservative statement might be that low temperature inhibition is not a result of reduced diffusion rates; clearly, shifting the temperature can differentially affect the rate and equilibrium constants of separate elementary steps in a multistep sequence such that what was rate limiting no longer is. In the present situation this might arise if microtubule integrity were required for both visible patching and endocytosis. One might ask what advantages this diffusion-collection process offers. Would not the trapping and internalization of soluble ligands be faster if receptors were permanently glued to coated pits, thus bypassing steps dependent upon inexorably slow lateral diffusion? Alternatively, does spreading receptor sites uniformly over the plasma membrane improve the total catch of soluble ligands? Theoretical calculations (Berg and Purcell, 1977; DeLisi, 1981; Shoup and Szabo, 1982) lead to the prediction that for average cell dimensions the capture rate of soluble molecules is near maximal for only a few thousand sites per cell;
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that is, the number of sites provided by coated pits alone should give efficient trapping. This does assume, however, that ligand binding by the cell is diffusion limited, and this assumption is not generally valid for all surface receptors (see Wank et al., 1983). It is known that several types of ligands can enter the cell through a single coated pit (Maxfield et al., 1978; Dickson et al., 1981; Willingham et al., 1981; Carpentier et al., 1982). Perhaps this sharing helps to explain the design of coated pit structure: There may be insufficient room inside a single coated pit to house several copies of all the different receptor types which interface with it. A multiple-user system may actually be simpler to coordinate than the evolutionary alternative of having many distinct kinds of coated pits, each with its own unique complement of receptors.
B. REACTIONS IN Two DIMENSIONS 1. Reduction of Dimensionality: Fact or Friction? a. Target Finding by Diffusion. Most membrane-mediated reactions fall into one of two categories: (1) Those involving water-soluble reactants and products which must find and depart the active site of a membrane-bound enzyme by some combination of two- and three-dimensional (2D and 3D) diffusion. (2) Reactions between more permanently attached molecules (e.g., integral enzymes, nascent polypeptides, and lipid-soluble isoprenoid coenzymes) where strict 2D interdiffusion governs collisional encounters. In 1968 Adam and Delbruck provided the first theoretical framework for analysis of membrane target finding via diffusion. They dealt specifically with the diffusion-limited trapping of water soluble molecules by a membrane-bound target which acts like a perfect sink (one must stretch the imagination a bit to picture enzymes and hormone receptors as infinitely absorbing, perpetually reusable traps). Based upon their calculations they suggested that a significant rate enhancement might accrue to membrane-mediated reactions if instead of finding the enzyme active site by purely 3D diffusive encounters a water-soluble substrate first adsorbed nonspecifically to the membrane and underwent a strictly 2D random walk until hitting the target. According to the theory this two-stage process, or “reduction of dimensionality,” during the diffusive search will increase the efficiency of target finding over that obtained in the absence of surface diffusion, provided that certain conditions are met. Namely, the adsorbed molecules must be sufficiently mobile (ratio of diffusion coefficients in 2D versus 3D: D,,lD3, L 0.01) and the ratio of diffusion space size to target size sufficiently small. For D,,lD,, 2 0.01 and an enzyme active site diameter of 20 A, the critical diffusion volume is about the size of a bacterium or a mitochondrion. For volumes much larger than this no advantage purportedly exists. Although in 1968 no lateral diffusion coefficients for any biomembrane components had yet been determined, Adam
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and Delbruck nevertheless ventured that reduced diffusion times-presumably faster reactions-confer a selective advantage on the intracellular membranous organelles of eukaryotes; reduced diffusion times theoretically result from confining reactants within a subcritical volume and promotion of two-step capture by membranous subdivision of the aqueous space inside an organelle. Of course, all of this presupposes that the reactions of interest are diffusion-limited or would be without dimensionality reduction. Adam and Delbruck’s idea received an interesting twist when a more general analysis of membrane target finding by soluble reactants (Berg and Purcell, 1977; DeLisi and Wiegel, 1981) revealed that, in principle, purely 3D target searching can be more effective than one would suppose. Berg and Purcell reasoned that, due solely to statistical considerations, once a molecule has found the membrane it is guaranteed a period of diffusion near the surface, and thus has several more tries at bumping into the target before mixing with molecules out in solution. In effect, an inert membrane has its own “trapping” ability, causing the reactant to hop around awhile in a “quasi-2D” search. For reactants that do adsorb to the membrane a truly two-dimensional search can occur; whether this leads to an enhancement depends on the conditions described above, the adsorption energy (Berg and Purcell, 1977), rate of dissociation from the membrane (Richter and Eigen, 1974), and other difficulty estimated factors like the relative orientations of membrane-bound vs free reactants. b. Reaction Rates in 2 0 versus 3 0 . Anomalously rapid association of the lac repressor with its operator on DNA (Richter and Eigen, 1974; Schranner and Richter, 1978) and reduced transient times of some multienzyme complexes (Welch and Gaertner, 1975; Mosbach, 1976) provide the strongest support for rate enhancement by guided diffusion (dimensionality reduction). There is also some empirical evidence that under appropriate conditions the rate of reaction of aqueous with membrane-bound species can be enhanced by prior adsorption to the membrane (Overfield and Wraight, 1980). However, the common notion that reactions are necessarily “faster” in or on membranes than in the cytoplasmic aqueous phase has yet to secure a firm theoretical foundation. As discussed in the last section, the efficiency with which soluble ligands find membrane bound targets cannot be attributed simply to a reduction from 3D to 2D diffusion. Furthermore, even given a favorable ratio of 2D to 3D diffusion rates (say 0.01), it is not at all clear whether a significant theoretical rate advantage actually exists for reactions completely confined to the membrane plane. How does one compare the efficiency of a 2D reaction with one in 3D? What biologically relevant parameter, and what values of that parameter will actually indicate a selective advantage? It is our contention that if cells could think they would probably be most concerned with the total number of a given molecule they could make per unit time. A meaningful parameter to test the sweeping argument for reduction of
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dimensionality might then be the net reaction rate ratio for equal numbers of reactant molecules in purely 2D and purely 3D reactions. One estimate of this ratio is obtained by comparing the collision frequencies of two reactants when confined to a membrane plane with that when contained within a 3D aqueous volume ensheathed by that membrane, i.e., the initial reaction rate of a totally diffusion-limited reaction (every encounter productive). According to Smoluchowski formalism, unlike in three dimensions, the rate “constant” for 2D diffusion-controlled reactions of the type A + B + C declines continuously with time (Emeis and Fehder, 1970; Naqvi, 1974) as it asymptotically approaches zero (Torney and McConnell, 1983). Hardt ( 1979) was apparently unaware of this in her comparison of reaction rates in one, two, and three dimensions, and her expressions for ID and 2D diffusion-limited rate constants are patently false. The only rigorous expressions suited to our calculation are those of Torney and McConnell (1983), and we employ these below. For simplicity, let us assume that the reaction involves two molecular species present in equal number; this is an arbitrary choice which should bias the result in favor of the membrane. To place the calculation into biological perspective, we take the reaction between cytochrome c and cytochrome oxidase in mitochrondria as an example. Estimates of cytochrome oxidase surface densities in mitochrondria range from about 1 per 50,000 to 1 per 590,000 A2 (Klingenberg, 1967; Hackenbrock and Hammon, 1975; Hochman et al., 1982), giving a total number of oxidase between 0.2 and 1.7 X lo4 for a spherical mitoplast of radius 0.5 km. Since the ratio of cytochrome c to the oxidase varies from about 0.8 to 1.7 (Capaldi, 1982), our assumption of equal numbers of each reactant is reasonable. The encounter radius is assumed to be 20 A. Making simplifying assumptions about the lattice spacing, for times greater than a small fraction of a second we find the ratio of diffusion-controlled reaction rates in 2D vs 3D to be:
RJR, = 83(D,/D,)/ln(AD,t) where A is of the order of l O I 4 cm-,. Recalling that membrane proteins in general diffuse two to three orders of magnitude more slowly than soluble proteins in buffers, and that the empirically determined diffusion rates of cytochrome c and the oxidase (see Section 111,B,2) are no exception, this maximum possible “enhancement” could actually be an impediment, especially at long times. In the above case, even if one inserts the greatest D, yet observed for a membrane protein (5 X 10W9 cm2/second) and a reasonable D, of lo-’ cm2/ second, the membrane-mediated reaction is still appreciably slower than the bulk phase reaction (R2/R, = 0.3). Notice that we have compared the hypothetical reaction rates for a maximum ratio of volume to surface area (spherical vesicle). Some cellular organelles contain flattened membranes, e.g., chloroplast grana stacks, mitochondria1
48
MICHAEL MCCLOSKEY AND MU-MING PO0
cristae, and pancake-shaped Golgi stacks. Reactions in the aqueous space between the appressed membranes can be thought of as approaching the extreme of reaction within a plane. Drawing on the previous result, since diffusion within these spaces might be as fast as in free solution, we suggest that diffusion-limited reactions between aqueous and membranous reactants may be enhanced somewhat therein. c. Anisotropic Rotation and Orientation Constraints. In contrast with diffusion-limited reactions between small molecules, it is unlikely that very many reactions between macromolecules are controlled simply by collision frequencies of the reactants. After all, large molecules with one active site are not uniformly reactive over their surface and not all collisions are expected to result in a favorable alignment of the reactive sites. For example, we can infer from results of Schmitz and Schurr (1972) that the surface domains involved in electron transfer from cytochrome c to peroxidase probably occupy 10% (or less) of the total surface area of both proteins. Similarly, only 7-15% of the surface area per subunit is inaccessible to solvent in protein complexes such as the insulin dimer, the ap dimer of hemoglobin, and the trysin-BPTI complex (Chothia and Janin, 1975). That this heterogeneous reactivity can have a large influence on the rates of diffusion-limited biochemical reactions is indicated by the very low probability (steric) factor (1.8 X for head-tail joining of bacteriophage T4D (Aksiyote-Benbasat and Bloomfield, 198 1). All indications are that large amplitude rotational motion of integral membrane proteins is confined to an axis normal to the membrane plane. This restricted rotation permanently aligns the reactive groups in a way that is impossible for soluble macromolecules, and one might expect this orientation to provide some rate advantage in reactions between large proteins. A rigorous theoretical treatment of the effect of orientation constraints on reaction rates is beyond the scope of this article, but a semiquantitative calculation based upon probability arguments is informative. Assume the two reacting proteins to be spherical in shape with radii a, and a, and each to have a circular reactive patch of radius b, or b, on its surface. For simplicity, let a1 = a, and b , = b,. Assume that reaction ensues when there is overlap of these sites during a collision. For proteins in aqueous solution the probability that a collision will result in overlap of reactive sites is the product of fractional areas occupied by the sites on each molecule. Next suppose the same proteins are immersed in a bilayer and constrained to rotate about an axis normal to the membrane-such that the reactive sites are correctly positioned to interact. In this arrangement the appropriate fractional area is the ratio of target area to the area swept out by the target in rotating through 360" about the axis normal to the membrane, and the probability of a productive collision is the product of this ratio for both proteins. The net ratio of probabilities in two dimensions to three dimensions is then, roughly
PROTEIN DIFFUSION IN CELL MEMBRANES
49
P21P, = (a/b)2 If we go back to the example of cytochrome c reacting with peroxidase this probability ratio is estimated to be about 10 (Schmitz and Schurr, 1972). This means that the actual rate ratio for 2D versus 3D might be noticeably greater than predicted solely on the basis of collision frequencies, and that reduction of the dimensionality of rotational diffusion (3D to 1D) may provide more of a rate advantage than reduction of dimensionality of translational diffusion (3D to 2D), at least when realistic lateral diffusion coefficients are used in the calculation. This simple (simplistic?) treatment considers only the probability of reacting upon the first contact, and neglects the possibility that an ineffective collision might be remedied by rotational searching within the encounter complex (Solc and Stockmayer, 1973; Simmons, 1975). d. Conclusions. We have shown in the above two sections that when reasonable diffusion rates and reactant concentrations are considered, standard collision frequency calculations do not substantiate the general conception that reactions are “faster” in membranes than in the aqueous phase-r that the speed of strictly membrane-bound reactions confers a selective advantage on intracellular membraneous organelles. However, we call attention to two previously unexplored aspects of dimensionality reduction. First, bulk phase reactions or membranous target searching could proceed faster within the intermembranous aqueous space of flattened intracellular membranes than in unbounded systems, without actual partitioning of the reactants into or on the membrane. Some potential candidates are plastocyanin-mediated electron transfer in thylakoid lumens, cytochrome c redox reactions along mitrochondrial cristae, and sorting of soluble proteins within Golgi stacks. Second, because integral proteins are held by the membrane in a reactive orientation, the probability that a given collision will be productive should be greater than for similar proteins in aqueous solution. Just what the magnitude of this effect actually is and whether it is great enough to override the characteristically slow lateral and rotational diffusion of membrane proteins in conferring a net 3D to 2D advantage remains to be seen.
2. Electron Transport a. Respiration. The mechanistic pathway of intra- and intermolecular electron transfer along the respiratory sequence is a challenging problem with several unresolved aspects. Exactly how these redox reactions drive ATP synthesis also remains a mystery. Those who subscribe to the chemiosmotic hypothesis cannot agree on whether a proton concentration gradient itself or the associated transmembrane electrical potential gradient is most important, and some nonsubscribers think that local (lateral) proton gradients and not transmembrane gradients are the most immediate link to ATP synthesis (Storey and Lee, 1981; Wikstrom, 1981; Williams, 1981; Haines, 1983). At the lowest level of resolution one is
50
MICHAEL MCCLOSKEY AND MU-MING PO0
concerned with the spatial organization of separate electron carriers within the inner mitochondrial membrane, and with its time dependence. Early concepts which equated the chemical sequence (decreasing redox potential) of individual components with a static physical sequence, or “respiratory chain” (Lehninger, 1959), have gradually given way to more dynamic models which picture at least some components as randomly distributed within the membrane plane and without any permanent binding between reaction partners. At the extreme of dynamicism are those who contend that the entire respiratory sequence, consisting of dehydrogenases, coenzyme Q, the cytochrome bc, complex, cytochrome c, and cytochrome oxidase (Fig. l), is completely jumbled and that electron transfer rates are determined solely by collision frequencies, and hence the lateral diffusion rates of separate molecules within the inner mitochondrial membrane (Hackenbrock, 1981). We devote extra space to this topic not only because of its paramount importance to living organisms, but also to document our assertion that meaningful interpretation of diffusion measurements is virtually impossible without a detailed knowledge of the biochemistry and ultrastructure of biomembranes. Early evidence on chain dynamics. One of the initial findings which weakened the physical “chain” concept was that complete electron transfer from NADH or succinate to oxygen could be produced in systems reconstructed from isolated complexes I-IV, cytochrome c , and ubiquinone (Hatefi, 1968). Klingenberg was the first to point out that individual cytochromes and flavoproteins (NADH and succinate dehydrogenases) do not occur in a 1:l mole ratio within inner membranes, and also that significant variation in the overall mole ratios exists between different tissues in one organism and between different species as well. Many investigators seem to consider this strong evidence against the existence of a specific supramolecular complex between different proteins of the respiratory sequence. Communication between physically discrete chains fed by succinate or NADH would have to occur at an early step, since succinate will reduce all the cytochrome be, molecules. The Q-pool behavior reported by Kroger and Klingenberg (1973a,b) was interpreted in just this way, that is, shuttling of electrons between separate chains by laterally mobile coenzyme Q. Relative lateral mobility of at least some portion of cytochrome c was implicated by the experiments of Wohlrab (1970), who found that membrane-bound cytochrome c can mediate the equilibration of iron oxidation states in cytochrome oxidase molecules located within separate “chains. ” Modulation of electron transport by exogenous lipids. In a series of ingenious experiments Hackenbrock and associates have manipulated the spacing between integral proteins of inner mitochondrial membrane preparations and studied the effects on rates of electron transport between separate portions of the respiratory sequence (Schneider et al., 1980). They found that when increasing numbers of sonicated lipid (soybean) vesicles are fused with inner membrane vesicles
PROTEIN DIFFUSION IN CELL MEMBRANES
--+C
SUCCINATE
51
Yt
FUMARATE
FIG. 1 . Mitochondria1 electron transport chain: Complex I , NADH dehydrogenase; Complex 11, succinate dehydrogenase; Complex 111, cytochrome bc, complex; Complex IV, cytochrome au3 complex (cytochrome oxidase); UQ, ubiquinone; Cyt C, cytochrome c.
(mitoplasts) from rat liver mitochondria the average spacing between intramembranous particles (IMPs) increases. Even though the separate complexes (I-IV) retain the same or show increased specific activities, electron transport from NADH and succinate to the bc, complex drops off appreciably with increased spacing between the IMPs. Schneider etal. (1980) concluded that electron transport between these molecules is a diffusion-controlled process, the increased interparticle distances reducing collision frequencies. Diffusion-mediated may be a more apt descriptor since all reactions (both diffusion- and activation energycontrolled) go slower when the reactant concentration is dropped. But even this conclusion is tempered by the results of another incorporation experiment where the average interparticle spacing was progressively reduced by cholesterol-induced lateral segregation of the inner membrane proteins (Schneider et al., 1982). Although cholesterol incorporation prevented a drop in rates of electron transport from NADH and succinate to complex 111, the rates never climbed above control levels-even though the average spacing within IMP rich domains became much shorter than in unmodified mitoplasts. If random encounters were the whole story, then shorter distances should generate faster reactions just as longer paths lead to slower reactions-assuming that electron transport remains diffusion-mediated. C. R. Hackenbrock (personal communication) suggests that imperfect lateral segregation led to entrainment of crucial electron carrier proteins in the proteinpoor domains. Another possibility is that ubiquinone remains uniformly dispersed as the proteins segregate, thus reducing the ratio of this coenzyme to complexes I-IV within the protein-rich region. Although these studies have correlated increased areas per IMP with slower reactions, they have not established a cause and effect relationship between the
52
MICHAEL MCCLOSKEY AND MU-MING PO0
two. Since respiration rates can only be lowered from native values by lipid incorporation, it is entirely possible that exogenous soybean lipids (which are structurally distinct from endogenous rat mitochondria1 lipids) cause progressive disruption of protein-protein interactions normally required for efficient electron transport. Such interactions may be more sensitive to the lipid environment than is tacitly assumed in most studies with reconstructed lipid-protein systems. This was vividly demonstrated by Siege1et al. (1981) in completely analogous vesicle fusion experiments with chloroplast thylakoid membranes, where dilution of endogenous lipids with soybean lecithin led to progressive detachment of light harvesting chlorophyll-protein complexes from photosystem I1 reaction centers and consequent decrease in energy transfer efficiency. Consideration of lipidmediated effects on the organization and function of membrane proteins is imperative in studying modified or reconstituted systems. Lateral difusion measurements. In contrast to NADH and succinate oxidase activities, Schneider et al. (1980) found little decrease in duroquinol oxidase activity as the amount of incorporated lipid was increased. (Duroquinol oxidase activity is electron transport from a water soluble hydroquinone to the bc, complex through cytochrome c and the oxidase to oxygen.) To rationalize this they suggested that cytochrome c either diffused very rapidly between physically separate bc, and aa3 complexes or that the three components diffused together as a unit. Since subsequent work showed that the oxidase and bc, complexes patch independently when inner membranes are treated with a mixture of anti-bc, and anti-aa, antibodies the former hypothesis was favored. To test this possibility Gupte er al. ( 1 983) measured diffusion of fluorescein-labeled cytochrome c on large inner membrane preparations formed by calcium induced fusion of mitoplasts. They found that the apparent lateral D value is markedly dependent on ionic strength: in 0.3 mM Hepes D = 4.0 X l o - " cm2/second; in 10 mM KP, D = 2.5 X 10WLocm*/second; in 25 mM KP, D = 2 X cm2/second. Electron transport from succinate or duroquinol to oxygen increased moderately on going from 0.3 mM Hepes to 10 mM KP, (factor of 1.2-1.5). Hochman et al. (1982, 1983) also estimated D values for rhodamine labeled cytochrome c over the surface of mitoplasts from megamitochondria of cuprizone-fed mice. Although the effect of ionic strength was not reported, the number obtained in 8 mM Hepes (1.6 X 10- lo cm2/second) is essentially the same as what Gupte et al. (1983) obtained in 10 mM KP,. In both experiments bound cytochrome c was present at 10 times the level found in intact mitochondria (endogenous cytochrome c was first depleted). One wonders if only 10%of the exogenous cytochrome c occupies sites that its natural counterpart does in a whole mitochondrion; appreciably faster or slower diffusion of this fraction might go unnoticed. It is interesting that while both groups find about the same D value at low ionic strength, one contends that lateral diffusion of cytochrome c is far too slow (at
PROTEIN DIFFUSION IN CELL MEMBRANES
53
least an order of magnitude) to account for observed reaction rates, while the other group favors the notion that electron transfer by cytochrome c is diffusion mediated. Calculations based upon mean free paths (Hochman et a l . , 1982) and Hardt’s equations (LeMasters et a l . , submitted) have been used to argue against or for the sufficiency of these measured D values in supporting completely diffusion-mediated electron transport. When we repeat these calculations using Adam-Delbmck (1968) or Torney-McConnell (1983) equations we find that lop9 cm2/second is apparently too slow to account for some of the more rapid rates of respiration reported for intact mitochondria (Moreadith and Jacobus, 1982), but perhaps sufficient to explain the intermediate to lower rates. However, this conclusion rests precariously upon input parameters such as the reaction radius, oxidase surface density (monomer-dimer?), steric factors, etc., and until these are defined more accurately the significance of this arithmetic remains obscure. The rationale for focusing on cytochrome c mobility was that motion of the integral complexes appeared too slow to support a collisional mechanism of electron transport between any parts of the sequence. Sowers and Hackenbrock (1981) were the first to measure lateral D values for these proteins; using a combination of postfield relaxation and freeze-fracture electron microscopy to quantitate IMP distributions they estimated an average diffusion coefficient of 8.3 X 10- l o cm2/second for all the IMPS in spherical mitoplasts from rat liver cm2/second mitochondria. The same group later found a D value of 4.0 X for complexes 111 and IV using photobleaching methods (personal communication). Hochman et al. (1983) also used FRAP to quantitate diffusion of complex IV in “megamitoplasts” from cuprizone fed mice and obtained a diffusion coefficient of 1.0 X 1 O - I o cm2/second. Again, whether any of these rates is sufficient to support a purely collisional mechanism of electron transport (i.e., 1/2 turnover per hit) depends rather critically on the distances involved, which are still uncertain (see Hackenbrock and Hammon, 1975). Suffice it to say that all these diffusion coefficients pertain to mitoplasts which have been rendered spherical by incubation in low osmolarity buffers, and that a substantial portion of the matrix proteins (240%) is lost during this process (Caplan and Greenwalt, 1966; C. R. Hackenbrock, personal communication). When one considers that the mitochondria1matrix contains between 0.35 and 1.05 g protein/ml (Capaldi, 1982) and that this range encompasses the composition of protein crystals, the loss of some 50% may lead to a major experimental artifact. Others (Srere, 1982; Capaldi, 1982) have already intimated that the motion and organization of integral proteins of the inner membrane may be affected by this dense matrix. One reason that cytochrome c lateral diffusion rates at physiological ionic strengths have not been reported is that the labeled protein does not stick to membranes very well in more concentrated buffers. The ionic strength inside a mitochondrion is likely to be greater than that of 0.2-25 mM buffers, and as
54
MICHAEL MCCLOSKEY AND MU-MING PO0
Gupte et al. (1983) point out, three-dimensional diffusion of cytochrome c within the intermembrane space of intact mitochondria “may be an important component of diffusion-mediated electron transfer. This is an interesting possibility since the translational diffusion rates of small proteins like cytochrome c in water are nearly three orders of magnitude greater than the largest rates measured for cytochrome c on membranes (Barisas et al., 1979; Cadman et al., 1981). While mitoplasts do carry out electron transport at low ionic strength (where all cytochrome c is membrane-bound), it should be noted that this is not necessarily at the maximal rates observed for whole mitochondria, and is therefore a weak argument against the contribution of aqueous phase diffusion to electron transport. However, it is not apparent from our crudely modified Berg-Purcell type calculations that even at cm*/second purely 3D diffusion would generate sufficient collision frequencies to explain the high electron transfer rates reported for rat heart mitochondria (Moreadith and Jacobus, 1982). This very approximate calculation ignores geometrical effects of the cristae folds; it is tempting to speculate that because these flattened membranes nearly confine the path of aqueous cytochrome c to lie in a plane that electron transport between bc, and aa3 enjoys the full kinetic benefit of 2D diffusion that Adam and Delbruck originally referred to, albeit for different reasons (Section III,B, 1). Biochemical/ kinetic evidence on be,-c-aa3 sequence. There are several other lines of evidence regarding the organization and dynamics of the bc,-c-aa, segment. It is now well documented that essentially the same amino acid residues on cytochrome c are involved in binding to not only the oxidase and reductase but also to other proteins such as cytochrome b, and cytochrome c peroxidase (Ferguson-Miller et af., 1978; Speck et al., 1979; Stonehuerner et al., 1979; Poulos and Kraut, 1980; Rieder and Bosshard, 1980; Azzi et al., 1982). These are positively charged lysines which encircle the heme crevice and form a complementary template to negatively charged carboxylate groups surrounding the exposed heme edge in the interacting proteins. The fact that bc, and au3 complexes bind to essentially the same residues on cytochrome c has been used to argue that the latter must diffuse laterally between these two components. However, limited rotation of cytochrome c within a ternary complex could also be a workable model-a model in concert with the observation that cytochrome c covalently bound to either the reductase or the oxidase is still capable of mediating electron transfer (Erecinska et al., 1980; Waring et al., 1980). In support of this scheme, the reduced form of cytochrome c dissociates from purified complex 111at least 10 times faster than the oxidized form. Ferguson-Miller er al. (1978) also envisage simultaneous complexation of cytochrome c by both the oxidase and reductase as being consistent with their kinetic results. Relevant to this model, Hackenbrock and Hammon (1975) made the interesting observation that monospecific antibodies against purified cytochrome oxidase will displace most of the endogenous cytochrome c from inner membranes, ”
PROTEIN DIFFUSION IN CELL MEMBRANES
55
The kinetics of electron transfer from cytochrome c through the oxidase to oxygen are complex, and exhibit two or three phases (Thompson et al., 1982); with purified oxidase and cytochrome c the polarographically determined rate of oxygen reduction is more than an order of magnitude faster than the net rate of appearance of oxidized cytochrome c as determined spectoscopically (FergusonMiller et al., 1978; Smith et al., 1979a). This coupled with other kinetic data has been interpreted to mean that the initial rapid phase of the reaction is due to multiple turnovers of bound cytochrome c prior to dissociation from the oxidase. In these experiments small molecular weight reductants like ascorbate and TMPD were used, and these have more ready access to the heme center than the physiological electron donor. Nevertheless, one is forced to seriously consider the possibility that once bound to the oxidase, cytochrome c turns over several times before the two become translationally separated. As far back as 1956 Chance and Williams discussed the general possibility that limited rotation or oscillation of separate components within a multiprotein complex could explain observed respiratory electron transfer rates. In fact, based upon spectroscopically measured kinetics in submitochondrial particles, Nicholls (1976) concluded that at least 80% of the endogenous cytochrome c in mitochondria is directly bound to the oxidase. Rotational diffusion measurements. Consistent with this idea, Dixit et al. (1982) found that a phosphorescent derivative of cytochrome c rotates at the same slow rate as cytochrome oxidase when bound to mitochondria1 membranes ( T= ~ 300 ksec). In this experiment the phosphorescent analog was substituted for native cytochrome c in an equivalent quantity to that present in vivo, although as with the fluorescent derivatives, subphysiological ionic strengths were necessary to prevent desorption. Saffman and Delbruck (1975) predicted that rotational diffusion coefficients of membrane proteins should be quite sensitive to molecular radii (D,cx r - 2 ) , and experimental findings are in accord with this (Cherry, 1979; Hughes et a l . , 1982). Since cytochrome c is much smaller than the oxidase it might be expected to rotate appreciably faster if it were nonspecifically bound as a monomer to the negatively charged lipid bilayer. The coincident rates may be fortuitous but they are suggestive of complexation between cytochrome c and the oxidase at low ionic strength. The data do not appear to exclude the possibility mentioned above of rapid, yet low amplitude rotation. Finally, we note that rotation rates of cytochrome oxidase reconstituted in lipid vesicles are not influenced by the simultaneous incorporation of the reductase (complex 111) (Kawato et a f . , 1981). Combining this with the relative independence of duroquinol oxidase activity and intramembranous particle density which Schneider et al. (1980) observed in lipid incorporation studies, Kawato and associates conclude that rapid lateral diffusion of cytochrome c between spatially discrete bc, and aa3 complexes is more likely than formation of a ternary complex of the three molecules. There is one obvious reason, however, why this reconstituted system may simulate with low fidelity the interactions within a
56
MICHAEL MCCLOSKEY AND MU-MING PO0
mitochondria1 inner membrane, namely, the lipid milieu is much different. As emphasized in a preceding paragraph, if lipid-mediated protein-protein interactions are involved in the maintenance of an intact, functional structure, then the arbitrary substitution of a synthetic lipid (fluid or otherwise) for the complex blend indigenous to a cellular organelle could disrupt that structure. Summary. While a large body of evidence weighs against the existence of a single static electron transport chain, much uncertainty remains regarding the dynamic structural organization of the terminal segment of the sequence. Several simplified or reconstituted systems which are amenable to direct measurements of protein diffusion have yielded interesting clues about the role of lateral diffusion in respiratory electron transfer. Nevertheless, for reasons made clear in the above, the relevance of measurements on these systems to the intact mitochondrion remains an open issue. b. Photosynthesis. Current dogma regarding the mechanism of photosynthetic electron transport and phosphorylation in green membranes is embodied by the so called Z-scheme (Fig. 2), which has at its heart the concept of noncyclic electron transport. According to this picture two independent photochemical reactions activated by light of different wavelengths operate in series to drive the endergonic transfer of electrons from water to ferredoxin and then NADP. The initial photoact (680 nm) generates an oxidant capable of stripping electrons from water and a weak reductant which donates electrons to a sequence of carriers including plastoquinone, the cytochromeflb6 complex, and plasto-
Q WATER
r
Lo
REDUCTION POTENTIAL
-
FIG. 2. Photosynthetic electron transport chain: PSI, photosystem I (trap WOO); PSII, photosystem I1 (trap P680); PQ, plastoquinone; Cyt blf, cytochrome bdfcomplex; PC,plastocyanin; Fd, ferredoxin; FNR, ferredoxin-NADP oxidoreductase.
PROTEIN DIFFUSION IN CELL MEMBRANES
57
cyanin. The second photoact (700 nm) produces a weak oxidant that accepts electrons from plastocyanin and a strong reductant capable of reducing ferredoxin, a high potential Fe-S protein. Energy released during the exergonic transport of electrons from Q to plastocyanin is somehow utilized to make ATP, which along with NADPH is consumed during fixation of atmospheric carbon dioxide in dark reactions (Calvin cycle). Photophosphorylation of ADP is also coupled to some cyclic electron flow driven only by the second photoact. The two photochemical events occur in chemically distinct photosystems, which are now known to consist of unique proteins containing one reaction center chlorophyll a and many (order of lo2) highly oriented antenna chlorophylls which transfer excitation energy to the reaction center, or trap. Photosystem I1 (trap P680) mediates water oxidation while photosystem I (trap P700) reduces ferredoxin in both cyclic and noncyclic electron transport. The bulk of the chlorophyll a and essentially all chlorophyll b is contained in a third pigment-protein complex which conducts no known photochemistry. As its name suggests, a primary function of this light harvesting complex (LHC) is to transfer excitation energy to the photochemically active pigment-protein complexes. It is now quite certain that the lateral distribution of various components of the thylakoid membrane is markedly nonuniform. Thus, the ATPase and photosystem I are totally excluded from grana partitions (the appressed regions of grana stacks), while photosystem 113, cytochrome b,,,, and the LHC are highly concentrated there (Anderson and Anderson, 1982, and references therein; Usharani et al., 1983). The antibody accessibility of ferredoxin-NADP reductase (Jennings et al., 1979) is consistent with an earlier suggestion (Berzborn, 1969) that it is located primarily in stroma-exposed regions of the thylakoid. On the other hand, the cytochrome f / b 6 complex appears to be partitioned equally between appressed and exposed thylakoid regions (Cox and Anderson, 1981; Anderson, 1982). Average diameters of grana stacks range from 0.3 to 0.5 p,m, and stroma lamellae can be somewhat longer. Thus, relatively long-range lateral separation of the two photosystems presents an interesting biophysical problem for the conventional Z-scheme, namely, is lateral diffusion of the intermediate electron carriers fast enough to cover such distances within their turnover times? Anderson and Anderson (1982) suggest that plastoquinone and plastocyanin should be capable of mediating long distance electron transport by a diffusional mechanism. Indeed, if one simplifies the argument by assuming that all the pho3Several groups have presented spectroscopic evidence for two distinct types of PS 11: PS I1 a and PS I1 (3 (Melis and Hohman, 1976; Horton and Croze, 1979; Thielen and Van Gorkom, 1981). PS I1 p (the minor fraction of PS 11) is supposed to have fewer antenna chlorophyll a molecules associated with it than PS I1 a.Anderson and Melis (1983) now report that PS I1 a is found exclusively in grana partitions while PS I1 p is restricted to stroma exposed thylakloids.
58
MICHAEL MCCLOSKEY AND MU-MING PO0
tosystem I is located in margins of the grana thylakoids, then a back of the envelope calculation is possible. With PS I1 uniformly dispersed in the appressed membrane, the minimum (average) distance a quinone must diffuse during one turnover (20 msec) can be taken as twice the radius of the stack, or 0.4 pm. This would require a minimum lateral diffusion coefficient of 2 x 10V8 cm*/second, which is about as fast as any lipid analogs have been observed to diffuse in biomembranes (at physiological temperatures). In fact, the requirements on diffusion rates are more stringent than we assume in the above. First, the actual diffusion paths may be substantially greater, since a significant portion of PS I is located not in the margins and end membranes but in the fret membranes. Second, plastoquinone (PQ) may diffuse much slower than proponents of the diffusion mechanism will allow. Nobody has ever directly measured the lateral diffusion rate of this rather long lipid molecule nor has anyone yet proven that it is not bound to a carrier protein. Third, turnover times for other redox components that are candidates for lateral electron transport, e.g., plastocyanin, are appreciably shorter than 20 msec (Haehnel et al., 1980), and this demands even faster d i f f u ~ i o n Clearly, .~ a rigorous analysis depends critically on the values of several variables, none of which is known with tremendous certainty. Even though the immediate prospect of incorporating diffusional parameters into a realistic model for photosynthetic electron transport is small, our current knowledge of the lateral segregation of the two photosystems dictates that noncyclic electron transport via the conventional Z-scheme must involve comparatively long-range lateral diffusion of plastocyanin and perhaps the cytochrome b/f complex. Arnon and associates have recently challenged the notion of noncyclic electron flow in higher plants. According to the current Z-scheme PS I1 cannot directly photoreduce ferredoxin; only PS I is supposed to be a strong enough reductant to accomplish this. Using different inhibitors of electron transport that block at plastoquinone, Arnon et al. (1981) find that exogenous ferredoxin is directly reduced even when PS I is poisoned. Further, using the aqueous polymer twophase partition method to isolate vesicles highly enriched in PS 11, direct electron transfer from water to NADP is again observed (Arnon et al., 1983). The authors postulate that ferrodoxin is used as an electron acceptor by both PS I and PS 11, and that two separate photoacts involving PS 11 drive electron flow through a circuit involving plastoquinone, cytochrome blf and PS I1 associated cytochrome 4Diffusion in the aqueous phase near the membrane may play an important role in energy transducing membranes (see Section III,B,I). Cox and Anderson (1981) attempt to account for the rapid cm2/second) in the aqueous turnover of plastocyanin by postulating that it diffuses rapidly (ca. medium within the intrathylakoid space. Based upon their FRAP studies, Gupte et al. (1983) advance a similar argument for rapid diffusion of cytochrome c in the aqueous phase between inner and outer mitochondria1 membranes.
PROTEIN DIFFUSION IN CELL MEMBRANES
59
b,,,. While this hypothesis places less stringent requirements on the lateral mobility of plastocyanin and plastoquinone, Arnon believes that these species must still carry “a trickle” of electrons between the two photosystems, for regulatory purposes. Therefore, both of the major working hypotheses on photosynthetic electron transport appear to require a corrolary hypothesis which invokes relatively long-range lateral diffusion of some component(s) of the electron transport chain. An interesting ramification of Amon’s proposal stems from the apparently nonuniform lateral distribution of ferredoxin-NADP reductase. Ferredoxin reduced by PS 11 must diffuse from the appressed to the stroma exposed thylakoids before it can reduce NADP. Ferredoxin is water soluble, and easily lost from chloroplast membranes during isolation procedures. What percentage of the ferredoxin in an intact chloroplast is actually adsorbed to the membrane and what percentage is free in the stroma is not known. Thus, whether it would have to diffuse laterally on the membrane or free in solution is a moot point; the latter would seem inimical to maximal production of NADPH, but ferredoxin does participate in other reactions involving soluble phase reactants (e.g., thioredoxin). Relevant to the mode of linkage of ferredoxin to the membrane it is interesting to note that Wagner et al. (1982) find a 40-fold reduction in the rotational rate of membrane bound ferredoxin-NADP reductase upon addition of ferredoxin. Because ferredoxin is small (MW 12,000) it is not expected to reduce the rotational diffusion rate of the reductase (MW 35-40,000) nearly so much upon binding; the authors speculate that ferredoxin mediates the formation of a trimolecular complex between PS I, ferredoxin, and the reductase.
C. BIOLOGICAL SIGNALING AT
THE
MEMBRANE
Signal is defined here as a piece of information transferred between a cell and its environment. In the following discussion we consider the possible relevance of lateral diffusion to both the reception and propagation of signals at the plasma membrane. Some topics, for example the putative role of hormone receptor mobility in cyclic AMP-mediated responses have already received considerable attention in other places. However, two subjects from neurobiology and immunology involve a previously unemphasized concept, namely, diffusion-mediated signaling within the plane of the membrane. 1. Transmembrane Signaling
a. Cyclic AMP-Mediated Hormonal Response. The literature is replete with references to the possible significance of lateral mobility in cyclic AMP (CAMP)mediated hormonal response, and the subject needs little introduction here. Adenylate cyclase systems consist of at least three physically separable membrane proteins which interact upon hormone or neurotransmitter binding to catalyze
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MICHAEL MCCLOSKEY AND MU-MING Po0
synthesis of CAMP:the catalytic unit, the hormone (or neurotransmitter)receptor, and the GTP binding protein. The bulk of evidence is most easily rationalized with a “mobile receptor” hypothesis, which postulates that collisional encounters between independently diffusing catalytic units and receptor- hormone complexes produce the catalytically active state. Two observations supply the best evidence for this hypothesis. (1) Hormone stimulated cyclase activity can be restored in heterokaryons, one partner of which lacks functional catalytic units and the other of which lacks functional hormone receptors (Orly and Schramm, 1978; references in Schulster, 1979); (2) after irreversible inactivation of 93% of the p-adrenoreceptors in turkey erythrocytes, the remaining 7% can activate all the catalytic units, though at a slower rate (Tolkovsky and Levitsky, 1978). It was originally envisaged that receptor-ligand complexes interact directly with catalytic units, and discussion centered on the relative lateral mobility of just these two components. The situation assumes interesting complexity now that the existence of a completely separate GTP binding unit (G unit) is fully recognized. Rodbell (1980, 1981) discusses evidence from target size analysis, and proposes that in some cell types the G units and receptors are copolymerized into oligomeric structures in the membrane; according to the model, hormone or neurotransmitter binding triggers dispersal and frees the regulatory (G) and receptor units for interaction with the catalytic unit. This hypothesis may be rather speculative at present, but we find the results of a recent FRAP study in striking concert with one of its predictions. Henis and Elson (1981) observed that on Chang human liver cells both antagonist-labeled and (one infers) unliganded preceptors are present as visually discernible, immobile clusters over the cell surface. The small mobile fraction has a rapid lateral D value of 1.4 X cm*/second at 23°C. Agonist binding causes slow (minutes) dispersal and mobilization of receptors without affecting lipid diffusion rates. On the other hand, the time course of mobilization is much slower than that of cyclase activation (seconds) but roughly parallels the kinetics of receptor loss and enzyme desensitization. The inescapable conclusion is that ‘‘adenylate cyclase activation by preceptors does not require macroscopic lateral mobility of the majority of preceptors”-at least in Chang human liver cells. Others have also speculated that the cAMP response to hormones might be regulated through control over the lateral mobility of cyclase components. Hirata and Axelrod (1980) for example, suggest that specific ligands can induce local enzymatic modification of the lipid milieu, and this is supposed to enhance cyclase activity by increasing the frequency of encounters between receptors and catalytic units, e.g., because of greater lipid fluidity. The finding that microtubule disassembly can increase hormonally stimulated cAMP synthesis is consonant with cytoskeletal regulation of the mobility of cyclase components (Insel and Kennedy, 1978; Rudolph et al., 1979). Rasenick et al. (1981) observed that
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colchicine and vinblastine not only enhanced the G unit mediated activation of cyclase activity in rat synaptic membrane preparations, but also loosened the connection of G units to the membrane sufficiently that a sizable portion could be removed after one rinse in the centrifuge. They concluded that “the ability of the G unit to diffuse laterally in the membrane is a limiting factor in cyclase activation. ” Cis-unsaturated but not saturated fatty acids also enhanced cyclase activity, but without knocking G units off the membrane. Combining these results with the ability of cis but not trans-unsaturated or saturated fatty acids to block capping in leukocytes (Klausner et al., 1980), a new interpretation may be suggested for the initial finding (Hanski et af., 1979) that the “fluidizing agent” cis-vaccenic acid speeds agonist-induced activation in membrane fragments from turkey erythrocytes. It was found that the “rate constant” for hormone activation of cyclase increased linearly by 2O-fold over less than a 2-fold variation in the bilayer microviscosity; perhaps this was not due so much to increased lipid fluidity as to decreased interaction of some cyclase component with the cytoskeleton. We can only conclude that future studies which directly probe the time dependence of rotational and translational diffusion of individual cyclase components during activation will help firm our grasp on the actual mechanism of this physiologically important response. Resolution of the coupling mechanism also hinges on more detailed structural and biochemical knowledge of the noncovalently linked components. Development of fluorescent reporters with specific affinity for the individual proteins (see, e.g., Haley et al., 1983) seems to be the primary hurdle both for photobleaching and for resonance energy transfer experiments aimed at measuring distances between the individual sites. b. Leukocyte Degranufation. Receptor clustering and dispersal have frequently been considered candidates for the initial actuating event in several different immunological, hormonal, and neurological responses. Speculation on this topic has been fueled by the following kinds of evidence: bivalent antibody induced patching and capping in lymphocytes, PMNs, and fibroblasts (dePetris, 1977); polyclonal stimulation of lymphocytes by mitogenic lectins, bacterial lipopolysaccharides, and other potentially multivalent ligands; the dependence of antibody response on antigen valency and state of aggregation (Dintzis et al., 1976, 1982, and references therein); clustering of informational macromolecules prior to internalization (Goldstein et al., 1979; Schlessinger, 1979);the ability of bivalent polyclonal antibodies against the insulin receptor to mimic certain biological responses produced by insulin (Kahn, 1979; Baldwin et al., 1980); potentiation of the response to low concentrations of insulin or EGF by bivalent antibodies against these hormones (Shechter et af., 1979a,b); stimulation of testosterone synthesis in Leydig cells by bivalent antibodies against the lutropin receptor (Podesta et af., 1983); data referred to above on the adenylate cyclase system. Should it ever be demonstrated conclusively that passive receptor ag-
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gregation (or dispersal) really is an essential activating step, then local translational (and rotational) diffusion of membrane proteins will be directly implicated in the elicitation of physiological response. Perhaps the least equivocal example where clustering of specific cell surface receptors is a sufficient condition for cell triggering is the antigen-stimulated degranulation of mast cells and basophils. These are highly specialized leukocytes (basophils) and connective tissue cells (mast cells) that form an integral part of the immediate allergic response to soluble stimuli. On their surface are high affinity, monovalent receptors for the Fc stem of IgE (Mendoza and Metzger, 1976); although serum IgE levels are very low (nanomolar) IgE Fc receptors are normally at least partially (20-100%) saturated in vivo (Malveaux et al., 1978). The interior of these cells is packed with membrane surrounded granules containing histamine (and/or serotonin), heparin, and protein. Interaction of multivalent antigens (like ragweed proteins) with cell borne IgE causes granules to fuse with each other and with the plasmalemma, thus releasing histamine into the bloodstream. Several biochemical reactions reportedly accompany (or precede) degranulation, including phosphatidylserine decarboxylation, N-methylation of phosphatidylethanolamine, phospholipase-mediated arachidonic acid release, prostaglandin and leukotriene synthesis, CAMPproduction, activation of two protein kinase isozymes, and calcium influx (reviewed by Ishizaka, 1982); but the primary event seems to be antigen-induced bridging of IgE Fc, receptor complexes (reviewed by Kagey-Sobotka et a l . , 1982). In fact, neither IgE nor cross-linking antigen is required. Bivalent antibodies against the purified receptor will cause degranulation (Ishizaka et al., 1971; Isersky et a l . , 1978), as will bivalent antibodies against IgE when applied to IgE-sensitized cells (Ishizaka and Ishizaka, 1978); chemically cross-linked dimers of IgE in the absence of antigen will also trigger mast cells (Segal et al., 1977). Degranulation does not require global changes in the membrane, since locally applied stimuli cause degranulation directly beneath the stimulus (Lawson et al., 1978; Diamant et al., 1970). Extensive mathematical modeling of this system has been performed, and if the competing process of desensitization is allowed for the calculations predict with reasonable accuracy dose-response curves for histamine release in the presence of structurally well-defined bivalent haptens (DeLisi, 1979; Dembo et al., 1979a,b; Chabay et al., 1980). Using a range (100-fold) of experimentally measured rate constants to work backwards from the model and predict a diffusion coefficient for the IgE Fc, receptor complex, DeLisi (1979) obtains numbers to 10- cm2/second, a region which straddles the FRAPin the range of determined value of 2 X 10- l o cm2/second (Schlessinger et al., 1976). c. Cell-Cell Communication via Diffusion-Mediated Trapping. There are some recent observations on basophil degranulation which may have far-reaching biological implications in terms of generalized signalling mechanisms in
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cell-cell communica$on. While it is certain that relatively long-lived and thermodynamically stable pairwise interactions between Fc, receptors are sufficient to generate the primary biochemical/biophysical events leading to degranulation of mast cells and basophils, they may not be necessary. Rat basophilic leukemia (RBL) cells bearing anti-dinitrophenyl IgE will release serotonin upon contact with either fluid or solid phospholipid vesicles containing a monovalent and most probably monomeric DNP lipid hapten (Balakrishnan et al., 1982). Assuming that dimerization or oligomerization of Fc, receptors remains an obligatory step in stimulation by the lipid vesicles, one would like to know what drives receptor aggregation in the absence of cross-linking reagents. Balakrishnan and co-workers suggest, among other possibilities, that diffusion-mediated localization and concentration of receptors in the vesicle-cell contact zone may contribute. We have observed substantial and rapid accumulation of fluorescent, cell-bound antiDNP IgE at the region of contact with DNP-derivatized polyacrylamide beads, even in the presence of 30 mM NaN,. Provided that a prior equilibrium exists between monomeric and “clustered” IgE receptors, locally high IgE receptor concentrations may shift the equilibrium in favor of “clusters” and thus initiate degranulation. If specific receptor clustering is a widespread and basic signaling device then diffusion-mediated trapping may offer a general mechanism whereby two mobile, independently diffusing components on opposing cells can participate in cell-cell communication. Bell (1979) proposed an analogous model to explain the interaction of histocompatibility-restricted cytotoxic T lymphocytes with virally infected target cells.5 In a provocative report Kampe and Peterson (1979) showed that histocompatibility antigens actually do localize in the contact zone between killer T cells and their targets, although the mechanism of localization was not investigated. One wonders if lymphocyte triggering by antigen presenting macrophages is mediated in part by passive contact-induced accumulation of antigen and receptors in the contact zone. Dintzis and associates (1976, 1982) find that the primary antibody response to T cell-independent antigenic polymers depends critically on the number of haptens per polymer chain, and propose that formation of a specific, well-defined cluster of lymphocyte receptors, or immunon, is an obligatory step in triggering antibody synthesis. They suggest that cell-cell contact between lymphocytes and “helper” cells with clustered surface-bound antigen could also generate the putative immunons; based upon the results of Balakrishnan e? al. (1982) it may be simpler to postulate that in the case of T cell5Local as well as long-range diffusion is implicated in this and other cell-cell interactions where specific receptors must bind to complementary species on an adjacent cell. As shown in studies on macrophage phagocytosis (McConnell, 1979; Lewis ei al., 1980) this is most evident at low receptor/ ligand densities, where the probability of molecular bond formation as well as the maximally attainable numb& of bonds can be enhanced by relative lateral diffusion of the interacting components.
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dependent antigens, monomeric antigenic fragments on the macrophage surface (bound to Ir gene products?) induce clustering of lymphocyte receptors by a combination of diffusion-mediated trapping and Le Chatelier’s principle. We stress that diffusion trapping is a passive process which in the above applications can only promote clustering if the intercellularly bridged receptors remain mobile. d. Contact-Induced Immobilization. At the opposite extreme, it seems that complete immobilization of a freely diffusing molecule by binding to an immobile species on a neighboring cell could also pass information through the membrane, and the response might be faster in this case. Imagine, for example, that the cytoplasmic concentration of some key molecule was kept relatively constant and spatially uniform by continual collision dependent activation of a plasmalemma enzyme like adenylate cyclase. The sudden and local immobilization of activator molecules would cause a quick and initially localized drop in the cytoplasmic level of this key molecule, provided the enzyme was relatively immobile. Immobilization of naturally occurring membrane-associated inhibitory substances, e.g., the peripheral protein inhibitor of mitochondria1 ATPase (Emster et a l . , 1979), GABA-modulin (Wise et al., 1983), certain glycosaminoglycans which directly inhibit adenylate cyclase (Cutler, 1982), would also polarize the cell interior. While this group of molecules does not actually possess all the requisite attributes, it does serve to illustrate the general idea of how local, contact-induced freezing of molecular motion might work to promote information transfer between cells.
2. Information Flow in the Membrane Plane a. Counting Synaptic Contacts. “Signaling” usually connotes specifically the transmembrane delivery of messages. In principle, however, two cells can communicate without information ever leaving the membrane plane, through membrane reorganization mediated by lateral diffusion of membrane components. Formation of synaptic connections provides an interesting example. In a developing nervous system each postsynaptic cell receives a certain number of nerve terminals, usually more than the number left in a mature nervous system. In neonatal skeletal muscle fibers, each fiber is innervated by several separate axons (Brown et al., 1976); as maturation proceeds all but one synapse is eliminated through synaptic competition. Concomitantly with establishment of synaptic transmission, receptivity of the muscle surface to further innervation is lost. Similar events occur during synaptognesis between neurons (Purves and Lichtman, 1980). It appears that muscle and nerve cells can somehow count the number of cell-cell contacts both in the embryonic multi-innervated state and in the mature state. After a preset number of synaptic contacts is made, the extrasynaptic region of the membrane loses receptivity to further contact. One simple mechanism that would account for counting is based upon diffusion-mediated
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trapping. If the initial recognition/adhesion between neurite and postsynaptic cell is mediated by some mobile species of recognition/adhesion molecule that accumulates and becomes trapped at the developing synapse, then concurrent depletion of the molecule from the extrasynaptic membrane will eventually prevent the recognition/adhesion with more neurites. Thus, by regulating the amount of the hypothetical recognition/adhesion molecule present on the cell surface (e.g., by controlling its rate of synthesis and degradation), the cell could determine the allowed number of contacts without the need for any signal as to where on the surface the contacts have been made. The receptivity of embryonic and denervated adult skeletal muscle fibers to innervation correlates well with the presence of high ACh receptor concentrations. Might not the ACh receptor itself be a recognition/adhesion mediating glycoprotein, in accordance with the above hypothesis? If so, nerve-induced accumulation of ACh receptors would then explain three separate phenomena of neuromuscular interactions: contact stabilization, localized channel activity, and control over the number of synapses (signaling). After maturation contacts may be stabilized by accessory interactions, such as with components of the basement membrane (Sanes et al., 1978). b. Immune Adherence. Macrophage Fc, receptors bind to IgG molecules that are present as opsonin on various phagocytosable particles and promote the attachment and engulfment of these foreign or altered-self targets. From a detailed mathematical analysis of macrophage-target binding kinetics (Lewis er al., 1980) coupled with independent observations from quantitative electron microscopic (Petty et al., 1981) and biochemical (Mellman et al., 1981) studies the following picture has emerged: Fc, receptors are irreversibly removed from the cell surface by the phagocytic internalization of plasma membrane. A reservoir of membrane is made available to the surface during this phagocytic challenge such that the total plasmalemma area remains nearly constant even though 50% or more may be internalized during a typical experiment. Fc, receptors remaining on the cell surface apparently undergo complete redistribution after particle uptake and reinsertion of fresh, receptor deficient membrane but prior to subsequent particle binding and uptake (this is true for phagocytosis of IgG opsonized 1 pm lipid vesicles by murine macrophages). That is, they rapidly diffuse into the receptor deficient patches. It may be that “down regulation” of IgG Fc receptors in response to phagocytosis is part of an appestat mechanism for reticuloendothelial cells, which by controlling the strength of immune adherence prevents phagocytes from overtaxing their finite digestive capacity for particles like opsonized Streptococcus and senescent erythrocytes. Perhaps rapid lateral redistribution of Fc, receptors permits fine tuning of the response by ensuring a uniform-rather than locally concentrated4istribution of attachment sites. Depending on the size and position of newly incorporated membrane patches, a nonuniform topography due to
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IgG Fc receptor immobility could adversely prolong particle adherence. Although there is independent evidence of membrane recycling in macrophages (Muller er al., 1980), we know of no reports defining the size of newly added vesicles. Should their diameter be significant relative to that of a bacterium (say 0.4 Fm) then the above receptor dilution hypothesis is reasonable. Michl et al. (1 983) find that unligated Fc, receptors on murine macrophages diffuse at faster than 2 X l o p 9 cm*/second at 37”C, a rate compatible with rapid redistribution and two-dimensional “signaling. ”
D. SELF-ASSEMBLY AND SORTING One of the basic problems in cell biology is to understand the underlying biogenic processes which give rise to differentiated structures so characteristic of living organisms. Self-assembly and sorting of cellular components, both in the membrane and the cytoplasm, are key elements in these processes. Many membrane components are multimeric structures which are most likely self-assembled in the membrane from individual subunits. ,Some pertinent examples are cytochrome oxidase, photosynthetic protein-pigment complexes, Na-K-ATPase, prothrombinase, the membrane attack complex of complement, acetylcholine receptors, and gap junction connexons. Although very little is known about the detailed molecular events leading to self-assembly of multisubunit integral proteins, it is axiomatic that short-range translational diffusion of the individual subunits is mandatory. On the other hand, sorting mechanisms must somehow overcome the countervailing tendency of diffusion to randomize molecular distributions. A complex example of this interplay is found in eukaryotes, where the Golgi stacks are actively engaged in sorting of many membrane components. While a continual influx of new (from ER) and recycled membrane impinges on the Golgi, a melange of newly made and recycled proteins is constantly sorted and re-routed to the ER, lysosomes, secretory vesicles, or plasmalemma. At one step or another, these proteins must undergo long-range lateral separation in the membrane plane. While there is no lack of hypotheses pertaining to sorting within the Golgi complex, in reality next to nothing is known regarding the molecular mechanisms employed. For reviews see Tartakoff (1980), Rothmann (1981), Rothmann et al. (1981) and Olden ef al. (1982). Somewhat more is known about the following three examples, which should highlight the complexity of sorting problems, and also shed some light on the importance of protein diffusion in same. 1. Virus Budding One of the most dramatic cases of membrane sorting occurs during the budding of a membrane-enveloped virus from an infected cell. The viral envelope
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forms from preexisting host cell membrane, yet it contains at most only trace amounts of host-determined proteins, the bulk (99+ %) of the envelope protein content being contributed by one or a few virally coded transmembranous glycoproteins (for review see Lenard, 1978). Enclosed by the envelope is an often highly symmetrical nucleocapsid consisting usually of single stranded RNA in close association with one or a few virus-determined nucleocapsid-, or N-proteins. With a few exceptions (the togaviruses), there is also a nonglycosylated matrix- or M-protein situated in the tight space between nucleocapsid and membrane. Both the M- and N-proteins are synthesized on “soluble” polyribosomes and reach the plasmalemma independently of the viral gl ycoproteins, which take the membrane-bound route via the rough endoplasmic reticulum and Golgi complex. In the final stage of assembly a preformed nucleocapsid, integral glycoproteins, and M-protein coalesce at the plasma membrane (usually) and a new viral particle buds off. In a positive or negative sense, protein diffusion is involved in at least three of the problems which attend virus budding: First, what generates the forces required to mechanically distort the membrane of a forming virion? Second, how are host cell membrane proteins selectively excuded from the budding virus? Third, how are virus glycoproteins selectively included in the budding virion? The magnitude of this “sorting” problem is not trivial; on the basis of published data on the protein content of typical plasma membranes and of vesicular stomatitis virus (VSV) envelopes (Cartwright et al., 1972), one can estimate that the surface density of host cell proteins can be less than one-tenth that in the precursor plasma membrane. Over the years several models for budding of membrane-enveloped viruses have been proposed (for references see Johnson et al., 1981). Regarding the mechanical properties of membranes, Evans and Buxbaum (1981) have shown that given sufficiently strong membrane-membrane attraction erythrocytes will passively and nearly totally engulf membranous vesicles as large as 1-3 pm. Willison et al. (1971) found that 0.12-pm latex spheres are partially swallowed by tomato protoplasts, and suggested that adhesive forces were sufficient to warp the plasmalemma passively in the early stages of endocytosis. Likewise, Haywood ( 1975) has demonstrated that liposomal membranes containing glycolipids which bind to Sendai virus will adhere to and tightly encase the virion. If a viral nucleocapsid had affinity for viral membrane glycoproteins and/or M-protein it would presumably nucleate a similar process when it made contact with the membrane; diffusion-mediated trapping or two-dimensional crystallization of M and/or glycoproteins around the “nucleus” would provide a ready explanation for exclusion of host cell proteins. That such simple mechanisms do not operate in VSV is evidenced by the presence of several temperature-sensitive (ts) mutants which form virus particles severely deficient in or entirely lacking nucleocapsids (Schnitzer and Lodish, 1979). Other ts mutants of VSV lack the membranous viral glycoprotein (Deutsch, 1976; Little and Huang, 1977) but
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contain normal amounts of M-protein and nucleocapsid. This coupled with the significant (nearly twofold) variation in glycoprotein concentration that can occur in viral envelopes (Lazarowitz et al., 1973) is inconsistent with (1) pure steric hindrance due to tight packing of viral glycoproteins as the mechanism for exclusion of host membrane proteins (in those viruses containing M-proteins); (2) a cooperative aggregation of viral glycoproteins elicited by high glycoprotein concentrations. Apparently, no M-protein-deficient mutants with the ability to bud have been observed (Lenard, 1978); in this regard, it is interesting that Mprotein is not detectable in the virulent, nonbudding form of the subacute sclerosing panencephalitis strain of measles virus but is present in wild type and nonvirulent SSPE strains, both of which form buds (Lin and Thormar, 1980). This is suggestive evidence that M-protein is a central organizer essential for budding and possibly also for excluding host proteins from the viral envelope. Perhaps Mproteins are analogous to clathrin, which polymerizes into virus-sized baskets which bind to the plasmalemma, and which may form part of the structure within coated pits which discriminates between trappable and untrappable membrane components (e.g., ligated and unligated hormone receptors). The minimum structuraUcompositiona1requirements for budding have yet to be determined. While diffusion-mediated trapping of viral glycoproteins is probably not a universal mechanism for exclusion of host proteins, it may well induce segregation of glycoproteins into virally molded domains. For VSV and other M-protein-containing viruses there is a large excess of viral glycoprotein in the plasmalemma during budding, and for wild-type VSV infections of baby hamster kidney cells and chick embryo fibroblasts this component is diffusely distributed and largely mobile (75%), with a moderate diffusion coefficient of ca. 6 X 10- lo cm*/second (Reidler et al., 1981; Johnson et al., 1981). Qualitatively at least, these results are consistent with a diffusion trap mechanism. Reidler et al. (198 1) have argued from their photobleaching experiments with wild type and M-altered VSV mutants that specific complexation of M-protein with the glycoprotein is a rate-limiting step in budding of wild-type VSV particles but that reduced interaction of the altered M-protein with nucleocapsid becomes rate limiting in the mutants. The nature and extent of this putative interaction were unspecified. In contrast to VSV, Sindbis virus (SV) glycoproteins in the host plasmalemma are mostly immobile soon after infection starts (Johnson et al., 1981). This correlates with electron microscopic evidence that SV nucleocapsids become attached to intracellular membranes prior to reaching the plasmalemma (Johnson and Schlesinger, 1980; Gottlieb et al., 1979; Birdwell et al., 1973), perhaps implying that for this and other togaviruses, redistribution of viral glycoproteins from a diffuse to an aggregated state begins intracellularly. Since M-proteins are absent from these viruses, a simpler assembly may occur than for viruses con-
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taining M-proteins-involving glycoproteins.
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direct binding between nucleocapsid and
2. Thylakoid Stacking Thylakoid membranes in chloroplasts of higher plants and green algae are differentiated into grana stacks and stroma lamellae. As noted previously (Section III,B,2), the lateral distribution of several components of the photosynthetic apparatus is markedly nonuniform along the plane of the thylakoid membrane. While the true physiological significance of this topography is unknown, current thinking is dominated by the notion that it facilitates adaptation of the photosynthetic apparatus to different qualities and intensities of light. Thus, shade plants typically have higher ratios of grana to stroma lamellae than sun plants (Anderson et al., 1973); the ratio of stroma exposed to appressed thylakoid membrane area is also greater in algae cultivated in low light intensity than in algae grown in bright light (Reger and Kraus, 1970). Chloroplasts from higher plants and green algae respond within minutes to shifting light regimes (e.g., 650 vs 710 nm) such that maximal quantum yields are sustained (Bonaventura and Meyers, 1969; Murata, 1969; Punnet, 1971); this “State” transition is accompanied by partial thylakoid destacking and lateral redistribution of the light harvesting chlorophyll a / b protein complex (LHC) (Punnet, 1971; Bennoun and Jupin, 1974; Biggins, 1982; Staehelin et al., 1982). The transition is thought to reflect a balancing of the distribution of excitation energy between the two photosystems such that rates of noncyclic electron transport are optimized (Wang and Meyers, 1974; Butler, 1978). It is well established that red light stimulates phosphorylation of the LHC, and recent experiments have confirmed that the phosphorylated LHC moves out of PS I1 territory and into PS I enriched domains (Kyle et al., 1983; Staehelin et al., 1982). A cogent argument has been advanced that the redox state of plastoquinone regulates the kinase responsible for this phosphorylation, and in this way controls the relative rates of excitation in PS I and PS I1 traps (Bennet et al., 1980; Horton and Black, 1980). For full details on this model and references to the literature consult the review by Haworth et al. (1982). Passive diffusion-mediated trapping of the LHC appears to direct the stacking process per se, and once local environments are formed they may exclude other species due to steric constraints or electrostatic repulsion. In greening plastids the appearance of LHC polypeptides parallels the formation of stacked membranes (Armond et al., 1976; Davis et al., 1976; Argyroudi-Akoyunoglou and Akoyunoglou, 1977); mutants deficient in the LHC have no grana stacks (Anderson, 1975; Arntzen et al., 1976; Burke et al., 1979). Purified LHC undergoes reversible cation-mediated aggregation to form two-dimensional sheets which superficially mimic the grana/stroma system seen in intact chloroplasts (Mullet
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and Arntzen, 1980). As noted above, partial unstacking accompanies the phosphorylation-induced migration of LHC into stroma-exposed membranes. In virro, chloroplasts lacking the envelope undergo reversible cation-dependent stacking and destacking concomitantly with lateral redistribution of the LHC and other components (Izawa and Good, 1966; Goodenough and Staehelin, 1971; Ojakian and Satir, 1974; Staehelin, 1976). A small trypsin releasable peptide from the hydrophilic surface of the LHC is an absolute necessity for salt induced stacking and lateral segregation of the LHC (Mullet and Artnzen, 1980; Staehelin et al., 1980). From the time evolution of energy transfer between PS I1 units during artificial stacking, Rubin et af. (1981) calculate lateral D values in the range of 3.1 X to 1.9 X 10-l2 cm2/second (30 to 10°C) for PS I1 complexes. These numbers seem surprisingly low for proteins embedded in membranes composed of such highly unsaturated lipids and lacking obvious extrinsic constraints. Since the calculations are based on the assumption of a diffusion-limited process, the low D values may reflect some other rate-limiting step; blind application of the 3D Smoluchowski formulation to diffusion-limited processes in two dimensions is also perhaps unjustified (see Section III,B,l). In any case, it is probably only a matter of time before FRAP is used to directly measure the lateral diffusion coefficients of thylakoid components, not only to study the stacking process but also to investigate the role of lateral diffusion in photosynthetic electron transport (Section III,B ,2). The intrinsic fluorescence of the three major pigment-protein complexes should facilitate this work, and also preclude interfering effects of large labeling reagents like antibody fragments. No one knows the exact chemical nature of the "trap" for LHC, and speculation runs the gamut from specific polypeptide-polypeptide binding (lock and key) to nonspecific hydophobic interaction made possible by cation screening of the negative charges on opposing membranes. Barber (1980) and Rubin et al. (1981) present mathematical models of the stacking/segregation phenomenon based upon colloidal aggregation theory. PS I1 in the appressed regions is bound to the LHC, and it is not hard to imagine that LHC acts as an indirect trap for PS 11. Likewise, it is easy to conceive of the large extrinsic portion (CF,) of the ATPase as too big to fit into the appressed regions (4 nm). What is less clear is how PS I might be excluded from the grana partitions; its total mass is not terribly different from that of PS 11, although the aqueous projection may be. Perhaps it is highly negatively charged. How smaller extrinsic proteins like the ferredoxin-NADP reductase are excluded from grana partitions is also an open question. In summary, during the past few years the chloroplast has yielded much insight into the connection between protein mobility and membrane differentiation, but central pieces of the puzzle are still missing. In particular, the mechanism for lateral exclusion of PS I remains to be determined. In vitro stacking and
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destacking may provide fertile ground for studying the role of lateral diffusion in membrane morphogenesis. 3. Phagocytosis There is an apparent sorting problem involved in leukocyte phagocytosis that passive redistribution models do not help to explain. While Fc, receptor activity is selectively lost from the murine macrophage cell surface during phagocytosis (Schmidt and Douglas, 1972; Petty et af., 1980a), other membrane proteins are retained, e.g., the receptor for complement component C3b, certain lectin receptors, and transport sites for lysine and adenosine (Tsan and Berlin, 1971; Ukena and Berlin, 1972; Oliver et al., 1974; Petty et al., 1980a). Although cell surface retention of a particular protein may be achieved through mechanisms as, for example, uptake followed by reinsertion of the same species from intracellular membranes or the unmasking of latent activities in the remaining membrane, it has been suggested that in some cases active cytoskeleton-mediated exclusion of selected proteins from the phagocytic membrane is involved (summary in Oliver and Berlin, 1982). Tsan and Berlin (1971) showed that irreversible inactivation of transport sites with a nonpenetrating reagent led to loss of transport activity (which was not recovered after phagocytosis), apparently indicating that new sites are not reinserted from inside the cell. Microtubule disrupting drugs prevent retention of adenine and lysine transport activities during phagocytosis (Ukena and Berlin, 1972), thus implicating some form of microtubule intervention. In a result bearing striking resemblance to that of Flanagan and Koch (1978) that cross-linked surface Ig binds to actin in lymphocytes, Jack and Fearon (1983) have now discovered that cross-linkage of C3b receptors in human PMNs leads to their association with a detergent (NP 40)-insoluble cytoskeleton fraction. Independent support for cytoskeletal control comes from the observation that initially randomly dispersed C3b receptors cluster and the clusters undergo oriented motion upon contact of polymorphonuclear leukocytes with various artificial substrata (Hafeman et a l . , 1982). Like anchorage modulation this is another global effect in response to local contact; clusters form over the entire surface upon attachment of one side of the cell to the substrate. Several lines of evidence thus substantiate the notion that active processes involving the cytoskeleton coordinate the exclusion of specific proteins from the phagocytic membrane.
IV. Closing Remarks Is diffusion of membrane proteins crucial for any known biological processes? The answer cannot be a resounding “yes” for all the membrane-directed events examined in this article. The problem stems from (1) a considerable uncertainty regarding the true diffusion rates of many proteins in cell membranes; (2) a lack
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of more concrete knowledge of the context within which the proteins are moving. Without a sharper picture of membrane microstructure, even the most accurate of diffusion measurements is difficult to interpret. Quantitative analysis of many problems involving “short-range’’ motion of proteins is particularly handicapped by the spatial resolution of currently available tools. We have considered how lateral diffusion of membrane proteins may directly facilitate a number of membrane-mediated biological functions, including selfassembly, cell-cell recognition and adhesion, enzymatic reactions, hormonal response, and other signaling processes. Yet it is obvious that motional phenomena such as lateral diffusion do not owe their existence to the fact that they might subserve various membrane functions; on the contrary, the diffusion of a membrane protein must to some extent be a mere reflection of the fluid lipid environment in which it resides. In fact, unbridled protein diffusion represents a potentially disruptive force which the cell must contend with in structuring membrane topography during growth, differentiation, and normal functioning. We have seen through our discussion of diffusion-mediated trapping that mechanisms may have evolved which can channel this potentially negative force and make it work for the cell. It is our feeling that this economical device is perhaps the most intriguing aspect of protein diffusion in cell membranes.
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INTERNATIONAL REVIEW OF CYTOLOGY, VOL. R7
ATPases in Mitotic Spindles M. M. PRATIDepartment of Anatomy and Cell Biology, University of Miami School of Medicine, Miami, Florida I.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11. Energy Requirements for Mitosis ............. 111. ATPase in the Mitotic Spindle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Cytochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
B. Dynein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Myosin.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Ca2 -ATPase E. Other ATP Hy Microtubules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Calmodulin Regulation of Spindle ATPase ..................... Spindle ATPase and Models of Mitosis.. . . . . . . . . . . . . . . . . . . . . . . Concluding Remarks . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . +
IV. V. VI.
83 84 86 86 87 90 91 94 95 96 101 101
I. Introduction Daniel Mazia suggested in 1965 that the study of mitosis was not exactly flourishing; “indeed the measure of its unpopularity is the fact that one can still almost keep up with the literature.” By this criterion, the study of mitosis is exceedingly popular today. The mechanism of mitosis has been probed morphologically, biochemically, biophysically, and theoretically; and the voluminous data have led to the generation of a number of possible models of mitotic apparatus (MA) function. For a concise discussion of these data and models, and an excellent bibliography, readers are referred to the recent review by Inoue (1981). Here, we will focus on one aspect of the biochemistry of mitotic spindles-the presence of adenosine triphosphate (ATP) hydrolyzing enzymes. With the introduction of techniques for the mass isolation of mitotic apparatus in 1952 (Mazia and Dan, 1952), biochemical studies of spindles became feasible. Mazia et al. (1961) were the first to describe an ATPase activity associated with isolated spindles, and since that first report, ATP hydrolysis in the MA has been studied by a variety of techniques including histochemistry, immunofluorescent microscopy, enzymatic analysis of isolated spindle proteins, and specific inhibition of chromosome movement. 83 Copyright 0 1984 by Academic Press. Inc. All rights of reproduction In any form reserved. ISBN 0-12-364487-9
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All of these areas of research will be covered to some extent in this article. We will be concerned primarily with the ATPases which seem to be present in the fully formed MA, and will mention only in passing the processes of chromosome condensation and metakinesis (see Dustin, 1978, for a discussion of MA formation). To a large extent, the discussion will center on the possible function of mitotic ATPases in chromosome movement and the current status of the search for a “mitotic motor.” While some historical background will be given, the emphasis will be on literature from the past 10-15 years. Since this article is somewhat narrow in scope, the reader is referred to a number of excellent reviews of mitosis which cover mitotic spindle isolation (McIntosh, 1979) and biochemistry (Petzelt, 1979a), ultrastructure of the MA (Fuge, 1977), calcium (Harris, 1978) and calmodulin in the MA (Nagle and Egrie, 1981), membranes in the mitotic apparatus (Hepler, 1977), an historical monograph on mitosis and cell division (Mazia, 1961), and a critical review of models of chromosome movement (Pickett-Heaps et al., 1982).
11. Energy Requirements for Mitosis Despite considerable investigation of the subject, the exact nature of the mitotic “motor” or “fuel” is not clear. Much of the early data was conflicting and three different hypotheses were developed concerning the energy for mitosis (see Mazia, 1961; Epel, 1963). (1) Chromosome movement is supported by an energy reservoir, stored during interphase (Bullough, 1952; Swann, 1957); (2) energy is stored within an activated MA, during its formation, and, perhaps, as an intrinsic property of its structure (Mazia, 1961); and (3) a continuous energy supply is required for chromosome movement (Epel, 1963). Support for the first two hypotheses came from experiments using respiratory inhibitors. In many cases, exposure of cells to metabolic inhibitors or low oxygen tensions prior to mitosis would prevent formation of the MA, or if the spindle did form, chromosome movement was hindered. However, if inhibitors were applied late in metaphase or during anaphase, karyokinesis continued in the supposed absence of a fresh energy source. Hence the suggestion was made that energy is obtained from a reservoir (for review, see Swann, 1957), or from an activated or elastic component within the MA (Mazia, 1961). Indeed, one current model of anaphase movement invokes an elastic component (Pickett-Heaps and Spurck, 1982a). The nature of the possible energy store was not addressed directly (Bullough, 1952; Swann, 1957), but it could be simply an ATP pool, though perhaps separate from the general cellular pool (Mazia, 1961). Another possibility is that the reservoir consists of creatine-phosphate (Cr-P). Koons et al. (1982) have
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localized creatine phosphokinase in the MA by immunofluorescence, and Silver et af. (1983) have shown that isolated sea urchin spindles contain creatine phosphokinase in sufficient quantity to generate millimolar levels of ATP. The third hypothesis, that energy must be generated throughout mitosis, gains support from a number of investigations showing that a continuous source of ATP is necessary for chromosome movement. Hoffmann-Berling (1954), demonstrated that ATP addition to primitive but elegant lysed cell “models” would induce cytoplasmic contraction and chromosome movement. Epel ( 1963) showed that carbon monoxide treatment at any stage could halt mitosis in sea urchin eggs, and that arrest was the direct result of lowered ATP levels. In addition, Goode and Roth (1969) demonstrated that elongation of the MA during isolation from giant amoeba required the presence of ATP in the isolation medium. There are several explanations for these apparently conflicting data. One is that the respiratory inhibitors have a variety of effects, in addition to lowering ATP levels. Amoore (1961a,b, 1963) showed that low oxygen levels caused the arrest of mitosis in pea root tips, but that the mechanism did not necessarily involve lower ATP levels. The most reasonable explanation is that in many of these studies insufficient concentrations of metabolic inhibitors were used (Mazia, 1961; Epel, 1963). The implication is that some residual ATP production remained in the inhibited cells, and this small, but continuous energy supply was sufficient to drive chromosome movement. This explanation is supported by recent evidence that the concentrations of metabolic inhibitors which inhibit chromosome movement have sharp, fairly high thresholds (Sawada and Rebhun, 1969; Pickett-Heaps and Spurck, 1982b). Pickett-Heaps and Spurck (1982b) have shown that treatment with M dinitrophenol (DNP) clearly affects mitotic movements in dividing diatoms; at 5 X M DNP, these effects are M DNP produces minimal alterations in cell divihighly diminished, and sion. Mitotic movements were also affected by similar, high concentrations of M ) , oligomycin ( l o p 5 cyanide ( l o p 3M ) , azide ( l o p 2 M ) , antimycin A M), and gramicidin M ) , though the thresholds were not always as sharp as that seen with DNP (Pickett-Heaps and Spurck, 1982b). The problem of residual ATP levels in the presence of metabolic inhibitors is further complicated by the fact that karyokinesis probably requires very little energy (Mazia, 1961; Nicklas, 1975; McIntosh, 1979; Pickett-Heaps and Spurck, 1982b). The force necessary to move a chromosome, at the observed slow speeds of less than 5 p,m/minute, is quite small, (Nicklas, 1965; Taylor, 1965) although Nicklas (1982) has recently shown that the MA can generate larger forces than necessary. Assuming reasonable energy efficiency, the power output of the spindle is at least two orders of magnitude lower than that of a contracting muscle or a beating flagellum. Nicklas (1975) argues, therefore, that if energy is being produced as in other known motile systems, a strong control
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mechanism must regulate the force produced, and, thereby, slow movement. It seems equally reasonable, however, that the number of force producers per mass unit in the MA may be small as compared with muscle or a flagellum. Thus, energy requirements and production during mitosis could parallel the measured small forces needed to move chromosomes. A final complication is that the energy requirements for chromosome separation may not be constant throughout anaphase. Chromosome separation can be divided into two kinds of movement-anaphase A, or chromosome to pole movement, and anaphase B, or pole separation (Mazia, 1 9 6 1 t a n d there is some indication that the mechanisms of the two are different. Ris (1949) found that chloral hydrate would block anaphase B but not anaphase A. Some current work (Cande, 1982) suggests that the energy requirements of anaphase A and B may be different. Using mildly detergent-lysed cell “models” (Cande and Wolniak, 1978) Cande (1982) reported that anaphase A continues in the absence of added ATP, and in the presence of an ATP depleting enzyme (apyrase), but that anaphase B is completely inhibited in the absence of ATP. These data led to the proposal that ATP hydrolysis is important only in the generation of pole separation (Cande, 1982). It should be noted, however, that earlier work by Cande and Wolniak (1978) showed that addition of metabolic inhibitors in the absence of ATP severely inhibited both anaphase A and anaphase B in the same type of cell model. The difference in these results can be explained by slightly different buffer conditions, or by any of the complications discussed above. In any case, the data do not decisively show that ATP is unnecessary for anaphase A, though the absolute levels of nucleotide required may vary with the stage of anaphase.
111. ATPase in the Mitotic Spindle
A. CYTOCHEMISTRY It has been shown, both cytochemically (in vivo) and biochemically (in vitro) that the MA contains enzymes for ATP utilization. In 1961, Mazia and coworkers demonstrated ATPase activity in spindles isolated from first cleavage sea urchin embryos. Miki (1963) reported a similar enzyme activity in another species of sea urchin. Her early report was followed by a cytochemical analysis of ATPase activity in whole cleaving embryos, confirming a localization in the mitotic spindle (Miki, 1964). Hartman (1964) has shown by histochemical staining that ATPase activity is localized in the MA of two mammalian cell lines, HeLa cells and sarcoma 180 cells. In this case, the ATPase activity appeared to be concentrated at the poles and in the interzone, and was distinguished from nonspecific phosphatase activity. It was further noted that the ATPase activity
ATPase IN MITOTIC SPINDLES
87
was suppressed by a sulfhydryl-blocking reagent (p-chloromercuriphenyl sulfonic acid) (Hartman, 1964). More recently, Lyubskii et al. (1979) have localized Ca2 -activated ATPase and 5’-nucleotidase in chromosomes of dividing mammalian cells. It is most likely that these ATPases have a role in chromosome condensation (or, perhaps, congression) and we will not deal with them in detail (see, however, Section 111,C). +
B. DYNEIN None of these cytochemical or early biochemical reports addressed the exact nature or identity of the mitotic ATPase, although many workers hypothesized that the enzyme activity might be similar to that seen in other contractile systems. As soon as it became clear that the major structural component of the MA is microtubules (Kane, 1962; Rebhun and Sander, 1967), the analogy to ciliary and flagellar function was obvious, and the hypothesis that a dynein-like ATPase might provide the force for chromosome movement soon emerged (McIntosh et al., 1969). There is now considerable evidence in support of such an hypothesis. Weisenberg and Taylor (1968) were the first to attempt to classify the ATPase activity of spindles. They prepared a soluble fraction from sea urchin eggs which was enriched for Mg2+-ATPase activity, and showed that the ATPase was associated with isolated mitotic spindles at first cleavage. The ATPase had enzymatic characteristics and large sedimentation coefficient ( 12s) in common with axonemal dynein ATPase (Gibbons and Rowe, 1965). In fact, it was suggested that this “egg Mg2+-ATPase” was likely to be a dynein precursor for later incorporation into blastula cilia. In retrospect, it seems likely that Weisenberg and Taylor (1968) described the same ATPase as was originally reported by Mazia et al. (1961). It is of some ironic interest, therefore, to note that when Gibbons (1963) first described dynein ATPase (isolated from Tetrahymena cilia), he indicated that the enzyme activity was more similar to that seen in mitotic spindles by Mazia er al. (1961), than to muscle-associated ATPase (myosin). Now that the unique enzymatic characteristics of flagellar and ciliary dyneins are more clearly defined (Warner and Mitchell, 1980, 1982; Gibbons et al., 1976), and the molecular composition of axonemal dyneins is becoming apparent (Pipemo and Luck, 1982; Sale and Gibbons, 1979; Bell and Gibbons, 1982; Tang et al., 1982), some workers have used these criteria to identify dynein-like ATPase in mitotic spindles. In an extension of Weisenberg and Taylor’s results, Pratt ( 1980) prepared an enriched fraction of Mg2 -ATPase from sea urchin eggs and embryos. This ATPase, called egg dynein or cytoplasmic dynein, was shown to have enzymatic properties, molecular weight, and polypeptide composition unique to dynein ATPase. Hisanaga and Sakai (1983) have now purified this enzyme to near homogeneity and confirmed the dynein-like character. Egg +
88
M. M. PRA’M
dynein appears to be specifically associated with the microtubular, fibrous components of the isolated sea urchin MA (Pratt et al., 1980; Fig. 1). Note that Mazia et al. (1961) also reported the association of ATPase activity with the “fibrous, structural components of the mitotic apparatus.” There is also physiological evidence for dynein ATPase in mitotic spindles. Cande and Wolniak (1978) used a lysed cell model to analyze anaphase chromosome movement. In this system, PtK, cells are permeabilized using two mild detergents, in series, in carefully controlled buffers. It has been determined that chromosome movement has ionic and nucleotide specificities similar to those for flagellar and egg dynein. In addition, separation of chromosomes is blocked by micromolar concentrations of vanadate ion ( + 5 oxidation state), a potent inhibitor of dynein ATPase, and the movement resumes when the vanadate is reduced to an inactive form (+4 oxidation state). Cande (1982) has also reported that anaphase movement can be blocked by EHNA (erythro-9-2,3-hydroxynonyladenine), another inhibitor of dynein ATPase (Bouchard et al., 1981; Penningroth et al., 1982). Since EHNA is also an inhibitor of protein carboxymethylase, these results may not bear directly on the presence of dynein in the MA. Other physiological evidence for dynein activity in mitosis comes from the only account of‘chromosome movement in isolated mitotic spindles (Sakai et al., 1976; Sakai, 1978a,b). Sakai and co-workers (1975, 1976) isolated first mitotic spindles, from sea urchin eggs, in the presence of glycerol. They reported that under appropriate buffer conditions, the chromosome continued to separate both by kinetochore-to-pole movement and polar separation. The rate of chromosomes movement was, however, less than one-fiftieth of that seen in vivo. Still, these workers reported that the movement was specific for ATP (over other nucleotides), as is dynein ATPase, and that the chromosome separation was inhibited by an antibody specific for flagellar dynein (Ogawa and Mohri, 1975). In a more recent study using the glycerol-isolated spindles, Sakai (1978a) has reported an increased velocity of chromosome movement (still well below in vivo rates) which is diminished by vanadate ion, an inhibitor of dynein ATPase. Anti-flagellar dynein antibodies have also been used to localize dynein in the MA. Mohri et al. (1976) have used immunofluorescent techniques to show that the antibody prepared to a fragment of flagelljr dynein (Ogawa and Mohri, 1975) specifically stains that mitotic apparatus of dividing sea urchin embryos. This type of staining is subject to artifact since the MA, as a region devoid of cellular organelles, tends to accumulate soluble molecules, for example, injected antibodies. However, these workers also report specific staining of the isolated sea urchin MA, thus reducing this particular problem. Immunofluorescent localization of dynein in PtK, and other mammalian mitotic apparatus has also been reported (Izutsu, et al., 1980). In contrast to these immunolocalizations, Zieve and McIntosh (1981) have reported that an antibody which recognizes bovine sperm dynein does not react
C. SPECIFIC ACTIVITIES OF FLAGELLAR DYNEIN, EGGDYNEIN, AND SPINDLE Mg2+-ATPase
K +-EDTA Mg2+ -ATPase Ca2+ -ATPase Flagellar dynein 0.211 2 0.004 0.169 2 0.027 Egg dynein 0.019 2 0.001 0.007 2 0.001 Spindle Mg2+-ATPase 0.019 2 0.003 0.016 2 0.007
ATPase 0.003 2 0 0 0.001 2 0
Mg2 -ATPase + oligomycin or oubain +
0.180 0.017 0.019
0.180 0.016 0.021
FIG. 1. Mitotic spindles isolated from first division sea urchin embryos in 5 mM EGTA, 0.5 mM MgC12, 10 mM PIPES, pH 6.8, 1% Nonidet P40; as seen in polarization (A) and phase (B)optics. (Photos by T. Otter). In (C), the ATPase activity of the whole isolated spindles is compared with that of flagellar dynein and a partially purified preparation of egg dynein under a variety of conditions. (From Pratt et a / . , 1980.)
90
M. M. PRATT
with mitotic spindle protein from a variety of cultured cells, in a precipitation assay. This antiserum was prepared against a complex antigen composed of electrophoretically separated high-molecular-weight proteins of bovine sperm, and the authors state clearly that it contains activity against a variety of antigens, including dynein. Thus, the failure of this reagent to precipitate dynein-like polypeptides from cultured cells, or to immunofluorescently stain the MA, may simply be due to a low concentration of dynein-specific antibodies in the antiserum, and/or the expected low concentration of dynein-like proteins in the cultured cells. C. MYOSIN
Although microtubules are the predominant structural components of the MA, actin has also been localized biochemically (see Petzelt, 1979a; Forer, 1978, for reviews), immunologically (Cande et al., 1977; Herman and Pollard, 1979), and histochemically (Sanger, 1975; Herman and Pollard, 1978) in spindles of many cells. Despite conflicting evidence that actin is not localized in the spindle (Aubin et al., 1979; Barak et al., 1981) many still favor the hypothesis that actomyosin-based contraction might be important in spindle function (see Forer, 1978, for discussion). There is little doubt that myosin like enzymes can be found in mitotic spindles. Lestourgen el al. (1975) demonstrated the presence of myosin in the MA of Physarum, electrophoretically and biochemically (enzymatic assay). However, the majority of the ATPase was found in association with chromosomes, and it was postulated that the enzyme has a role, along with actin, in chromosome condensation. On the basis of calcium-activated activity, it is possible that this ATPase corresponds to that localized histochemically in chromosomes (Lyubskii et al., 1979). It should be noted, however, that by enzymatic criteria, the vast majority of the ATPase found in isolated sea urchin spindles seems to be dyneinlike rather than myosin-like (Pratt et al., 1980; Fig. lc). Myosin localization in mitotic spindles has also been demonstrated immunohistochemically. Fujiwara and Pollard (1976, 1978) used a well-characterized antiserum to platelet myosin to study the localization of cytoplasmic myosin in a variety of human cell lines. The anti-myosin stained mitotic spindles, and in an elegant double-fluorescent antibody study, anti-myosin distribution was compared with anti-tubulin staining (Fujiwara and Pollard, 1978). The authors showed that the spindle microtubules seem to be surrounded by a “cloud” of myosin. Both the antiserum and the staining were extremely well characterized in this study. Thus, it seems clear that there is myosin in the region of the MA. However, there may still be some question as to whether the staining pattern actually reflects a concentration of myosin in the MA. The anti-myosin also stains the rest of the cytoplasm, and the MA staining is not nearly as bright as
ATPase IN MITOTIC SPINDLES
91
that in the cleavage furrow, a structure known to contain myosin. Since the MA staining with anti-myosin is very diffuse (as opposed to discrete anti-tubulin staining) and is only slightly more intense than overall cytoplasmic staining, it may be due to an accumulation of antibody in the organelle-free spindle region. In light of the data discussed above, it is interesting to note that the chromosomes do not stain with this anti-myosin antiserum (Fujiwara and Pollard, 1976, 1978). Antibodies have also been used in physiological studies of myosin-like activity in the MA. In every case, anti-myosin antibodies have been shown to have no effect on chromosome separation. For example, anti-myosin antibodies (Mabuchi and Okuno, 1977) did not inhibit anaphase movement in the glycerolisolated spindles described above (Sakai et al., 1976). Elegant work has been done by Mabuchi and Okuno (1977) and Kiehart et al. (1982) who used the same anti-myosin antibody (prepared against starfish egg myosin) in microinjection studies of mitosis in living marine embryos. Both groups clearly showed that anti-myosin had no inhibitory effect on karyokinesis while cytokinesis was completely inhibited. Kiehart et al. (1982) effected a dramatic demonstration of this result by microinjecting anti-myosin into only one cell of the first pair of blastomeres. While the uninjected cell went on to form a half-size blastula, the anti-myosin injected blastomere remained as one large cell, filling itself with normally mitotic nuclei, but unable to divide its cytoplasm due to inhibition of the contractile ring myosin (see Fig. 2). Further physiological studies of myosin in mitosis have been done by Cande et al. (1981) using the lysed PtK, cell model. In the permeabilized cells, reagents which inhibit the contraction of glycerinated muscle and cleavage furrow constriction have no effect on anaphase chromosome movement. These reagents include N-ethylmaleimide (NEM)-modified myosin subfragment-1, phalloidin, and cytochalasin B. These data are consistent with the antibody work, thus reinforcing the conclusion that myosin ATPase activity is not essential for chromosomal separation.
D. Ca2 -ATPase +
Mazia et al. (1972) were the first to identify a calcium-activated ATPase activity, in isolated sea urchin mitotic apparatus. Subsequently, Petzelt and coworkers identified the enzyme in plant tissues (Auel et al., 1980) and other marine embryos (see Petzelt, 1979a, Table I). The activity of the mitotic Ca2+ATPase has been shown to vary predictably with the cell cycle in sea urchin embryos (Petzelt, 1972a,b), even in parthenogenetically activated eggs (Petzelt and von Ledebur-Villiger, 1973; Petzelt, 1976). There is also an association of the mitotic Ca2 -ATPase activity with the cell cycle in mouse mastocytoma cells (Pezelt and Auel, 1977), and Physarum (Petzelt et al., 1980). The enzyme is most active just prior to mitosis and, since calcium inhibits microtubule poly+
FIG. 2. Anti-myosin injection into a sea urchin embryo at the two cell stage. The blastomere on the right was injected with anti-myosin just after f m t cleavage; the blastomere on the left is an uninjected control. (a) The cells are in early prophase, just after injection (oil droplet marks injected cell). (b) The cells are shown 10 hours after the injection, at which point the control cell has developed normally into a half-blastula. The injected cell has undergone normal karyokinesis but not cytokinesis, thus it appears as a single large cell filled with nucleii. Scale bar, 40 Fm. (From Kiehart era!., 1982.)
93
ATPase IN MITOTIC SPINDLES TABLE I SPECIFIC ACTIVITY OF THE MITOTIC Ca2 -ATPase<' +
~
Species
Tissue
Physarum polycephalum (rnixomycete) Paracentrotus lividus (sea urchin) Mouse Vicia faba (bean)
Specific activity (nmol Pi/mg protein/minute)
Plasmodium
12 ? 6
Unfertilized egg
27
Mastocytorna cells Root tips
30 2 8 24 ? 7
?
9
"From Petzelt er al. (1980)
merization, it has been suggested that the Ca2+-ATPase might play a role in controlling calcium levels during MA assembly. This hypothesis is supported by data which suggest that the mitotic Ca2+ATPase is active in calcium sequestration. Silver et al. (1980) and Nagle and Egrie (1981) have demonstrated that the sea urchin MA, when isolated with membranes intact, can accumulate added, radioactive calcium. Exposure of the isolates to Triton X- 100 (a nonionic detergent) disperses membranes, releases bound calcium, and eliminates calcium uptake (Silver et al., 1980; Egrie and Nagle, 1980). In addition, Kiehart (1981) has shown that the MA in living echinoderm embryos can sequester microinjected calcium very efficiently, apparently into vesicle compartments. The compartments can be visualized using fluorescent calcium-binding probes (Wolniak et al., 1980) and Wolniak el al. (1983) have very recently demonstrated fluctuations in the calcium content of these compartments, associated with the onset of anaphase. Finally, the mitotic Ca2 -ATPase has been localized by immunofluorescence (Petzelt, 1979b, 1980), using an antibody prepared against the purified enzyme (Petzelt and Auel, 1978), to components of the endoplasmic reticulum in the region of the spindle. The mitotic Ca2 -ATPase has been purified from Ehrlich ascites tumor cells, and has been partially characterized (Petzelt and Auel, 1978). One problem with the hypothesized role for the enzyme in calcium sequestration is that millimolar levels of calcium are required for optimal activity. For the enzyme activity to be physiologically relevant, modulation by calcium ions in the micromolar range would be required, since effects on microtubule integrity are seen at these low concentrations. Nagle and Egrie (1981) point out that the calcium optimum for the enzyme may be very different in vivo, where the ATPase appears to be integrally associated with a membrane, since that association could affect the calcium binding properties of the enzyme dramatically. +
+
94
M. M. PRATT
E. OTHERATP HYDROLYZING ENZYMESASSOCIATED WITH MICROTUBULES Many workers have described ATPase activities in cytoplasmic (brain) tubulin preparations which appear to be associated with microtubules (Bums and Pollard, 1974; Gaskin et al., 1974; Ihara et al., 1979; White et al., 1980; Tominaga et al., 1982). One of these enzymes is bound to a microsome fraction and may be only artifactually copurified with microtubules (Murphy et al., 1983a,b; Tominaga er al., 1982). Another brain ATPase is dependent on both tubulin and calcium for activity (Tominaga et af.,1982). White er al. (1980) have reported a tubule-associated ATPase which appears to be distinct from any previously described. ATPases of these types are also possible components of the MA, although there have been no direct investigations of them as yet. Analysis of these enzymes in the MA is hampered by (1) the fact that, if present, they are probably very small components of the spindle and (2) the lack of unequivocal techniques (inhibitors, for example) for identifying these ATPases. Another possible microtubule-associated substrate for ATP in the spindle is a protein kinase. The purified microtubule fraction from brain contains a kinase activity (Sloboda er al., 1975) which is a component of the microtubule associated protein-2 (MAP-2) molecule (Vallee et al., 1981). This cyclic-AMP-dependent kinase is of the RII type and the primary substrate is the MAP-2 molecule itself (Theurkauf and Vallee, 1982). The phosphorylated MAPs are more efficient at promoting microtubule assembly (Theurkauf and Vallee, 1982). Highmolecular-weight MAPs have been localized by immunofluorescense in the MA of fibroblasts (Sherline and Schiavone, 1978). In addition, Browne er al. (1980) have reported the immunofluorescent localization of protein kinase in the spindles of tissue culture cells. This enzyme has been identified immunologically as an RII, CAMP-dependent protein kinase, the same type as that associated with brain MAP-2 (Theurkauf and Vallee, 1982). The biochemical identification and role of this “spindle kinase” remain to be determined. Finally, there is some indication that ATP can affect tubulin-microtubule dynamics. Rebhun and Sawada (1969) showed that MA birefringence is abolished in sea urchin embryos after treatments which lower the ATP concentration M DNP or 5 X 10W3M azide). In addition, cells treated with metabolic inhibitors during microtubule regrowth (following nocodazole treatment) appear to be deficient in tubule organization (DeBrabender et al., 1981). These studies suggest that some ATP is necessary to maintain an ordered microtubule superstructure. On the other hand, exogenous ATP, added to crude rat brain extracts, causes the depolymerization of cold stable microtubules (Margolis and Rauch, 1981). The mechanism is probably indirect, involving a kinase, since a 64K MW MAP is concomitantly phosphorylated (Margolis and Rauch, 1981). It has also been reported that ATP can destabilize microtubules in vitro, as assayed by
ATPase IN MITOTIC SPINDLES
95
increased rates of monomer movement through polymer or “treadmilling” (Margolis and Wilson, 1979), although it is not clear whether this is due to a direct interaction of tubulin and ATP, or to an indirect mechanism involving MAPS. Kirschner (1980) suggests that nucleotide triphosphates may be used, via “non-energy-producing” hydrolysis, to regulate the organization of filaments (actin and microtubules) within cells. Thus, it is interesting to consider the possibility that direct binding of ATP (perhaps at an appropriate threshold concentration) to microtubules may be important in regulating the disassembly phase of mitosis.
IV. Calmodulin Regulation of Spindle ATPase Current data are insufficient to unequivocally assign a role to any ATPase in the mitotic apparatus, thus it may be somewhat premature to discuss possible regulatory mechanisms of an as yet undefined “mitotic motor.” The spindle, however, contains at least one regulatory protein, calmodulin, which may interact with mitotic ATPases. In addition, certain models of the mitotic mechanism may be rendered more plausible by the existence of appropriate regulatory systems. Calmodulin (CaM), or calcium-dependent regulatory protein, has been identified as a modulator of a large variety of enzyme activities (see Watterson and Vincenzi, 1980), and CaM is localized in the mitotic spindle (see Nagle and Egrie, 1981, for review). Anderson et al. (1978) and Welsh et al. (1978) used anti-CaM antibodies and immunofluorescent microscopy to demonstrate specific staining of the mammalian MA. Calmodulin has also been localized, using immunoelectron microscopy, to the poleward ends of spindle microtubules in mammalian cells (DeMey et al., 1980). In addition, the distribution of CaM in living cell, described by Hamaguchi and Iwasa (1980), includes some concentration in the spindle. These investigators microinjected fluorescently tagged, purified CaM into sea urchin eggs, and followed the rearrangement of CaM fluorescence through fertilization and first cleavage. The CaM was specifically arrayed in the MA, again showing an apparent concentration at the spindle poles. There are several possible regulatory roles for CaM in the spindle. First, it could modulate microtubule polymerization, possibly through regulation of the microtubule-associated enzymes mentioned above (Section 111,E). It has been reported that CaM can increase the calcium sensitivity of brain microtubule depolymerization (Marcum et al., 1978; Nishida and Kumagai, 1980). This possibility seems less likely in view of two reports that CaM has no effect on the calcium sensitivity of cytoplasmic microtubules prepared from whole sea urchin eggs (Nishida and Kumagai, 1980; Keller et al., 1982), or from sea urchin mitotic apparatus (Keller et al., 1982).
96
M . M. PRATT
The mitotic Ca2 -ATPase (see above) is another logical target for CaM binding and regulation. However, Egrie and Nagle (1980) made a direct study of this possibility and showed that CaM has no effect on the calcium-activated ATPase activity of the enzyme. They also demonstrated that the accumulation of calcium by the isolated spindle is not modulated by CaM. There are data to suggest that CaM may regulate myosin and/or dynein found in the MA. Adelstein (1982) has recently reviewed the process by which calmodulin regulates the calcium-dependent, actin activation, of cytoplasmic myosin. Briefly, a rise in the calcium concentration to approximately l o p 6 M allows the interaction of calmodulin with a myosin kinase. The kinase is thereby stimulated to phosphorylate the myosin light chain. Prior to light chain phosphorylation, the myosin ATPase activity cannot be stimulated by interaction with actin, but the phosphorylated myosin is subject to actin activation of Mg2+-ATPase. If a cytoplasmic myosin plays a role in mitotic function, it may be modulated by a similar mechanism. The relevant calcium concentration, micromolar, is significant since it is in the same range as that reported to depolymerize sea urchin cytoplasmic microtubules (Nishida and Kumagai, 1980; Keller et al., 1982), and to cause shortening of chromosomal spindle fibers (Salmon and Segall, 1980). There is also some evidence that calmodulin may activate dynein ATPase in a calcium-dependent manner. Blum et al. (1980) have studied the interaction of calmodulin and dynein prepared from Tetrahymena ciliary axonemes. They report that the calcium activated ATPase (Ca2+-ATPase) of one form of the dynein can be stimulated 10-fold by CaM. Perhaps even more relevant is the report that calmodulin can activate the Mg2+ -ATPase activity of egg dynein prepared from the sea urchin Hemicentrotus pulcherrimus (Hisanaga and Sakai, 1983; Hisanaga and Pratt, 1982; Fig. 3). The activation by CaM is dependent on calcium, and the threshold calcium concentration is, again, micromolar (Hisanaga and Pratt, 1982, 1983; Fig. 3). Although it can be shown that calmodulin interacts with both ciliary dynein (Blum et al., 1980) and egg dynein (Hisanaga and Pratt, 1982), it may be that the ATPase activation is not due to the direct binding. The activation may be indirect, possibly through a protein kinase, as discussed for myosin above. However, in preliminary experiments, this author has been unable to detect any phosphorylation of cytoplasmic dynein, in enzyme preparations which show CaM activation of Mg2 -ATPase activity (Hisanaga and Pratt, 1983). +
+
V. Spindle ATPase and Models of Mitosis Current models of mitosis can be conveniently divided into two categories on the basis of whether actin or microtubules are proposed transducers of force for chromosome movement. For a review of the important features and feasability of
A
Brain
1
20
FLG.3. CaC12
40
60
80 100 CaM (pg/ml)
120
140
160
Cah4 activation of sm urchin cgg dynein ATPase. (A) MgzZ'-ATPaseactivity at increasing CaM conccntrdtions in the presence of 0.2 mM
(m) or 0.2 mM EGTA (0). CaM isolated from sea urchin eggs or porcine brain gives the same result. (B) Calcium dependence of the dynein
or sea urchin cggs (a). The abscissa shows the free calcium ion concentration activation in the presence of I00 pglrnl CaM isolated fmm porcine brain (0) in EGTA buffered solutions. (From Hiranaga and Pratt, 1983.)
98
M. M. PRATT
current models of mitosis readers are referred to recent reviews by Inout (198 1) and Pickett-Heaps et al. (1982). We will consider which, if any, of the mitotic ATPases has an essential role in a particular model. Attention will be focused on the evidence that a given enzyme may provide force for anaphase chromosome separation. In the case of actin-based models, the proposal is, of course, that myosin ATPase supplies the force for chromosome movement. There is one recent report which suggest some similarity between the mechanisms of muscle contraction and mitosis. Sillers and Forer (1981) examined the effects of different wavelengths of ultraviolet (UV) light on spindles of live cells, on glycerinated myofibrils, and on reactivated cilia. They found that the action spectra for stopping chromosome movement and for inhibiting myofibril contraction were quite similar, while different UV wavelengths inhibited ciliary beating. Generally, however, the data concerning the presence of actin and myosin as integral spindle components are confusing and somewhat contradictory (see Section 111,C). Moreover, in view of considerable evidence that myosin-specific probes do not inhibit anaphase chromosome separation in the same cells where cytokinesis, an actomyosin based movement, is inhibited, it seems unlikely that this enzyme provides the force for anaphase motion. In the case of microtubule-based models for mitosis, the “dynamic equilibrium” model as first proposed by InouC (1959; see also InouC and Sato, 1967; Inoue and Ritter, 1975) is unique in that it does not invoke some kind of interaction between microtubules. Highly simplified, this model suggests that slow, controlled, depolymerization of kinetochore microtubules provides the force for pulling chromosomes to the poles, while the regulated polymerization of nonkinetochore tubules leads to polar separation. This model is elegant and particularly noteworthy since it was proposed years before microtubule structure and polymerization were described. The hypothesis remains consistent with much current data, although recent information concerning the lability of the various types of spindle tubules suggests that revision is in order (Salmon and Begg, 1980). A current suggestion is that microtubule depolymerization/ polymerization may primarily control the rate of chromosome movement without directly contributing any force (Salmon, 1974). In this model, the ATP utilizing enzymes which may modulate microtubule dynamics (Section III,E) are the type which could provide energy for, and regulation of tubule assembly and disassembly. There are at least three models for mitosis which suggest that interaction between spindle microtubules generates chromosome movement-the sliding hypothesis, the treadmilling model, and the zipper hypothesis. In the sliding hypothesis (McIntosh et al., 1969), it is proposed that spindle fiber tubules slide relative to one another. The sliding is thought to be generated by dynamic structures which “bridge” adjacent microtubules, and which may be composed
ATPase IN MITOTIC SPINDLES
99
of a dynein-like protein. The dynein ATPase, then, would provide the energy for force generation in a manner similar to that seen in cilia and flagella. The treadmilling hypothesis of mitotic movement combines elements of microtubule interactions with recent information about the kinetics of microtubule polymerization, in a model which is a unique hybrid of the sliding filament and dynamic equilibrium concepts (Margolis et af., 1978; Margolis and Wilson, 1981). In part, this model suggests that chromosome movement is brought about by the unidirectional loss of tubulin monomers from the polar end of kinetochore fibers, as the fiber microtubules slide past an anchor point at the poles (Margolis and Wilson, 1981). The zipper model also proposes that kinetochore fibers have important interactions near the poles (Bajer, 1973; Bajer and Molk-Bajer, 1975). In this case the hypothesis is that kinetochore microtubules transiently and sequentially interact with polar microtubules in a region close to the spindle poles. Interaction is thought (1) to be mediated by reasonably static bridges (possibly MAPS), and (2) to lead to the breaking of some kinetochore microtubules as the chromosome moves toward the pole. This hypothesis may be supported by recent experiments of Nicklas er al. (1982) which dramatically demonstrate tight interactions of kinetochore fibers with other spindle elements in the polar region. The data supporting the presence and apparent function of dynein in the MA (see section III,B) are consistent with a sliding filament model. There has been some concern recently that this hypothesis is rendered less likely by the demonstration that all of the microtubules in one “half spindle” are of identical polarity (Euteneuer and McIntosh, 1981; Telzer and Haimo, 1981; Euteneuer et al., 1982), since the model, as originally stated, depended on the generation of sliding between anti-parallel tubules (McIntosh et al., 1969). In addition, King et al. (1982) reported that sliding could not be induced between microtubules in spindles isolated from yeast. Haimo et al. (1979), however, have shown that dynein can crossbridge cytoplasmic microtubules, and Warner and Mitchell (198 1) have demonstrated that dynein will bridge both parallel and anti-parallel ciliary microtubules. In keeping with the analogy to axonemal movement, it should be pointed out that sliding of ciliary and flagellar outer doublets is between microtubules of the same polarity. Nevertheless, a currently favored hypothesis is that sliding is restricted to the interzonal region of the MA, where microtubules of different polarities overlap, and that sliding contributes primarily to polar elongation (anaphase B) (McIntosh, 1981). An important question is that of the ultrastructural localization of dynein within the spindle. The suggestion is that if a mitotic dynein functions similarly to axonemal dynein, it should be visible as “arm” or “bridge” structures arrayed along the mitotic fibers. While there are a few elegant examples of crossbridges between microtubules within the MA (Wilson, 1969; Hepler er af., 1970; InouC and Ritter, 1975), the fact is that regularly spaced arms are not typically seen.
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One possibility is that dynein crossbridges are present but are not visualized either because the molecular organization of the spindle is less regular than that of the axoneme, or because the dynein is not preserved by present fixation conditions. The latter suggestion seems less likely in view of the demonstration that microtubules of the isolated MA can be “decorated” with exogenously added dynein, which is easily visualized ultrastructurally (Telzer and Haimo, 1981). (Although the exogenous axonemal dynein could have different fixation properties than a mitotic dynein.) Moreover, the added dynein binds in a specific arrangement, indicating the presence of highly regular dynein-binding sites on the MA microtubules. While the binding of exogenous dynein may suggest that binding sites on mitotic tubules are not typically occupied by a mitotic dynein, it is also possible that endogenous dynein could have been extracted during spindle isolation. Another possibility is that dynein in the MA is not distributed along the microtubules. The differences between axonemal and cytoplasmic dyneins (Pratt and Stephens, 1978; Pratt, 1980) and tubulins (Stephens, 1978) would support this idea. For example, the dynein might be localized exclusively in the interzone, as mentioned above. In fact, bridges have been more consistently visualized in the interzone than in any other spindle region. Alternatively, I would like to suggest that a mitotic dynein may be primarily localized in the polar region of the spindle, possibly as a major component of the amorphous, pericentriolar material, or as a bridge between microtubules and spindle membranes (Hepler et al., 1981; also see Pratt et al., 1980). Such a distribution is consistent with data showing a concentration of immunofluorescent anti-dynein staining at the spindle poles (Mohri et al., 1976). It is intriguing that the possible dynein regulator, CaM, is also concentrated in polar regions of the MA (DeMey et al., 1980; Hamaguchi and Iwasa, 1980). Theoretically, a polar dynein could act as a bridge, providing the apparently greater interaction of adjacent microtubules in the spindle poles (Bajer and MolbBajer, 1975; Nicklas et al., 1982). With respect to the treadmilling model, dynein localized at the poles could serve as the postulated anchor/sliding site (see above; Margolis et al., 1978). For example, the dynein might compose a molecular “ratchet” upon which kinetochore fibers, and attached chromosomes, would be “reeled” into the aster (see Begg and Ellis, 1979, for a discussion of apparent reeling of chromosomes to the poles). That portion of the kinetochore fiber microtubules which had passed the “dynein ratchet” would depolymerize, possibly as a result of being released from tension (see Hays et al., 1982). This model is consistent with some current data and, more importantly, is directly testable in one aspect; the ultrastructural localization of dynein in the spindle can be determined by immunoelectron microscopy using one of the newly available dynein antibodies (Okuno et al., 1981; Asai and Wilson, 1982; Mitchell and Rosenbaum, 1982; Piperno, 1982).
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VI. Concluding Remarks It is overwhelming to think how complex a process is mitosis, and humbling to realize how little we know about even one component of the mitotic spindle-the ATPases. Nonetheless, the very existence of this article shows that new information is constantly being gathered, and progress is being made. We can now identify some of the ATPases in the spindle and begin to assess their function in chromosome movement. In terms of forces for the anaphase separation of chromosomes, the data clearly favor dynein-microtubule interactions over actomyosin, though the model is far from proven. In addition, there are some data regarding regulation of mitotic ATPase. A great deal of work remains to determine the exact roles of the various ATPases in mitotic function. Future research will continue to focus on the unequivocal identification of the “motor” for chromosome movement. In this regard, it is necessary to develop specific inhibitors, both biochemical and immunological, for the various ATPases, particularly dynein. Valuable information came from experiments using anti-myosin antibody, and antibodies to cytoplasmic dynein (Asai and Wilson, 1982) and the mitotic calcium ATPase (Petzelt, 1979b) could generate data about the function of these enzymes. Antibodies could also be used effectively in the ultrastructural localization of these enzymes in whole mitotic cells and isolated MA. Finally, the development of a reliable, well-defined, in vitro reactivated mitotic spindle remains the goal of many researchers. A model system would be invaluable in assessing the essential enzymatic components of a functional mitotic apparatus. ACKNOWLEDGMENTS
I would like to thank Drs. Dan Kiehart, Tim Otter, and Christian Petzelt who provided figures and tables. I would also like to thank a number of people for fascinating discussions, in which many of the ideas presented here were formulated. Those who contributed valuable time and thoughts were Drs. David Begg, Bany Eckert, Tom Hays, Peter Hepler, Ira Herman, Shin-ichi Hisanaga, Shinya Inout, Tim Otter, Mary Porter, Ted Salmon, Ray Stephens, and Rich Vallee. In addition, I am grateful to Dr. Ray Stephens and Dr. Barry Eckert for providing useful comments on the manuscript. During the preparation of this manuscript, Dr. Pratt was supported by National Science Foundation Grant PCM 81-19156. Some of the data presented was obtained while the author worked in the laboratory of Dr.H.Sakai, Dept. of Biophysics and Biochemistry, Tokyo, Japan, under the auspices of a Jean and Katsuma Dan Fellowship.
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INTERNATIONAL REVIEW OF CYTOLOGY, VOL 87
Nucleolar Structure GUY GOESSENS Institut d' Histologie, Universite' de I'Etat a Liege, LiPge, Belgium I.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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A. Morphology and Compos B. Functional Significance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Variations in the Ultrastructural Morphology. . . . . . . . . . . . . . . . . . . . A. During the Cell Cycle .................................. B. During Meiosis in Germinal Cells C. Under Some Physiological or Experimental Conditions . . . . . . . D. Functional Significance . . . . . . . . . . . . . . . . IV. Exportation of Nucleolar RNP . . . . . . . V. General Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . .....
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I. Introduction Our current understanding of the nucleolus has come from a synthesis of biochemical and structural studies. Biochemical studies have shown that ribosome formation takes place in the nucleolus and involves a well-established maturation process. First, the ribosomal RNA originally transcribed in mammalian cells as a 45 S molecule is methylated and assembled with specific nucleolar proteins. This complex constitutes a 80 S RNP' particle or so-called preribosome. Second, the 45 S RNA is sequentially cleaved and transformed into various intermediate species of lower sedimentation constant and finally into mature 28 S and 18s RNA. Thus the nucleolus is the site of synthesis and processing of preribosomal RNA and also assembly of proteins into the preribosomal particles. The chromatin containing ribosomal RNA genes, the primary gene products and derivatives of these products (pre-rRNA), their associated proteins (nucleolar or ribosomal proteins) and the enzymatic equipment [RNA polymerase I, RNA methylase(s), RNA process'Abbreviations: DNA, deoxyribonucleic acid; DNase, deoxyribonuclease; FC, fibrillar center; NOR(s), nucleolar organizing region(s); rDNA, ribosomal deoxyribonucleic acid; RNA, ribonucleic acid; RNase, ribonuclease; RNP, ribonucleoprotein; rRNA, ribosomal ribonucleic acid; rRNP, ribosomal ribonucleoprotein. 107 Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-364487-9
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ing enzymes necessary for synthesis, conversion, and assembly of ribosomes] involve the nucleolus. The formation of ribosomes as revealed by biochemical studies (review in Hadjiolov, 1980) is a complex process which also reflects on the structural organization of the nucleolus. Our knowledge on this structural organization has been principally due to a lot of electron microscopy studies. The results obtained in this field before 1970 have been summarized in the classical schematic drawing proposed by Bernhard and Granboulan (1968). During the last past years, some laboratories have turned to experiments with nucleolar ultrastructure. These investigations have now reached such a remarkable level which justifies the attempt to summarize and interpret the main lines of present knowledge in this field. However, the topic of this article is mainly restricted to recent ultrastructural information on the organization of the nucleolus. This article cannot cover all the contributions of all the authors investigating the nucleolar ultrastructure despite their importance in this field. Therefore the reader is referred to earlier reviews (Bernhard and Granboulan, 1968; Hay, 1968; Lafontaine, 1968, 1974; Busch and Smetana, 1970; Bouteille et al., 1974; Smetana and Busch, 1974) which cover the original literature on the nucleolus up to 1970 and also to the recent reviews of Fakan and Puvion (1980), Puvion and Moyne (198 I), and to the articles published in the volume entitled “The Nucleolus” and edited by Jordan and Cullis (1982). Thus the results summarized in the present article focus on information obtained during the past 10 years on the ultrastructural organization of the nucleolus and one main purpose will be to discuss the relationships between ultrastructure and function(s).
11. The Nucleolar Components
The nucleolus is seen to consist of structural components distinguishable by differences in staining properties and ultrastructural characteristics. The nucleolar body itself is essentially composed of fibrils and granules which appear as dark staining areas. These fibrillar and granular components are often, but not always, organized in a more or less reticular structure corresponding to the nucleolonema already observed by light microscopy (Estable and Sotelo, 1951). This network is separated by interstitial spaces whose content seems similar to the nuclear sap and generally referred to as nucleolar vacuoles. This explains the spongelike aspect of the nucleolus described at the photonic level. The fibrillar zones of the nucleolar body are generally composed of two distinguishable types of fibrous material. One is a very electron-opaque fibrous material (the fibrillar component). The other is a lighter fibrous material which often appears as small spheres surrounded by and intimately associated with the dense
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fibrillar component. This electron-lucent material enclosed by the fibrillar component is now generally called fibrillar center(s) (FC). The nucleolar body is surrounded by a layer of condensed chromatin of variable thickness. From this associated chromatin, structures penetrate into the nucleolar body and are called intranucleolar chromatin. So, the nucleolus consists at least of five structural components: the fibrillar center (s), the fibrillar component, the granular component, the nucleolar interstices, and the condensed associated chromatin. However, after the removal of these nucleolar components, a residual fibrillar meshwork persists. This residual nucleolar structure retaining the size and the shape of the original nucleolus is called the nucleolar matrix. These components are present in such a wide variety of tissues and organisms as to be considered universal. However, their spatial relationships one to the others are extraordinarily diverse. For instance, phytohemagglutinin-induced lymphocytic transformation leads to enlargement of the nucleolus and dispersion of nucleolar components. By contrast, the nucleolar structures in resting and maturing lymphoid cells are smaller or more segregated. On the other hand, nucleolar number, size, and shape vary widely depending on the organism, cell type, and physiological state of the cell. Since the changes in nucleolar structure may be interpreted in relation to the RNA metabolism of the cell, the nucleolus is one of the best models in which studies on the structure-function relationships at the cellular level have been carried out. In the present part of this article, we analyze the morphology and composition of a nucleolar model before studying later the variations in the ultrastructural aspect of the nucleolus and the relationships between structure and function(s). A. MORPHOLOGY AND COMPOSITION 1. The Fibrillar Center(s) a. Ultrastructure. The term fibrillar center (FC) has been proposed by Recher et al. (1969) to designate the loose fibrillar material associated with the dense fibrillar component. This term was chosen because of the central location of this substance within the dense fibrillar component and because of its fibrillar structure. More than 20 different names (review in Goessens and Lepoint, 1979) have been applied to designate this nucleolar component in many different types of plant or animal cells. For instances, in plant cells these lightly staining zones have been called lacunae or L-zones (for review see Lafontaine, 1974) and in animal cells, Smetana et al. (1971) observed that the morphology of the central light areas of ring-shaped nucleoli in lymphocytes is similar to the morphology of the FC in epithelial cells. In fact, the existence of spherical structures in the nucleolus has been known for more than a hundred years and has been referred to as nucleolini (Montgomery, 1898). Observations with the electron microscope
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revealed that the nucleolini already seen with the photon microscope correspond to the FC (Love and Soriano, 1971). Recently, FC has become the name most commonly used. The FC are regions of moderate density that contrast with the high electron density of the fibrillar component and appear to be fibrillar in texture (Figs. 1, 2, and 3). They are variable in size and shape and are seen to be restricted to the fibrillar component of the nucleolar body. They are quite different from nucleolar interstices which have an appearance indistinguishable from nucleoplasm (Fig. 4). Three-dimensional reconstructions based on serial sections of the nucleolus have demonstrated contrary to the suggestion discussed by Chouinard (1971) that the FC in the mouse oocytes do not represent transverse or oblique sections of a continuous structure but appear as distinct entities which are separated from one another by the fibrillar component (Mirre and Stahl, 1981). However, it was shown by Chouinard and Leblond (1967) and by Chouinard (1970) that structural continuity can be established between the content of certain of the FC and chromatin strands connected with the associated chromatin. In animal cells, the FC generally appear homogeneous. In plant cells, however, various aspects of FC can be observed. They consist of fine fibrillar material which sometimes contains a dark core which has similar contrast and structure of the extranucleolar chromatin (Chouinard, 1974; Medina et al., 1978; Mughal and Godward, 1979; Risueno et al., 1982). The FC were found in a large variety of cells both in animals and plants, in normal cells as well as in tumor cells, in vivo and in vitro (for review see Goessens and Lepoint, 1979). However, the FC are not easily demonstrable by electron microscopy in the nucleoli of every type of cell (Fig. 3). In some, they are not obvious but they become more conspicuous (Fig. 24) under some experimental conditions (Goessens, 1976a) and in others, for instance in Drosophila KCo cells, the FC are not visible and could be absent (Knibiehler et al., 1982). So, in spite of the abundant literature about the FC, some important items on FC remain open to question. b. Composition. DNA. In human cell nucleoli, Recher et al. (1969) showed that the FC are primarily composed of pepsin digestible proteins and contain fine fibrils that resist pepsin and ribonuclease digestions. Deoxyribonuclease was found to have no effect on glutaraldehyde-fixed cells but the possibility of DNA being present in such FC has not been excluded. While it was felt that this remaining material might be euchromatin it was not possible at this time to prove this by cytochemical and autoradiographic techniques (Recher et al., 1970). On the basis of enzyme digestions carried out on formaldehyde-fixed Ehrlich tumor cells, it was suggested that the FC are composed of proteins but also contain a small amount of DNA (Goessens, 1973). This observation has been confirmed on the same cellular type by autoradiographic techniques using tritiated thymidine (Fig. 7) or tritiated actinomycin D (Goessens, 1974, 1976b).
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Busch and Smetana (1970) had previously reported the presence of chromatin filaments within the central light areas (FC) of ring-shaped nucleoli of lymphocytes. It is now well established that the FC observed in animal and plant cells contain DNA. This conclusion is the result of much cytochemical investigation performed by means of enzymatic digestions, preferential or specific stainings (Figs. 5 and 6 ) , and autoradiography after uptake of tritiated thymidine (Fig. 7) or tritiated actinomycin D (Busch and Smetana, 1970; Goessens, 1973, 1974,
FIG. 1 . Nucleolus in an Ehrlich tumor cell. The FC (arrows) are surrounded by the fibrillar component (0. The granular component ( 9 ) is distributed at the periphery. Nucleolar associated chromatin (ch). Nucleolar interstices (i). X27,500. (From Goessens, 1976b, by permission of Academic Press.)
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I13
FIG.4. Portion of an Ehrlich tumor cell nucleolus. FC is quite different from nucleolar interstice (i) which has an appearance indistinguishable from the nucleoplasm. X49,OOO.
1976b, 1979; Lafontaine and Lord, 1973; Anteunis et al., 1975, 1979; Hubert, 1975; Pouchelet et al., 1975; Bertaux et al., 1978; Mirre and Stahl, 1978a; Pouchelet and Anteunis, 1979; Risueno et al., 1982). The FC are connected with clumps of condensed intranucleolar chromatin which are related to the perinucleolar shell of condensed associated chromatin (Ashraf and Godward, 1980). These connections are clearly visualized (Fig. 6) with oxidized diaminobenzidine combined with enzymatic digestions (Pouchelet and Anteunis, 1978; Anteunis et al., 1979; Goessens, 1979) or with staining by the ammine osmium technique (Mirre and Stahl, 1978a). The nature of the DNA contained in the FC could be studied by hybridization FIG. 2. An other Ehrlich tumor cell nucleolus in which the interstices (i) are more numerous. FC (arrows). Fibrillar component (0. Granular component (g). Nucleolar interstices (i). X 15,000. FIG. 3. Nucleolus of chick fibroblast cultured in v i m . The fibrillar (0 and the granular (g) components are organized in a more or less reticular structure. FC (arrows) are not obvious; they are included in and obscured by the fibrillar component (0. X20.000.
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1 I5
in situ with tritium-labeled 18 S and 28 S RNA. In a recent work, Arroua et al. ( 1982) demonstrated that the FC of human spermatocyte nucleolus contains
rDNA. This observation was possible because germinal cells of meiotic prophase are a suitable material since the FC is separated from the other nucleolar components. Thus the FC are visible in the light microscope as spherical structures located on one side of the nucleplus. Autoradiographs following hybridization in situ performed with tritium-labeled cDNA complementary to 28 S RNA showed a cluster of grains overlaying the FC (Arroua et al., 1982). RNA. Contrary to the opinion of Yasuzumi and Sugihara (1965), it is now admitted that the FC do not contain RNA since radioactively labeled RNA precursors cannot be incorporated into this nucleolar component (for a review see Fakan , 1978). Proteins. On the basis of enzyme digestion studies, many authors concluded that the FC are largely composed of proteins (Dutta et al., 1963; Schoefl, 1964; Smetana et al., 1968; Recher et al., 1969; Goessens, 1973; Mirre and Stahl, I 978a). Ag-NOR proteins. Since the end of the last century, several studies have indicated that nucleoli have a marked affinity for silver. Depending on the conditions of staining with silver, nucleoli appear uniformly stained or contained trabecular structures (Estable and Sotelo, 1951 ; Tandler, 1954; Lettr6 et al., 1966). In nucleoli silver stained according to Bloom and Goodpasture (l976), FC are found to be intensively stained (Figs. 9 and 10) whereas the fibrillar component exhibited less intense staining (Hernandez-Verdum et al., 1978, 1980b; Ellinger and Wachtler, 1980; Goessens, 1982). However, on interphasic neurons of rat superior cervical ganglia, the accumulation of silver grains is more pronounced on the fibrillar component than on the FC themselves (Pebusque et al., 1981b). The results obtained with this Ag-staining method depend upon pretreatment and fixatives used. Therefore differences in the results must be seen in relation to different preparation procedures. There is one-to-one correlation between the chromosomal sites showing hybridization of 28 S + 18 S rRNA (Hsu et al., 1975) and those staining with silver in nine mammalian cell lines (Goodpasture and Bloom, 1975). This correlation indicates that this silver procedure stains the NORs or the sites of ribosomal cistrons. For this reason, this staining method is currently designated as the Ag-NOR staining method. FIG.5. Nucleolus of Ehrlich tumor cell after EDTA staining. Nucleolar associated chromatin (ch) and FC are bleached but fibrillar and granular components are contrasted. X34,OOO. FIG.6. Nucleolus of Ehrlich tumor cell after RNase digestion and DAB staining. Fibrillar and granular components are not visible. Masses of condensed chromatin (arrows) are in connection with the FC. Nucleolar associated chromatin (ch). X21,OOO. (From Goessens and Lepoint, 1979, by permission of SociCtC FranGaise de Microscopie Electronique.)
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117
Recently, the simultaneous selective detection of DNA and Ag-NOR proteins was performed on cultured human cells. Derenzini et al. (1981) showed that the Ag-NOR proteins are restricted to the nucleolar body itself and are not associated with the condensed nucleolar chromatin but may be closely linked to intranucleolar. highly dispersed, chromatin structures. Histochemical tests have indicated that the silver staining arises from acidic nucleolar proteins whose carboxyl groups interact with silver ions (Schwarzacher et al., 1978; Olert et a l . , 1979). Furthermore, two major nucleolar proteins C23 and B23 have been identified; these bind silver in polyacrylamide gels under staining conditions similar to those applied to cells (Lischwe et al., 1979). Immunization of rabbits with the highly purified protein C23 resulted in the production of a specific antibody. When the immunoperoxydase method was used to localize protein C23 in cells, it was found in FC of nucleoli and in NORs of metaphase chromosomes (Lischwe et al., 1981). Protein of approximately MW 195,000, enriched in purified and washed oocyte nucleoli, has been found to be Ag stained with especially high intensity (Williams et al., 1982). This observation suggests that this high-molecularweight protein contributes to the Ag staining of the amplified nucleoli. It is interesting to note that the large subunit of the RNA polymerase type A or I which occurs in transcriptionally active nucleoli has been determined to be in the 195,000 MW range (Chambon et al., 1974). Moreover, Angelier et al. (1982) applied Ag-NOR staining at the molecular level on preparations spread on grids and observed that the silver grains overlap onto the transcribed part of the nucleolar transcriptional units and are not seen in apparently untranscribed spacer regions. On extended transcriptional units, silver grains are preferentially located along the axial region and are observed all along the fibrillar sequences both in their distal part and initiation point. This distribution was quantitatively evaluated. Examination of 40 transcriptional units revealed no marked difference between the number of silver grains located in the initial, medial, and final parts of the transcribed region of the units. These very nice and interesting observations (Angelier et al., 1982) suggest a close relationship between Ag-NOR proteins and the DNP axis where RNA polymerase 1 is situated. On the other hand, in differentiated cells stimulated to reenter the cell cycle (lymphocytes treated with phytohemagglutinin or chick erythrocytes reactivated by cell-cell hybridization), the size and the number of silver-stained areas increase (Arrighi
FIG. 7 . Nucleolus of an Ehrlich tumor cell incubated with tritiated thymidine. Note the focal labeling of the FC. Labeling is also localized over the nucleolar associated chromatin. X30.000. FIG.8. Incorporation of tritiated uridine in Ehrlich tumor cell nucleolus (10-minute incubation). The radioactivity is essentially associated with the fibrillar component around the FC. X 17,000 (From Goessens, 1976b. by permission of Academic Press.)
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et al., 1980). This change corresponds rather well to the increase in nucleolar RNA polymerase activity (Carlsson et al., 1973; Jaehning et al., 1975). Enzymes. Conversion of the initial products of transcription of the rRNA genes to the mature ribosomal RNA components involves a series of enzymes necessary for instance in the methylation of pre-rRNA or in endonucleolytic cleavages of rRNA precursors. Methylases and ribonucleases have been purified from Ehrlich tumor cell nucleoli (Obara et al., 1982; Eichler and Eales, 1982). The precise intranucleolar localization is known for some enzyme activities. Acid phosphatase that is active at pH 5.0 has been demonstrated in the FC of HeLa cell nucleoli (Soriano and Love, 1971). Fox and Studzinski (1982) have provided cytochemical demonstration of a strong Mg2 -dependent adenosine triphosphatase reaction in circumscribed areas of nucleoli which correspond to FC. It seems that the enzyme has a function in the unfolding-compaction cycle of rDNA. RNA polymerase I which is not inhibited by a-amanitin is restricted to the nucleolus (for review see Chambon et al., 1974). Gruca et al. (1978) showed that activity of RNA polymerase I is located within the fibrillar components of the nucleolus suggesting that these components contain the active template for rRNA synthesis in rat liver nucleoli. Metals and inorganic cations. The nucleoli have a high affinity for heavy metals (Tandler, 1954; Studzinski, 1965) and relatively large amounts of metals are contained in nucleoli of normal and malignant cells (Ca, Zn, Cu, Mn, Cr, Ni). Binding of Cr and Ni to nucleoli is more resistant than that of the other metals to treatment with nucleases (Ono et al., 1981). FC show coarse deposits after pyroantimonate-osmium fixation (Hardin et al., 1970; Parmley et al., 1977). Accumulation of deposits also occurs on the dense fibrillar component (Rodriguez-Garcia and Stockert, 1979). The dense precipitates which originate as a result of reaction between tissues and the pyroantimonate-osmium fixative are due to inorganic cations especially CaZ+ and Mg2+ (Tandler et al., 1970; Clarck and Ackerman, 1971). So, in the nucleoli, inorganic cations are mainly located in the fibrillar components (FC and fibrillar component) whereas granular component and condensed associated chromatin are free of antimonate precipitates. The significance of this accumulation within nucleoli and specially within FC could be related to their metabolic activity and to the presence of RNA polymerase I, the activity of which is dependent on divalent cations (Tres et al., 1972). Lipids. Dutta et al. (1963) and Esper (1965) using the Sudan Black B method +
Fics. 9 A N D 10. Ag-NOR staining in Ehrlich tumor cell nucleoli. The silver grains are localized in the FC. Fig. 9, X27.000. Fig. 10, x31,OOO.
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demonstrated bound lipids in nucleolar structures that Recher et al. (1972) considered equivalent to FC. Olivier (1967) observed phospholipids in nucleolar central areas of oocytes that seem to correspond to FC. The presence of phosphorylated compounds within FC could be responsible for the lead reaction observed on the fibrillar elements of nucleoli (Recher et al., 1972). 2. The Fibrillar Component a. Ultrastructure. The fibrillar component consists of a dense osmiophilic material composed of fibrils approximately 4 nm in diameter. These fibrils are packed into strands associated with FC suggesting a close functional relationship between these two nucleolar components (Figs. 1 and 2). The fibrillar component forms regions of high electron density which sometimes exhibit a reticulate appearance (Fig. 3). The resemblance of this network observed at the ultrastructural level (Bernhard et al., 1952) to Estable and Sotelo’s light micrographs (1951) explains that the term nucleolonema has been applied in both cases (Bernhard er al., 1955). b. Composition. Observations based on RNase digestions have shown that RNA is present in the fibrillar and the granular components (Bernhard and Granboulan, 1963; Marinozzi, 1963; Swift, 1963). Double digestions with RNase and pepsin demonstrated the RNP nature of these nucleolar components (Marinozzi, 1964). High resolution autoradiographic studies on monkey cells (Granboulan and Granboulan, 1965) and on amphibian embryos (Karasaki, 1965) indicated that tritiated uridine is first incorporated in the fibrillar component (Fig. 8). Since these original observations, it has been well established that the fibrillar component is the site of pre-rRNA synthesis (for review see Fakan, 1978). Kinetic data obtained by Royal and Simard (1975) in a correlated autoradiographic and biochemical study of RNA synthesis in nucleoli of Chinese hamster ovary cells suggested that the 80 S RNP containing the 45 S RNA are localized in the fibrillar component of the nucleolus. In addition lo RNP, the fibrillar component which in certain animal and plant cells consists of filamentous structures was found to contain DNA. This conclusion arises from numerous studies performed with enzymatic digestions, autoradiographic methods, or cytochemical stainings (La Cour and Wells, 1967; Lord and Lafontaine, 1969; Chouinard, 1970; Lafontaine and Lord, 1973; Anteunis et al., 1973; Ryser et al., 1973; Sanchez and Oberti, 1974; Pouchelet et a l . , 1975; for a review see Smetana and Busch, 1974). As shown by Brinkley and Berns (1974), the fibrillar and granular components were segregated into light and dark zones by treatment with actinomycin D. The light and dark zones do correspond to the fibrillar and granular nucleolar components. Following this segregation, the cells were treated with quinacrine hydrochloride which sensitizes the nucleoli to argon laser light; then the segregated nucleolar components
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were selectively irradiated with the laser microbeam. The data obtained indicated that selective damage to the light (fibrillar) area is generally more damaging than damage to the dark (granular) area illustrating that DNA is closely associated with the nucleolar fibrillar component. This conception confirms previous and controvertial observations suggesting that in several types of animal cells the nucleolonema stains with Feulgen procedure and represents a part of the nucleolar chromosomes (Lettrt et a l . , 1966; Ghosh et al., 1969). So, it appears clearly that DNA is present not only within the FC but also in the immediately surrounding dense fibrillar component. As previously mentioned, the fibrillar component can also be positive after Ag-NOR staining indicating the existence of acidic proteins.
3 . The Granular Component a. Ultrastructure. The granular component is formed of loosely packed granules approximately 15 nm in diameter which closely resemble cytoplasmic ribosomes. These nucleolar granules are more or less numerous. In normal nucleolus, they are situated around the fibrillar component or along and between the strands of fibrillar component (Figs:l, 2, and 3). In the latter condition, fibrillar and granular components are intermingled in rather coarse masses but in some experimental or physiological conditions, they tend to become segregated from one another. b. Composition. The granules are sensitive to pronase or pepsin and ribonuclease. So, as the fibrils, the granules are composed of RNP (Marinozzi, 1964). It has been demonstrated that after 5-minute pulse-labeling with tritiated uridine of tissue culture cells, silver grains are localized predominantly over the fibrillar component of the nucleolus (Granboulan and Granboulan, 1965). Granular component becomes labeled only after longer periods of radioactive incubation and/or after a cold chase. This demonstrates a migration of newly synthesized nucleolar RNA from the fibrillar into the granular component (Granboulan and Granboulan, 1965). These results were also confirmed after treatment of cells with actinomycin D provoking nucleolar segregation (Geuskens and Bernhard, 1966). By electron microscopic autoradiography on the nucleolus of Triturus gastrulae, Karasaki (1965) also showed that label appears first in the fibrillar component, later in the granular component, and finally in the cytoplasm. Biochemical studies of isolated nucleolar fibrillar and granular components also indicated that the fibrillar component is precursory to the granular component (Daskal et a l . , 1974). Ultrastructural, autoradiographic, and biochemical data demonstrated that the nucleolar granules are formed in close proximity to the fibrillar component, later
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to be released as preribosomal particles and finally transported to the cytoplasm as ribosomal subunits.
4. The Nucleolar Interstices a. Ultrastructure. Nucleoli frequently contain more or less spherical light areas of lower density than the surrounding nucleolar mass. They have been referred to as nucleolar vacuoles (Committee on Nucleolar Nomenclature, 1966) but are in fact interstices which are seen to be enclosed within the granular regions of the nucleolus (Figs. 2 and 3). The morphology, size, and number of the nucleolar interstices are variable and depend on the nucleolar type. b. Composition. The nucleolar interstices generally contain a material which exhibits a texture and an electron density matching those of the surrounding nucleoplasm which may suggest a continuity between these two nuclear compartments. They are quite different from FC (Fig. 4). However, Moreno Diaz de la Espina et al. (1980) observed that the interior of the interstices in plant cells shows a well-defined ultrastructure different from that of the ground nucleoplasm. The size and cytochemical properties of granules found in the interstices as well as their similarities with the preribosomal particles led these authors to identify them as mature preribosomal particles. When RNA processing is impaired by drugs, Moreno Diaz de la Espina et al. (1980) found in the interstices granules which could correspond to incomplete processed rRNA precursors as observed for similar conditions in animal cells and termed perichromatin-like granules (for review see Puvion and Moyne, 1981). According to Moreno Diaz de la Espina et al. (1980), the nucleolar interstices play a role in the storing and transport of preribosomal precursors. It is interesting to note that the appearance of interstices is correlated with the beginning of uridine incorporation into root cells of Zea mays (Deltour and Bronchart, 1971; De Barsy et a l . , 1974).
5 . The Nucleolar-Associated Chromatin a. Ultrastructure. Nucleolar-associated chromatin corresponding to the structure already described at the photonic level by Caspersson (1950) surrounds the nucleolus. This shell of perinucleolar chromatin separates the nucleolar body from the nucleoplasm and is connected with clumps of intranucleolar chromatin which penetrate into the nucleolar body (Fig. 6). Thus the nucleolar-associated chromatin is composed of dense peri- and intranucleolar chromatin. The FC have been shown to be attached to and continuous with this fully condensed chromatin (Goessens, 1979; Ashraf and Godward, 1980). The organization and the amount of the intranucleolar as well as perinucleolar chromatin seem to depend on the cell type. b. Composition. Cytochemical analyses (enzymatic digestions and autoradiography after tritiated thymidine incorporation) demonstrated the presence of
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DNA in the nucleolar-associated chromatin (review in Busch and Smetana, 1970). More recently, nucleolar DNA organization was studied in a few cellular types with oxidized diaminobenzidine (Anteunis et a l . , 1979; Pouchelet and Anteunis, 1979). This technique combined with enzymatic digestions enables these authors to observe a gradual decondensation of a portion of the perinucleolar DNA which it becomes spread into the fibrillar regions of the nucleolus. For instance, the nucleoli of guinea pig lymph node lymphocytes possess a central fibrillar zone (contrary to what is observed after conventional techniques, this area appears homogeneous after DAB treatment) with a few intranucleolar clumps at its periphery. These clumps are composed of densely packed DNA. The presence of this intranucleolar chromatin and its topographical localization connected to both the perinucleolar chromatin and the fibrillar zone led Anteunis et al. (1979) to suppose that these clumps should be directly related to some functional activity of this zone. Moreover the fact that the fibrillar zone appeared homogeneous and only faintly stained after DAB treatment indicates that all the nucleic acids present within this zone must be in an unfolded fibrillar form. Bachellerie et al. (1977) have developed a fractionation procedure for isolated nucleoli that results in the isolation of topographically distinct areas of nucleolar chromatin which appear to be differently involved in ribosomal RNA synthesis. The separated pen- and intranucleolar chromatin fractions have been assayed for ribosomal gene dosage. The preferential localization of ribosomal cistrons in intranucleolar chromatin has been clearly demonstrated in normally growing cells (Bachellerie et al., 1977). Using the osmium-ammine method (Cogliati and Gautier, 1973), Derenzini et al. (1982) were able to study the structure of ihtranucleolar chromatin in thin sections of rat hepatocytes fixed in situ. Besides chromatin with nucleosomal configuration, these authors described loose agglomerates of extended DNA filaments with a thickness of 2-3 nm. These latter structures appeared to be peculiar to nucleolar chromatin. The intranucleolar DNA not structured into nucleosomes observed in situ might represent the counterpart of the extended configuration of ribosomal DNA as visualized in spread preparations in transcribing units and in the spacer regions between them (Derenzini et a l . , 1982). 6 . The Nucleolar Matrix The cell nucleus contains various structures that are insoluble in high and low salt buffers and nonionic detergents and are assumed to provide karyoskeletal support to other nuclear components. These karyoskeletal elements include the residual nuclear envelope layer which still retains residual nuclear pore complexes, a residual interchromatinic matrix structure, and residual nucleoli (for review see Berezney, 1979). Thus, the nucleolus, like the nucleus, is now known to contain a scaffold or skeletal matrix material (Todorov and Hadjiolov, 1979).
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Franke et al. (1 98 1) have recently described in fractions enriched in amplified nucleoli from oocytes of Xenopus laevis, a meshwork of filaments resistant to various extractions in high salt buffers which contain only one predominant protein characterized by a polypeptide of 145,000 MW. This protein has not been found in frog erythrocyte nuclei which are known to be devoid of nucleoli. Krohne et al. (1982) reported that this protein specifically associated with nucleoli is chemically and immunologically different from other karyoskeletal insoluble nuclear proteins found in pore complex-lamina fractions or in nuclear sap and can be localized by antibody techniques to nucleoli of oocytes and other cells. Therefore it is concluded that this insoluble protein structure represents a skeletal meshwork specific for the nucleolus (for review see Scheer et a l., 1982).
B. FUNCTIONAL SIGNIFICANCE As postulated by Chouinard (1975), the nucleolus would correspond to a specialized structural device allowing for the formation, stabilization, and packaging of essential gene products derived from its intrinsic chromatin material. The nucleolus therefore is an active genetic site on a chromosome and has much in common with a puff or Balbiani ring on a giant polytene chromosome or a loop in a lampbrush chromosome. This conception confirms an already old assumption of Lettr6 et al. (1966, pp. 109-110): “If one assumes that the nucleolonema contains part of the nucleolar chromosomes, . . . its morphological appearance may be compared either to a puff formation in polytene chromosomes or to the loop formation in lampbrush chromosomes. The synthesized material can be either accumulated around the chromosomal filaments or may be released into the nuclear sap depending on the quantity and quality of the material produced.” The organization of the nucleolus can be viewed as a dynamic process in which the nucleolus can be considered a product of the activity of one or more chromosomes containing rDNA segments condensed or uncondensed depending on the cell demand of ribosomal RNA (Mirre and Stahl, 1981). Two parts can be distinguished in the nucleolus: the chromatin portion and the RNP products. It is clear that the chromatinic filaments included within the nucleolar fibrillar components (FC and fibrillar component) are connected with the nucleolarassociated chromatin (Pouchelet and Anteunis, 1978; Mirre and Stahl, 1978a; Anteunis et al., 1979; Goessens, 1979; Ashraf and Godward, 1980). This necessarily implies that the chromatinic intranucleolar filaments and the nucleolarassociated heterochromatin constitute a single unit which belongs to nucleolar chromosome(s). However, at the present time, we do not know the real function of condensed intra- and perinucleolar chromatin. It could correspond to NOR chromatin in a
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condensed state containing ribosomal genes for 18 S rRNA, 28 S rRNA, and 5.8 S rRNA but it could also contain other genes involved in ribosome biogenesis namely the genes coding for the structural ribosomal proteins and the genes for the specific enzymes and other proteins specialized in transcription, modification, and processing mechanisms leading to mature ribosomes. Practically nothing is known about the latter ribosomal genes (Hadjiolov, 1980). However characterization of chromatin from isolated nucleoli showed that only 10-20% are truly intranucleolar and still a small part (1%) contains rDNA (Busch and Smetana, 1970). It is now generally accepted that rDNA is situated in the FC and in the fibrillar component of interphasic nucleolus. In the FC, it is associated with more or less proteins which could correspond to ribosomal proteins, enzymes, and skeletal elements of the nucleolar matrix. Thus the size of the FC could especially depend on the amount of these proteins. The transcription occurs around the FC resulting in the formation of a layer of electron-opaque fibrils. Consequently the latter region (fibrillar component) cannot be dissociated from the FC since together they constitute a functional unit. Moreover, since the studies of Miller and Beatty (1969), it is known that in spread preparations highly active rDNA appears as tandem arrays of transcriptional units with a characteristic Christmas-tree pattern of lateral RNP fibers and that the nascent pre-rRNA does not immediately detach itself from the rDNA thus allowing observation of transcription units (review in Franke et a l . , 1979). By combining spreading technique with electron microscopic autoradiography it was possible to propose a parallel between the gradients of transcribed RNA fibrils (“Christmas tree”-like forms) and the nucleolar fibrillar component observed in situ in sectioned material (for review see Fakan and Puvion, 1980). It should now be admitted that the fibrillar component would be composed of both rDNA and newly synthesized RNP. Thus active rRNA genes could be obscured by the products of transcription and maturation (Miller and Beatty, 1969; Franke et al., 1979). For instance, compact nucleoli produced in hepatocytes by treatment with cycloheximide are characterized by a relatively uniform distribution of RNP components which mask an intranucleolar network consisting of proteins and fine DNA filaments (Smetana et al., 1980). The network within the nucleolar body of compact nucleoli was masked by the nucleolar RNP components since it was visible after their removal by partial digestion with pepsin and complete digestion with RNase (Smetana et al., 1980). On the other hand, it is well established that the two RNP nucleolar components namely the fibrillar component and the granular component relate to two stages in the formation of ribosomes. There is not only cytological or autoradiographic but also biochemical evidence that the fibrillar component is precursory to the granular component. For instance, Daskal et al. (1974) developed a method for fractionation of the nucleolus into granular and fibrillar components.
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The nucleotide composition of the RNA of the fibrillar and granular fractions was similar to that of ribosomal RNA. The kinetics of incorporation of [32P]orthophosphate into RNA in both fractions showed that the specific activity of the RNA of the fibrillar fraction was fourfold greater than that of RNA in granular region at 15 minutes. After 2 hours, the specific activity of the RNA in the granular region was 1.5 times greater than that in the fibrillar region. Twodimensional polyacrylamide gel electrophoresis showed that the fibrillar fraction contained fewer proteins than the granular fraction; some of the proteins were common to both the nucleolar granular particles and cytoplasmic ribosomes while others appeared to be unique to this fraction. These studies (Daskal et al., 1974) support other data suggesting that the granular elements are formed in close proximity to the fibrillar areas, later to be released as preribosomal particles and finally transported to the cytoplasm as ribosomal subunits. Since the fibrillar component is known to be the precursor of the granular one, the lack of granules therefore indicates either a time-lag between ribosomal precursor synthesis and the storage of the precursor granules after processing or a rapid export of the newly processed molecules (Hernandez-Verdun et al., 1980a). In conclusion, it appears evident that the fine structure and the size of the nucleolus are very sensitive to changes in ribosomal RNA syntheses. The nucleolus therefore serves as a cytological indication of the transcription of rRNA genes. But it seems likely that the normal nucleolar morphology also requires a balance among the rRNA syntheses, the production and accumulation of ribosomal and nucleolar-specific proteins, and the transport of ribosomal subunits out of the nucleolus. According to this conception and from the theoretical nucleolar model described in the present part of this article, it will be possible to interpret the variations in the nucleolar ultrastructure. 111. Variations in the Ultrastructural Morphology
Nucleolar number, size, and shape vary widely depending on the organism, cell type, and physiological state of the cell. For instance, nucleoli of actively synthesizing cells are often larger than those of less active cells. So a decrease in size of the nucleolus may be correlated with a decline in function. Such variations will be briefly analyzed with special regard to the cell cycle, some physiological maturation processes, and some experimental conditions. THE CELLCYCLE A. DURING 1. During Interphase a. Nucleolar Number. In chick fibroblasts cultivated in v i m (Bassleer, 1968) and in Ehrlich tumor cells (Bassleer et a l . , 1973; Lepoint and Bassleer,
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1978), the number of nucleoli does not vary during interphase but the total number of nucleoli which appear in both daughter cells in posttelophase is usually twice as high as in the mother cell. These data are in agreement with observations performed in 1931 by Heitz who concluded that the number of nucleoli arising in telophase is always constant and is characteristicof the species as is the chromosome number. However fusion of nucleoli could occur early in telophase. Thus the number of nucleoli can be much lower than the number of NORs because of a marked tendency for nucleoli to fuse. Anastassova-Kristeva (1977) found that in various cultured human cells the number of nucleoli per nucleus varies according to the stage in the cell cycle. At the end of mitosis and the beginning of G , , multiple nucleoli exist. The maximum number of nucleoli is connected with the gene expression of rDNA of the 10 NORs in man. The nucleoli gradually fuse until at the beginning of S there is only one nucleolus per nucleus. A significant reduction in nucleolar number between G, and mitosis due largely to fusion was also found in synchronous strain L cells (Gonzalez and Nardone, 1968) and in lymphocytes after stimulation by phytohemagglutinin (Wachtler et al., 1982). Nucleolar fusion was demonstrated in binucleate cells of Allium cepa by Ghosh (1976). Although the sister nuclei in these binucleate cells induced through caffeine treatment have almost identical nucleolar development, some of the cells show a different nucleolar number in the sister nuclei but the nucleolar area in the nuclei indicates that the difference in the number is the result of nucleolar fusion in one of the sister nuclei. Other evidence for a relationship between the nucleolus and the chromosomes comes from studies of polyploids. De Mol(l926) showed an increase of nucleolar number with degree of ploidy. The number of nucleoli per nucleus in the normal liver increases in parallel with the increase of ploidy (Hirano et a l . , 1981). In Ehrlich tumor cells, the number of nucleoli depends on the number of genomes. Hyperdiploid line cells contain 45 chromosomes and 2 nucleoli and hypertetraploid line cells contain 90 chromosomes and 4 nucleoli (Bassleer and Goessens, 1972). However, the possibility of various degrees of expression of nucleolar organizers as well as a possible tendency of the nucleoli to fuse or to divide cannot be excluded and can explain the variations observed in nucleolar number (Hsu et al., 1967). b. Nucleolar Volume. If the number of nucleoli does not vary during interphase in chick fibroblasts culture in vitro, the total nucleolar volume and the total nucleolar dry mass per nucleus double before prophase starts (Bassleer, 1968). In Ehrlich ascites tumor cells blocked in G,, the total volume of the nucleoli is increased but the number of nucleoli is unchanged (Bassleer et al., 1973). In Physarum polycephalum, Matsumoto and Funakoshi (1978) observed parallel changes in the increase curves of the nuclear and nucleolar areas in the mitotic cycle. Increases in nuclear and nucleolar size seem to be highly coordinated with the progress of the cell stage. Noel et al. (197 1) have also observed an
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enlargement of the nucleolar volume by a factor of two from the beginning of G I to the middle of S. A combination of several techniques including velocity sedimentation, autoradiography, cytophotometry, and stereology allowed quantitative data about the growth of the nucleolus and different nucleolar components in the course of interphase in Ehrlich tumor cells (Lepoint and Goessens, 1982). It was observed that the nuclear and nucleolar volumes double during the interphase (Table I) and that all the nucleolar components participate in the doubling of the nucleolar volume during interphase (Table 11). In Allium cepa meristematic cells, Sacristan-Garate et al. (1974) observed that the amount of the fibrillar component of the nucleolus is roughly constant in the course of interphase while growth of the nucleolus seems to take place exclusively in its granular portion. TABLE I NUCLEARAND NUCLEOLAR VOLUMESO
Nuclear Nucleolar
325.07 49.18
620.48 92.89
“Since the cellular volumes obtained by the Coulter counter are expressed in pm3, the different relative volumes determinated by stereological methods are easily converted into absolute volumes (from Lepoint and Goessens, 1982, Exp. Cell Res. 137, 456-459). TABLE I1 DENSITYVOLUMES~ OF THE NUCLEOLAR COMFQNENTS~
Vvgf(nu) Vvfc(nu) Vvvac(nu)
87.24 2 3.53 9.34 f 1.96 3.42 f 2.46
88.45 f 1.62 8.58 2 2.64 3.07 f 1.08
OThe results are expressed as percentages of the nucleolar volume. bThe volume fractions of the nucleolus occupied by the fibrillar and the granular components [Vvgf(nu)], by the fibrillar centers [Vvfc(nu)], and by the nucleolar “vacuoles” [Vvvac(nu)] were determined by stereological methods (from Lepoint and Goessens, 1982, Exp. Cell Res. 137, 456-459).
NUCLEOLAR STRUCTURE
129
However, nucleoli of various cell types do differ in the percentage of the total nucleolar area occupied by each ultrastructural component. In cultured diploid human fibroblasts, Jordan and McGovern (198 1) observed that the number of FC depends on the level of cell activity, the number rising with increasing cell activity. Their volumes are inversely proportional to their number per nucleolus indicating a possible fusion with cell inactivation. In normal liver cells, the granular and the fibrillar components comprise 77 and 19%, respectively, of the total nucleolar area; in Walker tumor cells, the granular component occupies 93% and the fibrillar component 3% of the total nucleolar area (Smetana et a l . , 1966). In neuronal cells of rat trigeminal ganglia (Hardin et a l . , 1970), the granular component comprises 54.5%, the fibrillar component 25.2%, the FC 8.0%, and the interstices 12.3%. The significance of all these differences is probably related to differences in rate of RNA synthesis. 2. During Mitosis During mitosis as the process of chromosome condensation proceeds, RNA synthesis either ceases completely or decreases sharply. The onset of new RNA synthesis is revealed in late telophase. These RNA synthesis modifications explain that the disappearance of the nucleoli at the end of prophase and their reconstitution in daughter cells during telophase are nearly universal events during somatic cell division. The behavior of the nucleoli during mitosis has been studied in a lot of cellular species (for review see Jordan and Cullis, 1982) and among others in Ehrlich tumor cells (Goessens and Lepoint, 1974). a. During Prophase. The changes in shape and the gradual reduction in size of the nucleolus is accompanied by the disappearance of the fibrillar component and the dispersion of the granular component in the surrounding nuclear sap (Figs. 11 and 12). The fibrillar component disappears at first; this is the morphological sign of the RNA synthesis cessation because it is known that the fibrillar component contains newly synthesized RNA and that nucleolar fibrils represent the origin of the nucleolar granules (Marinozzi, 1964; Granboulan and Granboulan, 1965; Geuskens and Bernhard, 1966). b. During Metaphase and Anaphase. After dispersion of the granular component, nucleolar material is difficult to distinguish with conventional staining techniques from other nuclear and cytoplasmic structures but, in Ehrlich tumor cells, the FC are detectable throughout mitosis and such structures are always associated with chromosomes (Figs. 13 and 14). However, material derived from the nucleolus, in other words nucleolar remnants which is represented by ribosome-like particles, is attached to metaphase chromosomes in various cell types for instances in root meristematic cells of Vicia faba (Lafontaine and Chouinard, 1963), in grasshopper neuroblast cells (Stevens, 1965), in Chinese hamster cells (Hsu et a l . , 1965; Noel et al., 1971), in mammalian materials (Heneen and Nichols, 1966), and in HeLa cells
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NUCLEOLAR STRUCTURE
131
(Terauchi, 1973). Moreno Diaz de la Espina et al. (1976) studied the nucleolar cycle in Allium cepa and demonstrated that the nucleolar material dispersed at the beginning of mitosis becomes incorporated into the metaphase chromosomes. Recently using silver impregnation technique, Paweletz and Risueno ( 1982) confirmed these observations and showed that some parts of the nucleolar material are forming a sheath and some are incorporated into the interior of the chromosomes during prophase and metaphase. In certain species of plants (Brown and Emery, 1957; review in GimenezMartin et al., 1977) or in various animal cells (Love and Suskind, 1961; Hsu et al., 1965; Heneen and Nichols, 1966; Sheldon et al., 1981), nucleoli have been found to persist during mitosis. These nucleoli have been called persistent nucleoli. Takeuchi and Takeuchi (198 1) consider that persistent nucleoli observed in embryonic ectodermal cells are merely remnants of disappearing nucleoli found in rapidly proliferating embryonic cells. Distinct nucleolar remnants can be in contact or not with chromosomes during mitosis. c. In Telophase. In telophase, the nucleoli arise at specific loci on chromosomes usually bearing a secondary constriction. The relationship between nucleoli and chromosomes is largely due to the classical works of Heitz (193 1 ) and McClintock (1934). As shown in Ehrlich tumor cells (Goessens and Lepoint, 1974), the fibrillar component reappears in close association with the FC (Figs. 16 and 17). Shortly afterward the granular component is also present and the structure of the nucleoli is soon identical with that usually observed in interphase cells. The reappearance of the fibrillar component before that of the granular component provides morphological support for the fact that fibrils represent the origin of the granules. The sequential appearance of the fibrillar and granular components of the nucleolus during the process of nucleologenesis is a common event in plant and animal cells (see among others: Lafontaine and Chouinard, 1963; Goessens and Lepoint, 1974; Lafontaine and Lord, 1974; Chouinard, 1975; Hernandez-Verdun et al., 1980a). The most interesting observation performed in Ehrlich tumor cells consists of the reapparition of the nucleolar components around and in contact with the FC. This is the demonstration that the FC is in fact the NOR, the term indicating that the nucleolus originates in that region. The origin of the material which constitutes the reappearing nucleoli is still a
FIG. 1 I . An Ehrlich tumor cell in prophase. The nuclear envelope is still present and surrounds the nucleolus (arrows) and the chromosomes. X7000. (From Goessens and Lepoint, 1974, by permission of Academic Press.) FIG. 12. Nucleolus in a prophase cell. The nucleolus is composed of the granular component (g) and the FC. The fibrillar component has disappeared. X23.000. (From Goessens and Lepoint. 1974, by permission of Academic Press.)
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NUCLEOLAR STRUCTURE
133
matter of uncertainty. At least two theories exist concerning the mode of nucleolar formation. (1) The nucleoli arise from material carried through mitosis providing material for nucleolar reformation. (2) The reformation occurs as DNA cistrons in the NOR are activated for transcription. To examine this problem, several authors have studied the process of nucleolar formation under conditions of RNA synthesis inhibition. Telophase reconstruction of the nucleolus in Chinese hamster cells and in meristematic cells of Allium cepa does not require simultaneous RNA synthesis and occurs at the expense of RNA that has been synthesized prior to mitosis (Semeshin et al., 1975). In cultured mammalian cells RNA synthesis after mitosis is not necessary for organization of nucleoli after mitosis. However, inhibition of nucleolar RNA synthesis before mitosis renders the cell incapable of forming nucleoli immediately after mitosis (Phillips and Phillips, 1973). On the contrary, Stockert et al. (1970) and Gimenez-Martin et al. (1974) demonstrated that nucleologenesis in plant cells is dependent on simultaneous nucleolar transcription. Following these conceptions, the question remains open. However, in the light of more recent results obtained on plant cells (De La Torre and Gimenez-Martin, 1982) or in animal cells (Lepoint and Goessens, 1978), it appears that the reformation of the nucleoli in telophase is due to an accumulation of RNA molecules around the NOR. These RNA molecules are synthesized for one part during the last G, of the preceding cell cycle and for the other part during telophase. d. The NOR. According to Heitz (1931) the site of nucleolus formation (NOR) is an achromatic part of chromosome. McClintock (1934) also observed that the nucleolus is attached to the “nucleolar stalk” but she reported that the NOR is localized in a heterochromatin part. Two alternatives with respect to the chromosomal position of NOR and rDNA exist (Anastassova-Kristeva et al., 1977). (1) The secondary constriction (achromatic part of chromosome) is the site of the NOR and the rRNA cistrons. (2) The NOR comprises the secondary constriction and an adjacent heterochromatic chromosome segment in which at least a part of the rDNA is located. In various cell types, the location of the rRNA genes has been made possible by cytological hybridization of 18 S and 28 S rRNA to metaphase chromosomes FIG. 13. An Ehrlich tumor cell in anaphase. In the middle of a chromosome a secondary constriction (NOR) is visible. X9000. FIG. 14. The secondary constriction corresponds to the FC of the interphasic nucleolus which persists during mitosis included in some chromosomes. X 22,000. (From Goessens and Lepoint, 1974, by permission of Academic Press.) FIG. 15. Ag-NOR staining on an anaphase chromosome. The silver grains are localized in the secondary constriction which corresponds to the FC. X 30,000.
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NUCLEOLAR STRUCTURE
135
and in most cases the autoradiographic silver grains have been observed in correspondence with the secondary constrictions (Henderson et al., 1972; Hsu et a l . , 1975). On the other hand, there is some evidence for a positive correlation between the size of the secondary constriction and the number of rRNA genes (Miller and Brown, 1969; Elsevier and Ruddle, 1975; Anastassova-Kristeva et a f . , 1977; Maggini and De Dominicis, 1977). Silver staining methods (Goodpasture and Bloom, 1975; Howell et a l . , 1975) have been widely used to detect NORs in the karyotypes of several organisms. There is evidence from studies in somatic cell hybrids that these NORs stained by silver were actively engaged in the synthesis of rRNA during the preceding interphase (Miller et a f . , 1976). As previously discussed, the material which positively stains with silver is not the rDNA itself but an acidic protein associated with the rRNA transcribed at the ribosomal DNA sites (Goodpasture and Bloom, 1975; Howell et a l . , 1975). The loci of Feulgen-negative secondary constrictions coincide with those of the grain clusters after in situ rRNA/DNA hybridization and those of silver precipitation after silver staining in Vicia sativa (Burger and Knalmann, 1980). Electron microscopic studies of secondary constrictions have shown that the achromatic regions are not really constrictions (Hsu et a l . , 1967; Lafontaine, 1968; Brinkley and Stubblefield, 1970; Goessens and Lepoint, 1974). The width of the secondary constriction at metaphase is the same as that of the chromosome arms. However, the chromatin fibers are lossely condensed in the secondary constrictions presumably due to the fact that these fibers are decondensed as late as early prophase when the nucleolus is still active in rRNA syntheses. In Ehrlich tumor cells (Fig. 14), the secondary constrictions which contain neither granular nor dense fibrillar components not only have the same morphology as the FC of the interphase nucleolus but are also positive as the FC are after silver staining (Fig. 15). On the basis of their morphology, their silver staining properties and their behavior during mitosis, it is clear that the FC are the counterpart of the secondary constrictions. However there are exceptions (Hsu et a l . , 1975); some secondary constrictions do not offer to bind tritiated ribosomal RNA and some regions which bind ribosomal RNA do not appear as secondary constrictions in metaphase chromosomes. In Alfium cepa, the NOR is constituted of chromatin differentiated in high and low density zones. Esponda and Gimenez-Martin (1972, 1974, 1975) suggested FIG. 16. An Ehrlich tumor cell in telophase. New nucleoli (arrows) are in formation. X8500. FIG. 17. In late telophase, the fibrillar and the granular components reappear around the FC and the nucleolus acquires progressively its interphasic ultrastructure. X50,OOO. FIG. 18. Ag-NOR staining on a nucleolus in late telophase. The silver grains are localized in the FC. X53.000.
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a pattern for the NOR in which areas of functional chromatin would appear alternating with areas of nonfunctional chromatin. An argon laser microbeam was used by Berns and Cheng (1971) to irradiate regions outside and inside the secondary constrictions of nucleolar organizing chromosomes. From the results obtained it appears that there is a region adjacent to the secondary constriction that in some way is involved in nucleolar formation. Thus it is possible that during mitosis but also during interphase the NORs are not restricted only to uncondensed chromatin. As suggested by Jordan and Luck (1976), the NOR could be considered as a chromatin region with a tendency of incomplete condensation giving rise to the lightly staining zone and showing different extents of complete condensation giving rise to different proportions of darkly staining condensed chromatin regions. Nucleolar organizers may show varying proportions of the two zones and even appear to be entirely of one form or the other. B. DURING MEIOSISIN GERMINAL CELLS If the evolution of the nucleolus during meiosis in germinal cells presents analogies to the mitotic evolution, meiotic prophase I constitutes a choice material for study the genes coding for ribosomal RNA (Stahl et d.,1980). During early and middle pachytene phases, the nucleolus is not visible; it reappears only at late pachytene in the form of a tiny spherule pressed against a chromocenter. During diplotene the nucleolus develops considerably in contact with a chromocenter. Thus germinal cells present advantages for studying nucleolar chromosomes and their relationships with the nucleolus. These relationships have been analyzed in a series of clear papers published by Mirre and Stahl and by Stahl et al. (for review see Stahl, 1982). Observations in quail oocytes (Mirre and Stahl, 1976) indicated that the nucleolus is formed in connection with definite chromosomal structures from which DNP fibers penetrate in a pale fibrillar zone (FC) surrounded by electron-dense fibrillar strands. Beyond them the main nucleolar mass is formed of fibrils and granules, the latter being more peripheral. Gradually as the nucleolus enlarges, it has at first a vacuolar then a reticular appearance. This morphology is analogous to that observed by Jordan and Luck (1976) in the meiocytes of Endymion non-scriptus (L). Thus at the very onset of formation, the nucleolus displays a very simple structure, i.e., it is composed of a FC surrounded by a layer of electron-dense fibrils. Cytochemical studies (Mirre and Stahl, 1978a) suggested that the FC contains the rDNA fibrils and that their transcription occurs in the peripheral electron-opaque layer. The morphological features of the latter result from superposition of rDNA fibrils and newly synthesized rRNA. Just like during mitosis, the newly nucleolus synthesized during meiosis displays a gradient distribution of its components. From the FC to the opposite
NUCLEOLAR STRUCTURE
137
extremity of the nucleolus the latter is fibrillar, then fibrillogranular, and finally granular. A remarkable morphological study in the mouse oocyte realized by Mirre and Stahl(1978b, 1981) leads to the description of three steps in nucleolar formation. In the first step, the nucleolus is in the form of FC surrounding by a layer of electron-dense fibrils. During the second step, the nucleolus undergoes development and is extended by strands of fibrillar component which become more fibrillogranular distally, and the third step is characterized by the development of the nucleolonema which coincides with the appearance of numerous small FC. These observations clearly illustrate the fact that the nucleolus is not a static formation and explain the great ultrastructural variability of the nucleolus which from a compact configuration can become developed with formation of a nucleolonema and acquisition of a reticulated structure. These observations and interpretations concerning the organization of the nucleolus are summarized in Fig. 19. OR EXPERIMENTAL CONDITIONS C. UNDER SOMEPHYSIOLOGICAL 1. During Early Embryogenesis in Animals The early development of many embryos is characterized by absence of nucleolar activity and synthesis of rRNA; protein synthesis is carried out by stored ribosomes produced during oogenesis. Most of the nuclei in two-cell mouse embryos contain several compact, agranular round nucleoli. In some species, these atypical agranular nucleoli have been referred to as nucleolus-like bodies (Karasaki, 1968; Naus and Kidder, 1982). During mouse embryogenesis, rRNA synthesis begins at the four-cell stage (for review see Epstein, 1975). The beginning of nucleolar RNA synthesis coincides with morphological changes in the nucleoli which become progressively reticulated and develop nucleolonema in which the different nucleolar components are intermingled (Szollosi, 1971 ; Fakan and Odartchenko, 1980). Raveh et a/. (1976) mentioned that the evolution of chick nucleolus during early embryogenesis is strikingly analogous to the behavior of the nucleolus at telophase in Ehrlich tumor cells as observed by Goessens and Lepoint ( 1974).
2. During Activation of Quiescent Cells in Plants Ultrastructural modifications of the nucleolus occur during activation of the cells of plant storage organs in Helianthus tuberosus (Jordan and Chapman, 1971), in Daucus carota (Jordan and Chapman, 1973), in Zea mays (Deltour and Bronchart, 1971; De Barsy et a l . , 1974; Deltour et a l . , 1979), and in Allium cepa (Risueno and Moreno Diaz de la Espina, 1979). The most striking modification is the movement of the NOR to a deeper position in the nucleolus. This NOR (called L-zone by some authors due to its
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a P
3
Fic. 19. Diagram of the organization of the forming and developing nucleolus in the mouse oocyte. (a) At mid-pachytene, the newly formed nucleolus is composed of a fibrillar center (C) surrounded by an electron-dense fibrillar layer. Later, a granular component appears in the most peripheral zone. At this stage, the fibrillar center and its surrounding electron-dense fibrillar layer contain the whole amount of rDNA which is located in the chromatin fibers emanating from the secondary constriction region of the bivalent. The surrounding electron-dense fibrillar layer corresponds to the rDNA transcription site. HC, Centromeric heterochromatin; Bi, synaptonemal complex of the bivalent. (b) The size increase of the nucleolus is mainly the consequence of extension of the electron-dense fibrillar component. The rDNA formerly compacted in the fibrillar center uncoils and is actively transcribed in the fibrillar component. (c) At late pachytene, the two nucleoli fuse together by their distal fibrillogranular regions. (d) At diplotene, the paired nucleolar chromosomes separate. The uncoiling of the DNA has progressed with extension of the fibrillar component of the anastomosing strands which constitute the nucleolenemal reticulum. Secondary fibrillar centers appear within the nucleolonema. Transcription of rDNA occurs in the whole electron-dense fibrillar component. The fibrillar centers are distinct entities where the rDNA remains locally compacted. The strands of the nucleolonemal fibrillar component containing uncoiled rDNA are in continuity with the layer of
NUCLEOLAR STRUCTURE
139
lightly staining characteristics and corresponding to FC) changes its position moving from the periphery to the fibrillar component of the nucleolus. At the same time, it becomes less clear and could disappear. The NOR observed in radicular cells of quiescent maize embryos by Fakan and Deltour (1981) is very similar to the FC described in various types of animal cells. In the maize however a typical NOR can be detected only in quiescent cells or during the first hours of germination. The fact that the NOR appears exclusively at the beginning of germination might be related to the low rate of pre-rRNA synthesis which is observed during this period. In more advanced germination stages when prerRNA is synthesized at a high rate no typical NOR or FC can be identified (Fakan and Deltour, 1981). In addition when maize seeds are transferred to 4"C, prerRNA synthesis is drastically decreased and the NOR reappears in the nucleolus (Crevecoeur et a l . , 1983). In quiescent embryo cells, the nucleolus is compact and predominantly fibrillar. With cell activation, it becomes more granular and vacuolated. The appearance of interstices is correlated with the beginning of uridine incorporation (Deltour and Bronchart, 1971). De Barsy et al. (1974) have quantified this process of nucleolar vacuolation in root cells of the embryo of Zea mays. They suggested that the appearance of interstices within the nucleolus is the result of a quick and significant loss of granular component in the nucleolus. 3. Seasonal Modijkations Seasonal variations have been observed both in the size and the structure of nucleoli in terminal buds of proximal branches and in root tips of Scoth pines, Pinus sylvestris L. (Kupila-Ahvenniemi and Hohtoloa, 1979, 1980). Marked differences are noticed between the samples collected between the end of October and February and the samples collected at the end of April. The nucleoli with fully intermingled fibrillar and granular components dominate in actively proliferating tissues collected in April whereas this type is almost lacking during the winter when the prominent feature is the loosening of the nucleolar structure with an individualization of the NORs which become visible as distinct often ball-like entities located at the surface of nucleoli. The other time of transition, the time of nucleolar reactivation in the spring, is first marked by a change in the nucleolar structure, by an increase in nucleolar size, and by the embedding of the NORs in the nucleolar material. The latter phenomenon resembles those described in the case of seed germination (Jordan and Chapman, 1971, 1973; Deltour et al., 1979). electron-dense fibrils surrounding every fibrillar center. The rDNA describes in the nucleolus a sinuous course where the fibrillar centers represent zones of inactivity of the ribosomal genes or an expression of reserve capacity. (From Stahl er al., 1980, Reproducrion, Nutrition et Dkveloppemenr, 20, 469-483.)
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4. Circadian Rhythm In the last few years, Pebusque and Seite (1980, 1981) and Pebusque et al. ( 1981a) have realized exciting ultrastructural quantitative investigations on rat sympathetic neurons. During the light period the nucleoli of sympathetic neurons appear reticulated. Several small and more or less round-shaped FC can be observed (Fig. 20). The latter are most often surrounded by a continuous layer of dense fibrillar component. In animals, sacrificed between 2400 and 0100 hours, the nucleoli retain their reticulated structure. The most striking feature is the occurrence of very large FC, some of them being positively giant (Fig. 21). Stereological data established that the mean nucleolar volume increases up to 1.7-fold from the light to the dark period and the mean volume of the FC increases up to 13-fold (Pebusque and Seite, 1980). This result does not confirm the opinion according to which the relative size of the nucleolar components would be stable for a given cell type (Hardin et a l . , 1970). Pebusque and Sei'te noted that such marked development of FC during the night temporally coincides with a high increase in the metabolic and physiologic activities of the rat. Rhythmic variations of nucleolar volume in neurons from the superior cervical ganglion were also studied in rats under artificial synchronization (Pebusque et a l . , 1981a). The results of this study demonstrate that the more of less pronounced variations in the nucleolar volume of the interphase nuclei are related to a diurnal rhythm. In fact such a circadian rhythm concerns the nucleolar components. Both mean volumes of FC and fibrillar, granular, and intersitial components reach their zenith at 0100 hours during the dark period (Pebusque and Seite, 1981). On the basis of these spectacular results, these authors were not able to define whether the rhythmic ultrastructural variations observed are correlated to the level of transcriptional activity of the nucleolus. However, the nucleoli of rat sympathetic neurons constitute an excellent model system for morphological and functional investigations of the nucleolus.
5 . During Cell Differentiation, Maturation, and Aging a. In Epithelial Cells. Epithelial cells of the small intestine arise by mitosis in the crypts of Lieberkiihn to the tips of the villi and are cast off into the lumen of the gut. During this time their nucleoli undergo complete reorganization from ~~~
FIG.20. Nucleolus in sympathetic neuron of superior cervical ganglion of an artificially synchronized rat sacrificed during the daytime at 1500 hours. Small-sized FC (arrows) are seen surrounded by the fibrillar component which takes the form of a well delimited network. X21,OOO. (From Pebusque and Sei'te, 1981, by permission of Academic Press.) FIG.21. Nucleolus of superior cervical ganglion neuron of an artificially synchronized rat sacrificed during the darkness at 0100 hours. A highly voluminous FC and small ones (arrows) can be seen. X25,OOO. (From Pebusque and Selte, 1981, by permission of Academic Press.)
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141
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the reticulated aspect in young cells of the crypts to the compact structure in old cells of the tips of the villi (Adamstone and Taylor, 1972). Nucleolus and its components were quantitatively analyzed in columnar cells at six levels of the rat jejunal epithelium corresponding to stages of cell migration from crypt base to villus top (Altmann and Leblond, 1982). In columnar cells of crypt base, the nucleolus is large and reticulated and actively produces rRNA. As cells migrate up the crypt, the nucleolus decreases in size, becomes spherical and compact, and ceases producing rRNA. Most nucleolar components also decrease in size. In contrast, the total area of the FC remains constant. Nucleoli in the cells of other renewing systems undergo similar evolution, e.g., during the formation of keratinocytes (Karasek et al., 1972), of sebaceous cells (Karasek et al., 1973), or in epithelial cells of the kidney (Adamstone and Taylor, 1977). b. In Avian Red Cells. Immature as well as mature avian red cells represent a very convenient model for studies on nucleolar changes and RNA synthesis in the course of cell differentiation and maturation (Likovsky and Smetana, 1975; Smetana et al., 1975; Smetana and Likovsky, 1978). Early erythroblasts are characterized by the presence of nucleoli with nucleolonema and compact nucleoli. Subsequent maturation stages are characterized by the presence of an increased number of ring-shaped nucleoli and micronucleoli. Since compact nucleoli or nucleoli with nucleolonema synthesize RNA and ring-shaped nucleoli or micronucleoli reflect a decrease or inhibition of this process, these observations indicate the gradual decrease and inhibition of nucleolar RNA synthesis in maturing chick embryo. Lava1 et al. (1981) have examined the final stage of the nucleolar cycle in chick embryo erythrocyte nuclei whose chromatin is almost completely repressed. These chick erythrocyte nuclei are at a final stage of morphological differentiation and do not exhibit developed nucleolar structures but resting nucleolar structures in the form of fibrillar component containing in some cases a few granules. No RNA synthesis is detected in the resting nucleolar structures. No RNA polymerase I activity is detected in isolated erythrocyte nuclei but when specific NOR silver staining is applied to chick erythrocytes, these resting structures are positively stained (Hernandez-Verdun et al., 1980b). This result indicates the persistence of specific nucleolar acidic proteins (Lischwe et al., 1979; Olert et al., 1979) in these inactive structures which might constitute part of the nucleolar matrix proteins. However, resting nucleolar structures could constitute a repressed state of the FC around which nucleologenesis takes place during nucleolar reactivation (Dupuy-Coin et al., 1976; Hernandez-Verdun and Bouteille, 1979) or during telophase (Goessens and Lepoint, 1974; Lepoint and Goessens, 1978; Hernandez-Verdun et al., 1980a). c. In Lymphocytes. The thymus is regarded as a suitable model for studies of nucleolar changes during cell differentiation and maturation because of a rapid
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turnover of lymphocytes (Potmesil and Goldfeder, 1972, 1973). The majority of the thymic lymphoblasts and prolymphocytes contains dense and trabeculate nucleoli rich in RNP and exhibits intensive incorporation of tritiated uridine. A fraction of prolymphocytes enters into a dormant state and these lymphocytes are morphologically characterized by ring-shaped nucleoli. These results have suggested gradual restriction of the nucleolar function with respect to RNA synthesis in the maturative process of the majority of thymic lymphocytes (Potmesil and Goldfeder, 1973). On the other hand, nucleoli of lymphocytes undergo a typical sequence of structural changes after stimulation by phytohemagglutinin (Tokuyasu et al., 1968; Wachtler et al., 1980, 1982). The lymphocytes have one ring-shaped nucleolus composed of a distinct FC surrounded by the dense fibrillar component and little granular material. With phytohemagglutinin stimulation, large nucleoli with nucleolonema devoid of FC are seen. These nucleoli are later transformed to give compact nucleoli containing one or more FC. These changes form ring-shaped nucleoli into nucleoli with nucleolonema and the changes from nucleoli with nucleolonema into ring-shaped nucleoli were interpreted by Smetana and Busch (1974) as reflecting either nucleolar activation or an inactivation, respectively. According to Wachtler et al. (1980) the disappearance followed by a reappearance of FC suggest that the latter may serve as a store of specific nucleolar proteins which may be used up and disappear from the nucleoli during the early periods of stimulation giving rise to nucleoli with nucleolonema. d. During Aging in Vitro. Changes occur in the ultrastructure of mouse fibroblasts during in vitro aging (Evans et al., 1978). The most striking difference between old and young nuclei lies in their nucleoli. Those of early passage cells have the typical structure. In old cells, many nucleoli have a condensed and segregated appearance. Clear zones (FC) also appear in the nucleoli. Autoradiography of cells labeled with tritiated uridine reveals a 70% reduction in nucleolar RNA labeling in late passage fibroblasts. Evans et al. ( 1978) have suggested that an age-related repression of nucleolar activity induces these nucleolar modifications. 6. In Heterokaryons As previously mentioned, no nucleolar structure can be detected in mature erythrocyte nuclei although a small number of resting structures called micronucleoli (Smetana et al., 1975) have been described. These nuclei can be introduced by cell fusion into thd cytoplasm of active host cell. They are then reactivated, the erythrocyte nuclei resume ribosomal RNA synthesis as newly nucleolar structures become morphologically identified (Dupuy-Coin et al., 1976; Hernandez-Verdun e f al., 1979). This nuclear reactivation model was investigated by means of quantitative analysis of the various aspects of the
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nucleolar structures as the course of reactivation progressed. This was combined with a three-dimensional approach and a high resolution autoradiography after tritiated uridine incorporation. The data allowed Hernandez-Verdun et al. (1979) to propose a model representing the steps of the nucleologenis (Fig. 22). In this model, the FC is the first part of the nucleolus which is observed and appears as the initiation structure for late stages. 7. Under Other Experimental Conditions The action of physical and chemical agents on nucleolar ultrastructure has been repeatedly discussed and surveyed (Simard, 1970; Simard et a l . , 1974). Consequently, we will only discuss some particular and recent data. In segregated nucleoli induced by actinomycin D in porcine thyroid cells, the FC can be seen in contact with chromatin pedicle which connects the FC to the dense chromatin associated with the nuclear envelope (Vagner-Capodano and Stahl, 1980). On the other side, the FC is always in continuity with the dense fibrillar component (Figs. 23 and 24). These preferential relationships, particularly conspicuous during nucleolar segregation, are spontaneously visible in germ cells during meiotic prophase (Jordan and Luck, 1976; Mime and Stahl, 1976). In general, FC become prominent when there is some abnormality of RNA synthesis (Fakdn, 1971; Love and Soriano, 1971; Daskal e t a l . , 1976; Goessens, 1976a). So, the appearance of FC in some nucleoli might be related to repression of rRNA synthesis and to a redistribution of RNP structures. This conception was confirmed by quantitative analysis performed in human fibroblasts under six different culture conditions. From this study, Jordan and McGovern (1981) concluded that in all the cells with lower activity there is a higher proportion of FC in the nucleoli which indicates that it is a structural or essential component in contradistinction to a product. On the other hand, it is generally accepted that biosynthesis of pre-rRNA requires the morphological integrity of the nucleolus and numerous attempts were made to relate different nucleolar constituents to specific functions involved in pre-rRNA transcription and maturation. But Connan et al. (1980a) realized the surprising observation that treatment of RSV-transformed cells with a-amanitin causes fragmentation of their nucleoli but does not inhibit RNA synthesis suggesting that the morphological integrity of nucleoli is not required for transcription of pre-rRNA. Persistence of transcription was also observed in the same cell type with concomitant nucleolar segregation induced by actinomycin D (Connan et a l . , 1980b). 8. In Cancer Cells Differences in nucleolar size, number, and morphology between normal and neoplastic cells have been the subject of much interest from both the point of
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4
t t FIG. 22. Schematic illustration of the nucleologenesis and uridine incorporation during the course of chick erythrocyte reactivation. ( 1 ) The fibrillar center (FC), which corresponds to the NOR and appears as the initiation structure for later stages, is associated with the condensed chromatin (CC). (2) In this prenucleolus, the fibrillar component is not labeled, due, for instance, to a low level of activity. (3) In such a prenucleolus, the fibrillar component close to the fibrillar center is labeled (black dots). It is the morphological substrate of RNA synthesis as first detected. (4)In this developed nucleolus, the fibrillogranular component is differentiated and nucleolar vacuoles appear. Uridine incorporation (black dots) is found in the fibrillar and fibrillogranular component of the nucleolus. (From Hernandez-Verdun and Bouteille, 1979, by permission of Academic Press.)
view of diagnosis of neoplasia and that of fundamental studies on nucleolar function. Many observations have demonstrated nucleolar abnormalities in malignant cells as compared to corresponding nonneoplastic cells (review in Busch and Smetana, 1970). These abnormalities are reflected in modified nucleolar size and shape and organization of nucleolar components as well as increased nucleo-
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lar biosynthetic activities. Recent immunocytological studies on a variety of animal and human malignant cells indicate that their nucleoli contain antigen(s) detected by antitumor nucleolar antibodies (Busch and Busch, 1977; Davis et al., 1979). Using indirect immunofluorescence, antisera to HeLa nucleoli were found to detect nucleolus-localized antigens in a wide range of tumor cells but not in most nontumor tissues (Busch er al., 1979, 1981; Davis et al., 1979; Smetana er al., 1979). Although the nucleolus has been found in the 1930s to exhibit morphological pleomorphism and therefore is useful in diagnosis of malignancy, these results recently summarized by Busch et al. (1982) provide the first satisfactory evidence for a difference in the nucleolar antigens of normal and tumoral cell nucleoli.
D. FUNCTIONAL SIGNIFICANCE Variations in the nucleolar ultrastructure during nucleologenesis has been studied in various experimental systems such as telophase in mitotic reconstituted cells, in germ cells during meiosis, and in heterokaryons. In all these studied systems, the morphological features are very similar. The first step is the differentiation of RNP fibrillar component around or in contact with FC. It is noticeable that the nucleolus in an early stage of development (e.g., in telophase) but also in a final stage (e.g., in late prophase or in erythrocytes) is characterized by the presence of FC. Since the FC is a part of the genome of the organisms, the persistence of the FC as compared with other parts of the nucleolus is easily understood. As the development of the nucleolus proceeds, the newly synthesized RNP fibrillar component is transformed into RNP granular component. The main finding that emerges from these observations is that the nucleolus shows a pattern of organization which is polarized with respect to the FC. When the ribosomal cistrons are fully active, the fibrillar and granular components are abundant and two typical nucleolar configurations can be observed: cells whose nucleoli exhibit well-defined FC and cells with no evident FC. From this review concerning the variations in the nucleolar ultrastructure, it clearly appears that in nucleoli with no evident FC the latter can be obscured or masked by transcriptional RNP products but can be visualized when there is some abnormality in rRNA synthesis or some disturbance in maturation or transport of ribosomal precursors. FIG.23. Typical nucleolar segregation in Ehrlich tumor cell. Granular component (g), fibrillar component (0, and FC are easily visible. FC is connected with the fibrillar component. x44,OOO. FIG.24. Nucleolar segregation in chick fibroblast. Whereas FC is not obvious in control cells (see Fig. 3). it becomes visible under this experimental condition. The fibrillar component (f) is still attached to the FC. Granular component (g). X74,OOO.
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Because no transcription occurs within the FC but is initiated at their periphery, Wachtler et al. (1980) pointed out that the correspondence between NOR and FC does not prove identity. This conception may not be correct since NOR may be transcriptionally inactive (e.g., in metaphase). In the same way, Knibiehler et al. (1982) concluded that the FC do not seem to be an essential structure of nucleoli since they are not visible in Drosophila KCo cell nucleolus. This conclusion is also wrong. In fact, when no FC is evident it is not necessarily absent. As previously discussed FC may be masked by RNP products and we must keep in mind that FC contain not only rDNA but also a quantity of proteins. These proteins are used for transcription. When the stock of proteins is high the FC can be conspicuous; when there is no reserve of these proteins the FC are not visible but the NORs are still present. In conclusion, all the results presented here can be interpreted as follows. The shape, the size, and the morphology of the nucleolus are determined by the balance between the rate of production and stockage of the precursors of ribosomal RNP by the NORs and the demand for ribosomal RNP by the cytoplasm.
IV. Exportation of Nucleolar RNP The morphological manifestations of rRNP particles transfer from the nucleolus to the cytoplasm can be visualized at three steps: the nucleolar interstices, the contacts between nucleoli and nuclear envelope, and the nuclear bodies. As previously discussed, nucleolar interstices can play a role in the storing and the transport of preribosomal precursors (Deltour and Bronchart, 1971; De Barsy et al., 1974; Moreno Diaz de la Espina et al., 1980). In addition recent studies have demonstrated the existence in nucleolar interstices of perichromatin-like granules in cultured cells under conditions of impaired rRNA maturation. This special kind of perichromatin granules was considered as storage or degradation forms of preribosomal RNP (Puvion et al., 1979; Puvion and Moyne, 1981). It is well known that the nucleolus of eukaryotic cells are often found during interphase in the vicinity of the nuclear envelope (for review see Bouteille et al., 1982). In certain particular cell types, e.g., in human endometrium (Clyman, 1963, Terzakis, 1965), in Novikoff hepatoma (Babai et al., 1969), and in crayfish oocytes (Kessel and Beams, 1968), nucleolar channel systems or nucleolar canalicular structures have been observed. They consist of invaginations of the inner nuclear membrane. These formations appear to be different from the cytoplasmic inclusions which are always lined by a double membrane. Serial sectioning and high voltage electron microscopic techniques have shown that the channel system of the endometrial glandular cells is a dynamic structure with a cyclical pattern of evolution and disappearance and with a varying association with nuclear membrane and nucleolus (More and McSeveney, 1980).
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As suggested by several authors, these membranous nucleolar components may play a role in an intranucleolar transfer of the rRNA precursor molecules following their transcription. Recent quantitative ultrastructural analysis of nuclear organelle distribution in random sections provided evidence that in the cell types investigated, the nucleoli are always bound to the nuclear envelope either directly or through a nucleolar channel (Bourgeois et al., 1979). This spatial relationship was confirmed by the analysis of stereopairs of nuclei by computer reconstruction of bidimensional data from serial sections (Dupuy-Coin et al., 1982). Nuclear bodies have been observed in various cell types and a nucleolar origin of the nuclear bodies (Fig. 25) has been reported by several authors (review in
FIG. 25. In nucleus of porcine thyroid cell cultured in the presence of TSH, the nucleolus presents a large encapsulated bud attached to the nucleolar surface by a pedicle. These buds separate from the nucleolus and become characteristic nuclear bodies. (From Vagner-Capodano et a / ., 1980, by permission of Academic Press.)
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Bouteille et al., 1974; Vagner-Capodano et al., 1980). However, every structure which has been named nuclear body does not have a nucleolar origin (Le Goascogne and Baulieu, 1977). Do the nuclear bodies play a role in rRNP transport? The role of nuclear bodies in the transport of nucleolar RNP toward the cytoplasm is suggested by autoradiographic experiments performed by Vagner-Capodano et al., 1982).
FIG. 26. Hypothetical diagram of nucleolus. In this diagram, the shape and the number of the fibrillar centers have not been considered. In the middle of the model: one fibrillar center containing some fibrils of rDNA. The transcription of the rDNA occurs only at the periphery of the fibrillar center and is visualized as dense fibrillar component. Around the fibrillar center and the fibrillar component: granular component, nucleolar interstices, and some clumps of intranucleolar chromatin which penetrate from the nucleolar-associated chromatin into the nucleolar body even to the fibrillar center. (From Goessens and Lepoint, 1979, by permission of SocMtb Francaise de Microscopie Electronique.)
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V. General Conclusion It can be concluded that the nucleolar chromosomes have specialized regions (the NORs) which not only contain rDNA but also proteins involved in pre-rRNP synthesis and maturation. When these genetic sites are transcriptionally active, they appear as FC surrounded and sometimes obscured by the products of the transcription and the associated processing (the RNP fibrillar and granular components); the nucleoli can then be well identified. In favorable circumstances, the NOR of the interphase nucleolus appears as FC surrounded by fibrillar component. The FC and its associated fibrillar component constitute the nucleolar core from which Miller and Beatty (1969) have extracted the DNA molecules with repeated cistrons coding for ribosomal RNA and visualized as “Christmas trees” (Fig. 26). The morphological characteristics of the nucleoli reflect the intensity of ribosome precursor molecules formation. When rRNA synthesis stops in metaphase no nucleolus, as an individual nuclear entity, is discernible. The nucleolus reappears with the reinitiation of transcription. This nucleolar cycle is schematically summarized in Fig. 27. Thus, as previously mentioned, “the nucleolus is an
FIG. 27. Nucleolar cycle. In metaphase the FC persists and corresponds to the secondary constriction of the nucleolar chromosome. This chromosomal region contains uncondensed chromatin and proteins. When rDNA becomes transcriptionally active again, the fibrillar component first reappears around the FC. Shortly after the granular component is also present and the nucleolus acquires its interphase ultrastructure.
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active genetic site on a chromosome and has much in common with a puff or Balbiani ring on a giant polytene chromosome or a loop in a lampbrush chromosome. ”
ACKNOWLEDGMENTS The author is grateful to the “Fonds de la Recherche Scientifique MCdicale” for its financial support and is much indebted to Mrs. D. Germai, Mrs. D. Scabers, and Miss F. SkivCe for their technical assistance and Mrs. P. Dubois for the preparation of the manuscript.
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INTERNATIONAL REVIEW OF CYTOLOGY. VOL. 8 7
Membrane Heterogeneity in the Mammalian Spermatozoon W. V . HOLT Gamete Biology Unit, Department of Reproduction, Institute of Zoology, Zoological Society of London, London, England I. 11.
111.
IV.
V. VI.
VII.
Introduction . . . . . . . . . . . . . . . . . . . . . Summary of Sperm Structure and Function.. . . . . . . . . . . . . . . . . . . . Structural Differentiations in Sperm Membranes . . . . . . . . . . . . . . . . . A. The Sperm Head B. The Sperm Tail . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Developmental Aspects of Mammalian Sperm Membranes . . . . . . . . Biogenesis of Sperm Membranes . . . . . . . . . . . . Epididymal Contribution to Sperm Surface Heterogeneity . . . . . . . . . Functional Aspects of Membrane Heterogeneity in Spermatozoa. . . . A. Sperm Surface Modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Membrane Heterogeneity and Fusion Interactions . . . . . . . . . . . . Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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160 161 161 168 171 171 179 182 182 I83 188 189
I. Introduction Mammalian spermatozoa, after leaving the testis, are subjected to a sequence of environmental changes which involve passage through the epididymis, temporary storage, then further transport through the female reproductive tract. Testicular spermatozoa, or spermatozoa isolated from proximal regions of the epididymis, are unable to participate in fertilization, but presumably as a result of their epididymal transport, those from the cauda epididymidis or from an ejaculate can be induced to fertilize an egg. Use of the term “induced” is important in this context, as it must be recognized that epididymal transit confers only the potential to participate in fertilization, further modifications being necessary before gamete fusion will occur. During the past two or three decades numerous studies have aimed both to recognize the nature of the developmental changes undergone by spermatozoa, and to assess their physiological significance. In many cases these studies have tended to uncover further complexities, especially when species diversity is considered, however, certain findings of underlying significance have transcended the species barriers to indicate some of the factors involved in sperm development. I59 Copyright 0 1Y84 by Academic Press. Inc.
All rights of reproduction in any form reserved. ISBN 0-12-3644x7-Y
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Ultrastructural studies have shown that the sperm cell is surrounded by a plasma membrane, the interface between the cytoplasmic and external environments. Clearly, changes in the external environment are likely to affect plasma membrane properties such as permeability or stability, and thus influence events within the cell. As the main events in gamete union extensively involve membrane fusion, preparation of sperm membranes for this process, either by preventing premature membrane destabilization or by conferring the capacity to undergo triggered destabilization when necessary, is likely to be a crucial feature of sperm development. Detailed structural and cytochemical studies have shown conclusively that the plasma and acrosomal membranes are subdivided into discrete zones, or domains. Individual domains appear to be of unique composition or organization, factors which probably promote disparate physical properties and functions. Aspects of sperm membrane heterogeneity have been reviewed previously by Bedford and Cooper (1978), Koehler (1978b), and Friend (1982a,b). It is the purpose of this article to describe the salient points of the highly developed membrane heterogeneity encountered in spermatozoa. Attention will be focused mainly on the plasma and acrosomal membranes as these are most clearly involved in fusion interactions, before and during fertilization. Comparative aspects will be considered where relevant, as general features of underlying importance are unlikely to be limited to individual species.
11. Summary of Sperm Structure and Function
Spermiogenesis moulds the spermatozoon into the highly specialized shape required for reaching and penetrating the egg; upon completion of this initial stage of development the main structural characteristics of the sperm cell are readily apparent. In essence, the spermatozoon consists of two functionally independent compartments, the head and the tail. The head contains all the genetic material, but also carries an apparatus, the acrosome, for penetrating the egg investments and fusing with the vitelline membrane. The tail is mainly concerned with propagating the flagellar wave, thereby assisting in egg penetration; two morphologically distinct regions of the tail are readily apparent, the middle piece with its mitochondria1 spiral, and the more posterior regions of the flagellum which consist largely of axial filaments and associated fibers. The whole cell is surrounded by a plasma membrane, but the acrosome, nucleus, and mitochondria are also individually encapsulated by their respective membranes. For a more detailed account of sperm morphology, see the review by Fawcett (1975). As a phenomenon of widespread occurrence among mammals, the maturation of spermatozoa in tandem with their progression through the epididymal duct is
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now extensively documented. The increasing fertility of spermatozoa during epididymal transit has been noted in a number of species (guinea pig, Young, 1931; rat, Blandau and Rumery, 1964; rabbit, Bedford, 1966, 1967; OrgebinCrist, 1969; boar, Holtz and Smidt, 1976; ram, Fournier-Delpech et al., 1977; Hammarstedt ef al., 1982; human, Hinrichsen and Blaquier, 1980; marmoset, Moore, 1981) and the correlation of this effect with an increased capacity for progressive motility has similarly been noted (Blandau and Rumery, 1964; Fray et al., 1972; Gaddum, 1968; Fournier-Delpech et al., 1977). Earlier observations had established that the acquisition of fertility was also correlated with the migration of the cytoplasmic droplet, a bead of residual cytoplasm, away from its position at the neck of the cell, toward the distal part of the middle piece (Branton and Salisbury, 1947; Rao and Hart, 1948; Mukherjee and Bhattacharya, 1949; Hancock, 1957). Microscopic studies of spermatozoa at different stages of maturation revealed that morphological changes were also associated with the increased fertility, there being a tendency for the acrosome to decrease in size (Mukherjee and Bhattacharya, 1949; Bedford, 1963a, 1965; Jones, 1971). In some species, notably the guinea pig, dramatic changes in acrosomal shape were observed to occur during epididymal transit (Fawcett and Hollenberg, 1963). Prior to fertilization, but following sperm storage in the female reproductive tract for a number of hours, dependent on species, the outer acrosomal and plasma membranes fuse, causing the release of acrosomal enzymes and the exposure of a new membrane surface, the intraacrosomal aspect of the inner acrosomal membrane. This process has been termed “the acrosome reaction.” Attachment of sperm to the zona pellucida, followed by adhesion to and fusion with the vitelline membrane, seems to involve two specialized regions of the sperm head surface, the equatorial segment of the acrosome and the postacrosoma1 region of the sperm head. Both of these remain covered by plasma membrane upon completion of the acrosome reaction, implying strongly that these areas possess unique fusogenic and surface interactive properties.
111. Structural Differentiations in Sperm Membranes A. THE SPERMHEAD
When considering the format for this article it was evident that little purpose would be served by attempting to reiterate here all the complex, fine structural details of sperm membranes, which have been amply described in a number of freeze-fracture and surface replica studies. Illustrated accounts of sperm membrane structure in individual species are listed in Table I and an overall view of a typical mammalian sperm head is presented in Fig. 1. Instead, attention will be
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TABLE I REFERENCES TO FREEZE-FRACTURE, FREEZE-ETCHING, A N D SURFACE REPLICASTUDIES IN MAMMALIAN SPERMATOZOA FROM DIFFERENT SPECIES Species Guinea pig Rat Mouse Bull Ram Opossum Rabbit Human Water buffalo Hamster Boar Vole Degu Nonhuman primates Baboon Macacque Rhesus monkey
Referencesca
I , 2, 10-17, 23, 25, 34, 35. 14, 15, 25, 29, 34, 35, 39. 34, 38. 4, 6, 20, 35, 36. 5, 6. 31, 32. 8, 21, 25-27, 30, 35. 18, 21, 22, 33. 24 19, 34, 35. 37, 40, 41. 28. 3, 35. 7 9, 33. 35.
“ 1 , 2, Bearer and Friend (1980, 1982); 3, Benios et al. (1978); 4, 5, Bradley et al. (1979, 1980); 6, Bustos-Obregon and Flechon (1975); 7, Chretien et al. (1977); 8, Flechon (1974); 9, Flechon and Hafez (1975); 10-13, Friend (1977, 1980, 1982a.b); 14, Friend and Fawcett (1974); 15, Friend and Rudolf (1974); 16, Friend and Heuser (1981); 17, Friend et al. (1977); 18, Jamil et al. (1982); 19, Kinsey and Koehler (1976); 20-28, Koehler (1966, 1970b, 1972, 1973a,b, 1975a,b, 1976, 1978a); 29, Mortimer and Thompson (1976); 30, Motta and Van Blerkom (1975); 31, Olson et al. (1977); 32, Olson (1980); 33, Pedersen (1972); 34, 35, Phillips (1975, 1977); 36, Plattner (1971); 37, Russell et al. (1980); 38, Stackpole and Devorkin (1974); 39.40, Suzuki and Nagano (1980a,b); 41, Suzuki (1981).
FIG. 1. Freeze-fracture replica of an ejaculated spermatozoon from a Blackbuck, Antelope cervicapra. showing a general view of the head region. Distinct regions of the fractured plasma membrane are visible in this preparation, reflecting mainly the topology of the underlying structures. Thus the acrosomal region displays a typically undulating but smooth plasma membrane; the equatorial segment (ES) is characterized by an array of longitudinally directed ridges, and the postacrosoma1 region (PAR) is flat and smooth at this magnification. A collar of striated rods encircles the anterior neck region of the spermatozoon. The dashed lines in this illustration indicate the demarcations between the three main membrane regions. x 12,850. FIG. 2. Freeze-fracture micrograph of part of the head of a ram spermatozoon, cleaved in the region of the equatorial segment. The inner (IAM) and outer (OAM) acrosomal membranes display ordered, periodic structure which is emphasized in this illustration by the use of dashed lines. The nuclear membrane (NM) displays no such ordered structure; irregularly fractured faces such as this are a frequent observation. X75.000.
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drawn to the existence, properties, and possible functions of various membrane domains, consideration being given to the modifications they undergo during epididymal transit, capacitation, and fertilization. Light microscopists have long recognized that mammalian sperm cells are differentiated into various discrete regions; for example, Marza (1930) studied the distribution of lecithin and plasmalogens, among other components, in spermatozoa from eight mammalian species, and published color illustrations which clearly show the distinctions between the acrosomal and postacrosomal regions of the sperm head. The equatorial segment is also evident in some cases. Later work has confirmed and greatly extended these findings, particular success being achieved by the use of freeze-fracture and freeze-etching in combination with the transmission electron microscope. Koehler ( 1966) pioneered such freeze-etching studies and quickly demonstrated marked differences in the two-dimensional appearance of various membrane domains in bull spermatozoa. The sperm head plasma membrane was readily recognized as being divided into two main regions, one overlying the acrosome while the other covered the postacrosomal region; a transition zone representing the equatorial segment was also apparent. 'The features of the equatorial segment were unclear at that time, but because of its importance in fertilization (see, Bedford and Cooper, 1978) detailed studies of this structure have since been performed. Phillips (1977) examined the surface of the equatorial segment in replicas, and found, in eight mammalian species, that it was covered by rows of hexagonally packed particles. While in the bull, rabbit, hamster, guinea pig, degu, and rhesus monkey these particles were restricted to the equatorial segment, in the mouse and rat they covered much of the outer acrosomal membrane. Phillips (1977) interpreted these results as showing a degree of analogy between the equatorial segment in some species, and the more extensive region of the outer acrosomal membrane in the mouse and rat. Patterning of the outer acrosomal membrane in the mouse had previously been noted in freeze-fracture studies (Stackpole and Devorkin, 1974), and Friend and Fawcett ( 1974) similarly reported patterning of the equatorial segment in the guinea pig spermatozoon. These authors also noted, however, that the hexagonal patterns of the outer acrosomal membrane were not limited to the equatorial region. Hexagonal configurations of particles in the equatorial segment of the boar spermatozoon were noted by Suzuki (1981), who found that the center-tocenter spacing of these particles, 17 nm, matched the figure reported earlier (Phillips, 1977). In the boar, the remainder of the acrosomal membrane displayed a random array of intramembranous particles; similar results were found in this (Russell et al., 1980) and other species (rabbit, Koehler, 1970b; bull, Plattner, 1971; human, Koehler, 1972). Russell et al. (1980) investigated the structure of the equatorial segment in
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more detail, using freeze-fracture and degradative procedures. They showed that a series of intermembranous bridges linked the inner and outer acrosomal membranes in this region, and it was suggested that such bridges were represented in freeze-fracture preparations by the hexagonally packed particles; the data obtained supported an earlier proposal (Bedford et al., 1979) that the bridges acted to stabilize the structure of the equatorial segment. Linear patterns of the inner and outer acrosomal membranes of the equatorial segment are visible in freezefractured ram spermatozoa (Holt, unpublished observation; see Fig. 2). In the plasma membrane overlying the anterior of the equatorial segment, Friend and Fawcett (1974) observed by freeze-fracture a characteristic pattern of obliquely oriented rods, evident as alternating elevations and depressions. In thin sections these rods appeared as regularly spaced regions of electron density. Similarly serrated structures were earlier observed in the rabbit (Koehler, 1970b), and they are a conspicuous feature of spermatozoa in some ungulates (Fig. 3). Although their function is unclear, their positioning at a focal point for membrane fusion during fertilization, together with their occurrence among different species suggests that they fulfill some specific role. One interesting but highly speculative possibility is that the rods may function during gamete fusion by presenting the oolemma with a number of microdomains whose radius of curvature is significantly smaller than elsewhere on the sperm surface. Theoretical computations led Poste (see Poste and Allison, 1973) to suggest that close (molecular) apposition of membranes was possible only if regions of sufficiently small radius of curvature, less than 0.1 pm, were involved. The structures in question might meet such a requirement as they are approximately 0.1 pm in width and appear semicircular in profile. One of the most striking features reported by Koehler (1966) in his investigations of bull spermatozoa, and subsequently found in the water buffalo (Koehler, 1973b), rabbit (Koehler, 1970b; Flechon, 1974), ram (Bradley et al., 1980), and boar (Suzuki, 1981), was the arrangement of parallel striations situated near the base of the sperm head, immediately anterior to the posterior ring. These structures, together with the annulus, which delineates the boundary between the middle and principal pieces of the flagellum, appear to be highly stable membrane specializations, capable of effectively isolating contiguous membrane domains as well as fulfilling purely structural roles. The relatively smooth appearance of the acrosomal and postacrosomal regions of the plasma membrane, with their randomly dispersed intramembranous particles, in ejaculated bull (Koehler, 1966), ram (Bradley et al., 1980), rabbit (Flechon, 1974), and boar spermatozoa (Suzuki, 1981) contrasts with the highly organized appearance of the guinea pig sperm plasma membrane (Friend and Fawcett, 1974). Here, regularly spaced, contiguous circles, a feature of the glycocalyx, were seen in tangential sections of the sperm head. In freeze-fracture preparations, the anterior region of the sperm head was divided into separate
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plaques of hexagonal patterns, interspersed by areas of randomly dispersed particles and pits. This difference becomes of more interest when considered in the light of two studies (Suzuki and Nagano, 1980a; Suzuki, 1981), in which epididymal sperm maturation was examined by freeze-fracture in the rat and the boar. Regular geometric arrays were a transient feature of the acrosomal plasma membrane in the boar, being evident only as the cells passed through the distal region of the caput epididymidis. Hexagonal arrays of membrane particles, distinct from the previous geometric patterns, then developed in certain areas of the plasma membrane as the spermatozoa approached the cauda epididymidis only to disappear almost completely upon ejaculation. These hexagonal arrays were initially restricted to the marginal thickening of the acrosome, but extended to the postacrosomal region prior to their dispersion. The study of rat spermatozoa yielded somewhat similar results; while no regular patterns were discernible in spermatozoa from the initial segment and proximal caput epididymidis, plaques of parallel rows of 9-nm particles appeared as the cells progressed past this region of the duct. Later, when the spermatozoa reached the proximal cauda epididymidis these patterns largely vanished. Although evidence for a limited amount of hexagonal patterning in the rat sperm plasma membrane had been presented earlier by Friend and Fawcett (1974), Suzuki and Nagano (1980a) suggested that this may have been artifactual, caused by the mixing of mature with immature spermatozoa, during sampling. There is a case for believing that some of the observed membrane patterning is due to influences exerted by the glycocalyx. Friend (1980, 1982b) pointed out that the quilt pattern of guinea pig spermatozoa is diminished by treatments which remove the outer cell coat, for example, short incubation in Tyrode’s solution. Similarly, Suzuki and Nagano (1980a) found a correlation between the disappearance of the regular patterns of particles in the rat sperm plasma membrane, and the loss of the glycocalyx, which, in the rat, occurs while the cells are still in the epididymal duct. FIG. 3. Freeze-fracture electron micrograph of a Blackbuck spermatozoon showing a detail of the plasma membrane in the region of the equatorial segment. The most conspicuous structures are the regularly arranged series of elevations and depressions which lie at the anterior end of the equatorial segment. [The anterior direction (AR) is indicated on the micrograph.] In this species, numerous intramembranous particles are a feature of the sperm head plasma membrane. X80,OOO. FIG. 4. Freeze-fracture electron micrograph of part of a human spermatozoon, showing a cleavage plane through the neck region of the cell. An array of nucleopores is visible within the posterior nuclear membrane, obscured in places by the basal plate. The posterior ring (PR) separates these structures from more anterior regions of the nuclear membrane, and also from the postacrosomal region of the plasma membrane. X46,450.
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Fawcett (1975) proposed that regularly arranged geometric arrays in the plasma membrane were unnecessary for the occurrence of the acrosome reaction. The opposite may indeed be true, since the presence of stable, crystalline membrane domains would be indicated by these patterns, an unlikely prelude to such a membrane fusion event. Such membrane fusion phenomena are generally regarded as a consequence of membrane destabilization; this point will be discussed further in a later section of this article. Anterior to the nuclear ring, the nuclear membrane fractures irregularly, the plane of fracture frequently crossing from one leaflet to the other (Friend and Fawcett, 1974) (Fig. 2). Consequently, few features of interest have been noted here, although this uncommon phenomenon is itself indicative of unusual membrane properties. During spermatogenesis, the nuclear membrane is often seen in close association with chromatin, a relationship which presumably affects its mechanical characteristics. More interesting structures are found within the nuclear envelope posterior to the nuclear ring. Prominent in this region are the numerous nucleopores associated with that part of the membrane sometimes known as the redundant nuclear envelope (Fig. 4) (Friend and Fawcett, 1974; Stackpole and Devorkin, 1974; Mortimer and Thompson, 1976). Although the functions of these pores are unknown, attention should be paid to their possible role in nuclear-cytoplasmic communication. This is probably most important during spermatid elongation and nuclear condensation when the pores become concentrated in the posterior region of the nuclear membrane (Fig. 5); it is also relevant to note that Courtens and Loir (1975a,b) showed that spermatid development in the ram was accompanied by the orderly removal of somatic type histones from the nucleus, apparently via the posterior region of the nuclear membrane.
B. THE SPERMTAIL A comprehensive review of structural specializations within the membrane of the guinea pig sperm tail has been presented by Friend (1982b). This description is for the most part applicable to spermatozoa from other eutherian mammals; however, some points of particular interest from a comparative viewpoint will be considered here. 1. The Middle Piece
One of the most remarkable species differences in the structure of the sperm tail concerns the variation in membranous particle distribution over the middle ~~
~~
~~~
~
~~
~~~
~
~
FIG.5. Transmission electron micrograph of an elongating spermatid from the common marmoset, Callithrix jucchus. At this stage of maturation the nuclear ring (arrows) is present as a thickened specialization of the plasma membrane. From this stage onward the nuclear membrane posterior to the nuclear ring is occupied by numerous nucleopores, some of which are indicated here by open circles. x 18,750.
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piece of the opossum spermatozoon, when compared to eutherian mammals. Olson et al. (1977) demonstrated parallel, longitudinally arranged rows of aggregated membranous particles in the opossum sperm middle piece. The particles became aligned during epididymal transit (Olson, 1980). In thin sections the plasma membrane appeared scalloped, presumably reflecting the transverse sectioning of the rows (Olson and Hamilton, 1976; Olson et a l . , 1977). Negatively charged groups of the membrane surface showed similar alignment into rows (Bedford and Cooper, 1978), perhaps representing the exposed, glycosidic regions of these protein aggregates. Whether this pattern can be considered representative of marsupials as a group remains to be seen; however, it clearly differs from the membrane organization seen in placental mammals where no such parallel rows are seen. Instead, strands of particles, parallel to the pitch of the mitochondrial helix, cover the surface of the middle piece. Since these become randomized during capacitation (Friend, 1977) it would be of interest to see whether similar dispersion of the parallel rows of particles in the opossum also occurs prior to fertilization. Examination of deep-etched specimens of guinea pig spermatozoa has recently revealed an unsuspected array of highly ordered patterns (Friend and Heuser, 1981). The outwardly facing aspect of the outer mitochondrial membrane, i.e., that facing the plasma membrane, possessed randomly dispersed rods, each composed of two to four 70- to 80-A particles; over the inwardly facing aspect, however, the rods were regularly arranged in the form of a parallel series of ladders. The significance of these findings is obscure at present, but these ordered arrays might represent vectorial systems of enzymatic or transporting activities. The cytoplasmic droplet is an interesting but enigmatic feature of the middle piece. According to Friend (1982a,b) it fluoresces with filipin, stains with adriamycin, a probe for anionic lipids, and incorporates merocyanin-s540. As merocyanin is thought to be a probe for regions of membrane fluidity (Schlegel et al., 1980), these findings suggest that the cytoplasmic droplet plasma membrane is specialized for processes such as fusion and secretion. This is a provocative suggestion which must encourage new research into the function of this organelle, previously regarded merely as a redundant bead of cytoplasm. 2. The Principizl Piece The annulus divides the middle piece from the principal piece; here, as discussed previously, a potential barrier to the lateral migration of membranous components is provided by the rows of large particles anchored to an underlying, submembranous collar. A region possessing micron long lines of 15-nm E-face intramembranous particles separates the annulus from the region where the “zipper” begins. This is a double row of 9-nm particles which overlies fiber 1 of the axoneme. Friend and Fawcett (1974) described the zipper in guinea pig and rat spermatozoa, but it
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has subsequently been identified in other species. One function of the zipper is to hold the plasma membrane tightly against the fibrous sheath; apart from this, other functions, presumably fundamental because of its wide occurrence, have yet to be found. Degradative (Friend et al., 1979) and lectin-binding studies (Enders et al., 1981) have established that the particles constituting the zipper are integral tranmembranous proteins. They include four which dissolve in Triton X-100, and which possess exposed sugar residues.
IV. Developmental Aspects of Mammalian Sperm Membranes
BIOGENESIS OF SPERMMEMBRANES In common with other biological membranes, those of spermatozoa conform to the principles of the “fluid-mosaic’’ model of membrane structure (Singer and Nicolson, 1972). Thus the membrane is envisaged as a phospholipid bilayer in which integral protein molecules are intercalated; the proteins possess some ability to move two dimensionally, unless constrained in some way. That sperm membranes conform to this model is important for any consideration of posttesticular sperm development, since reference to studies of membrane synthesis and function in other cells would appear to place certain limits upon the way in which processes such as sperm maturation and capacitation may be viewed. It is thus instructive to consider whether the regional differentiation of the sperm surface might originate during spermatogenesis. Although the plasma membrane of the spermatozoon undergoes compositional changes during maturation and fertilization, it is likely that these modifications, which involve the appearance and disappearance of antigenic determinants, lectin-binding abilities, and charged groups, do not involve the cell in de novo synthesis of new integral membrane proteins. Currently, studies of the biosynthesis of secretory and integral membrane proteins in vitro, suggest that assembly of such proteins occurs in tandem with the extrusion of the lengthening peptide chain across the membrane. This model for the synthesis and secretion of proteins, known as the “signal hypothesis” (for reviews, see Blobel et al., 1979; Kreil, 1981), proposes that free ribosomes are initially involved in the synthesis of a short oligopeptide, the signal sequence, composed of about 25 amino acids. Recent work (Walter et al., 1981; Walter and Blobel, 1981a,b) has shown that a specific protein, termed “signal recognition protein” (SRP), recognizes and interacts with the signal sequence, and participates in the subsequent attachment of ribosomes to “translocation competent sites” on the endoplasmic reticulum membrane. Protein synthesis is then resumed, and the resultant polypeptide can either be partially retained by the membrane, or released on the luminal side for eventual secretion. The absence of protein synthesis in mammalian spermatozoa, except for lim-
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ited mitochondrial activity (Bragg and Handel, 1979), would preclude the operation of such a cotranslational system of membrane protein insertion. In mature spermatozoa, and probably during the later stages of spermiogenesis, no new integral membrane proteins could therefore be produced in this way. In the light of this argument it would be reasonable to conclude that integral membrane proteins of spermatozoa are synthesized during and before spermiogenesis, when the cellular apparatus for protein synthesis is still active. Some mitochondrial and chloroplast proteins are incorporated into membranes once translation has been completed but these appear to be exceptional. A recent study (Sabatini et a l . , 1982) has described the first case of a eukaryotic transmembrane polypeptide, the catalytic subunit of sodium, potassium ATPase, which reaches the plasma membrane without previous cotranslational insertion into the endoplasmic reticulum. Sabatini et al. (1982) commented that specific mechanisms might exist for the thermodynamically unfavorable uptake of a protein into a lipid bilayer. Identification of such mechanisms would be of particular relevance to epididymal function, where a potential exists for the uptake of epididymal secretions. Membrane lipids, like the proteins, are synthesized in the endoplasmic reticulum (for review, see Thompson, 1980) which can thus be regarded as the source of new membrane components. Transport of the new membrane components to their functional sites is a continuous process involving the participation of membrane vesicles, which are constantly budding away from, and fusing with, the endoplasmic reticulum, Golgi apparatus, and plasma membrane. These vesicles therefore represent a mode of transport by which membrane components can be recycled between organelles, to be inserted, removed, or modified as required. Again, spermatozoa possess none of this apparatus, which upholds the proposal that sperm membrane proteins are synthesized during spermatogenesis, when these components are present. This concept has recently been reviewed by Pearse and Bretscher (1981). 1. Biogenesis of the Plasma Membrane
Morphological examination of the various cell types involved in spermatogenesis makes it apparent that the origin of the sperm plasma membrane can be traced back continuously, through the various stages of spermatid elongation. This is particularly true of that portion of the sperm plasma membrane which covers the sperm head (Fawcett et a l . , 1971; Baccetti et a l . , 1978). In turn, the plasma membrane surrounding the young spermatid possesses continuous identity with that of its predecessors, the primary and secondary spermatocytes. Ultrastructurally this is readily apparent, more so when it is considered that features such as the “junctional” or “ectoplasmic” specializations (Russell, 1977) of the spermatocyte/Sertoli cell interface, which develop during spermatocyte differentiation, persist until late in spermiogenesis.
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Whether or not the flagellar plasma membrane is directly derived from that of the spermatocyte is a matter of contention. Fawcett et al. (1971), reviewing the events of spermiogenesis, regarded the formation of the flagellar canal as a consequence of the caudal flow of cytoplasm which accompanies spermatid elongation. It would appear implicit in this view that since the anterior limit of the flagellar canal is determined by the position of the annulus, membrane proliferation should occur as axonemal elongation progresses. The site(s) of assembly of such new membrane components is at present unclear. The scheme of flagellar plasma membrane biogenesis proposed by Fawcett et al. (1971) is somewhat at variance with the observations of Sapsford et al. (1967, 1969) for the bandicoot. These authors noted that during the earliest stages of spermiogenesis the axonemal complex was in direct contact with the spermatid cytoplasm. This finding is inconsistent with the view that the flagellar canal is formed by a caudal flow of cytoplasm, since no such direct contact would thus be possible. Sapsford et al. (1967, 1969) further observed the alignment of membranous vesicles parallel to the axial filaments, and proposed that the fusion of such vesicles would lead to flagellar canal formation. Similar findings have been reported by Baccetti et al. (1978) in human, bull, and rat spermatids. These authors made the additional suggestion that the membranous vesicles were derived from the Golgi apparatus, since, like the Golgi elements, they exhibited thiamine pyrophosphatase activity. Although these proposals seem to be at variance, the truth probably represents a compromise between them, especially as Sapsford et al. (1967, 1969) observed that vesicle fusion was completed once the centrioles were situated at the abaxial pole of the nucleus. As this process occurs early in spermiogenesis, caudal flow of cytoplasm would probably follow this stage. a. Immunological Aspects of Plasma Membrane Biogenesis. A number of recent immunological studies have lent support to the view that although the plasma membrane persists as an intact structure during spermatogenesis and spermiogenesis, various classes of proteins are inserted into the membrane in an ordered sequence. Membrane properties and characteristics would therefore be expected to change during differentiation. Considerable synthesis of new membrane antigens, presumably mainly glycoproteins and glycolipids, takes place once the leptotene and early zygotene primary spermatocytes move past the Sertoli cell junctions into the adluminal compartment of the testicular tubule. Further elaboration of new proteins, both membrane and cytoplasmic (Kramer, 1981; Kramer and Erickson, 1981; O’Brien and BellvC, 1979), continues and increases as spermiogenesis progresses, despite the apparent lack of messenger RNA synthesis in the middle to late classes of spermatids (Monesi, 1965, 1971; Moore, 1971; Kierszenbaum and Tres, 1975; Soderstrom and Parvinen, 1976). Certain of the antigens detected by antisera against isolated pachytene spermatocytes (Millette and Bellvt, 1977; Tung and Fritz, 1978) or type B sper-
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matogonia (Millette and BellvC, 1980) were found to be masked or selectively removed after being expressed for a short period. Some membrane proteins, present in pachytene spermatocytes or round spermatids, and characterized by Millette and Moulding (1981a,b), showed similar behavior. Such transient membrane components are thought to represent differentiation antigens which may be involved in the highly complex, but ordered, sequence of cellular rearrangement and realignment which accompanies spermatocyte differentiation and spermatid elongation. Of particular relevance to the subject of the present article are those studies which provide some insight into the mechanisms by which the surface of the differentiating germ cells becomes organized into the various macro- and microdomains, which are such a conspicuous feature of the spermatozoon. Millette and BellvC (1980) provided evidence that certain membrane antigens could be partitioned selectively into the plasma membrane surrounding the residual body, a structure which sequesters redundant cytoplasmic elements late in spermiogenesis. These authors argued that the selective lateral translation of surface components would be the most plausible explanation for this effect, and quoted as supportive evidence similar phenomena seen during erythropoiesis (for references, see Millette and Bellv6, 1980). The observation that translational mobility of antigenic components takes place on the surface of spermatogenic cells (Romrell and O’Rand, 1978; Tung et al., 1979; O’Rand and Romrell, 1981) encourages this view. While these investigators showed that “patching” or cap formation could be induced upon incubation at 37”C, Lingwood and Schachter (1981), who studied the behavior and distribution of galactolipids, found that patching could also be induced at 4°C. Interestingly, Tung et al. (1979) commented that the position of the caps formed upon young spermatids bore no relationship to cellular polarity, thereby showing that physical constraints to translational diffusion were absent in these cells. That constraints to the lateral diffusion of membrane proteins are apparently imposed during the middle or late stages of spermiogenesis has been indicated by O’Rand and Romrell(1980), who showed, in the rabbit, that certain regions of the spermatid surface, the middle piece in particular, could be preferentially labeled by this stage of development. b. Morphological Correlates of Domain Formation. If the organization of the spermatid plasma membrane into various domains is a process involving the lateral mobility and restriction of membrane components, then mechanisms by which interdomain boundaries might be set up are of considerable interest. Little is currently known of this process, however, consideration of some ultrastructural features of spermatid development in conjunction with factors which are believed to control the lateral mobility of membrane components allows some potential mechanisms to be identified. Early in spermatid development, two dense structures, the annulus and nuclear ring, are formed as specializations of the plasma membrane. The participation of
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these two structures in spermiogenesis has been described by Fawcett et al. (19711, and in essence the nuclear ring is formed in the neck region of the spermatid, while the annulus appears at the anterior end of the flagellar canal. The annulus undergoes caudal migration later in spermiogenesis, this process being followed by the positioning of mitochondria around the middle piece. The nuclear ring is situated at one end of an array of microtubules, the manchette, which surrounds the posterior region of the developing spermatid like a sheath (see Fawcett et al., 1971; and Fig. 5). The intimate connections between the spermatid plasma membrane, the nuclear ring, and the manchette suggest strongly that a boundary to the lateral migration of membrane components would be set up by the assembly of these structures. A number of precedents leading to this supposition have been described, most of these concerning the associative interactions between surface antigens and cytoskeletal elements (for reviews, see Nicolson, 1979; Edidin, 1981). The annulus might also be expected to provide a barrier to lateral diffusion between two membrane domains, that destined to cover the middle piece and that which overlies the principal piece. The annulus appears in thin sections as an electron-dense ring, formed in association with elements of the chromatoid body (Fawcett et al., 1970). Support for this suggestion has been provided by Baccetti et al. (1978) who, as previously discussed, regarded the periaxonemal plasma membrane as being newly formed, in contrast to other regions which derive from the preexisting plasma membrane. Baccetti et al. (1978) were unable, however, to demonstrate any differences in the concanavalin A-binding properties of the two regions. The possibility that the annulus segregates two major membrane domains was also considered by Friend ( 1982b), who noted that the characteristic, geometric array of intramembranous particles was not established until annular migration was complete. Upon completion, the annulus became anchored to a dense, wedge-shaped, aggregation of submembranous material, effectively isolating the middle and principal pieces of the flagellum. Development of the spermatid during spermiogenesis is achieved while the cell is tightly grasped by a rigid ectoplasmic specialization of the Sertoli cell plasma membrane (Russell, 1977). This structure is initially found facing regions of the pachytene spermatocyte plasma membrane, its main feature being an array of numerous actin filaments situated within the underlying cytoplasm. In view of their involvement in the control of lateral migration among membrane components, the presence here of actin filaments raises the question of their participation in the organization of the sperm surface. The evidence for such a role in this situation is at present poor, especially as no structural connections have been observed to span the intercellular space between the germ cell plasma membrane and that of the Sertoli cell (Russell, 1977). Despite this, there are now several reports of the presence of actin on the surface of the sperm head in a number of
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species, with the exception of the rat (Clarke and Yanagimachi, 1978; Talbot and Kleve, 1978; Campanella et al., 1979; Tamblyn, 1980). As yet it is unclear whether this actin is an integral component of the sperm head, or merely a remnant of the ectoplasmic specializations. A particularly interesting structure, the tubulobulbar complex, which is also a specialized connection between the germ cell plasma membrane and the Sertoli cell was recently described in a series of publications by Russell (Russell and Clermont, 1976; Russell, 1979a,b). These structures are formed late in spermiogenesis, shortly before spermiation, and consist of narrow, elongated, hollow tubes of spermatid plasma membrane, which penetrate the Sertoli cell cytoplasm, to terminate in a bulbous swelling (Fig. 6). The membranous tube is situated within a complementary recess of the Sertoli cell plasma membrane. Diminution of the cytoplasmic contents of spermatids was shown to accompany the process of formation and resorption of several generations of tubulobulbar complexes (Russell, 1979b). This phenomenon would also provide an excellent opportunity for excess membrane components to be preferentially resorbed by the Sertoli cell, in the way that partitioning into the residual body was noted by Millette and Bellvt (1980). 2 . Biogenesis of the Acrosomal Membrane In contrast with the rapid growth of information about the development of the plasma membrane, much less is known of the acrosomal membrane. Morphological studies have provided some circumstantial evidence suggestive of developmental mechanisms and a brief review will be presented. Spermiogenesis, at least in its early stages, appears to be a conservative feature of testicular function. The young spermatids of widely differing species possess remarkably similar morphological features, the Golgi apparatus giving rise to a series of membranous vesicles, which coalesce to form a proacrosomal vacuole. Enlargement of this vacuole is followed by its movement toward one pole of the spermatid nucleus to which it adheres. The vacuole then becomes flattened to form a cap over approximately half of the nuclear surface. FIG. 6. Transmission electron micrograph showing part of a mature spermatid from the common marmoset, Cul[irhrixjucchus: the tubulobulbar complex shown here has formed by uptake of a region of spermatid plasma membrane into the adjacent Sertoli cell. Ac, Acrosome; SpPM, spermatid plasma membrane. X76,300. FIG. 7. Sagittal section through the head of an ejaculated ram spermatozoon which is undergoing fusion with a hen erythrocyte in the presence of glyceryl monooleate. Fusion between the sperm and erythrocyte plasma membranes has occurred at the anterior region of the equatorial segment (ES), and the spermatozoon is embedded in erythrocyte cytoplasm. X72.000. FIG. 8. Sections of elongated spermatids from the golden hamster, at a late stage of spermiogenesis. Staining with aqueous phosphotungstic acid (pH 2.6) reveals a characteristic pattern within the acrosomes, shown here in various planes of sectioning; the outer acrosomal membrane is selectively and differentially stained by this method. X 16,300.
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At this point, two cytochemicaliy distinct acrosomal compartments are evident, the acrosomal granule, a thickened region overlying the site of initial attachment to the nuclear membrane, and the thinner acrosomal cap, the remainder of the acrosomal cytoplasm. Cytochemical techniques using phosphotungstic acid (Courtens, 1978; Holt, 1979a), thiocarbohydrazide-silver proteinate (Courtens, 1978), and lectins (Yamamoto, 1982) all show that these two acrosomal regions contain different components; segregation of glycosylated components appears to take place between the two acrosomal compartments. The differences in morphological appearance of the acrosomal cytoplasm were sufficient to prompt Burgos (1974) into postulating the existence of at least two synthetic routes for the acrosomal contents. He suggested that acrosomal granule components were derived via the Golgi apparatus, while the proximity of the endoplasmic reticulum and the acrosomal cap was suggestive of direct transfer of material, bypassing the Golgi apparatus. Recent work on glycoprotein synthesis in the testis has demonstrated that isolated pachytene spermatocytes and round spermatids are capable of incorporating radiolabeled fucose into more than 16 different glycoproteins (Grootegoed et al., 1982). Autoradiographic observations suggested that the Golgi apparatus was involved in this process; moreover, it was proposed that a pool of acrosomal proteins, recognized by the incorporation of labeled leucine, might be synthesized during the pachytene stage of spermatocyte development, ready for glycosylation and incorporation into acrosomal membranes and components. If a pool of partially glycosylated proteins exists prior to spermatid development, the suggested dual route for acrosomal component synthesis becomes more real, with preformed proteins either being processed by or bypassing the Golgi apparatus. The existence of two such varying acrosomal compartments would suggest indirectly that the acrosomal membrane is regionally differentiated at this early stage. Separate membrane domains would possess characteristic classes of receptors for interaction with cytoplasmic elements of different origin, as would occur if the scheme proposed by Burgos (1974) were correct. A third possible route for the synthesis of acrosomal components was proposed by Mollenhauer and Morri ( I 978), who noted the occurrence of polyribosomes bound to the outer acrosomal membrane of the cap region, but not the granule, of guinea pig spermatids. This evidence, as yet unsupported by similar findings in other species, would appear to indicate that direct transmembrane protein synthesis takes place upon certain regions of the outer acrosomal membrane, again illustrating the requirement for a differential distribution of receptor sites. Recently, direct evidence has been obtained for the occurrence of a transfer mechanism involving clathrin-coated vesicles, for the movement of acrosomal components from the distal face of the Golgi apparatus to the acrosome. Griffiths
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et al. (1980, 1981), using antibodies against clathrin-coated vesicles, demon-
strated reactivity with the Golgi apparatus, the coated vesicles, and parts of the acrosome. This study is a little disappointing because although the authors noted that guinea pig acrosomes were fluorescently labeled using their antibody, they were unable to find much corresponding label at the ultrastructural level. The involvement of a vesicular transport system from the Golgi apparatus to the acrosome had previously been inferred from morphological and enzyme cytochemical studies (Susi et al., 1971; Seiguer and Castro, 1972; Sandoz, 1970), where the apparent fusion of vesicles with the outer acrosomal membrane had been observed. Further investigations into the synthesis and transport of hydrolytic acrosomal enzymes have been published recently (Clermont et af., 1981; Tang et al., 1982). Later in spermiogenenesis a complex series of structural transitions within the acrosome lead to the elaborate differentiation of the acrosomal membrane (Holt, 1979a). For instance, hamster mature spermatids and caput epididymal spermatozoa display characteristic staining affinities for phosphotungstic acid (Fig. 8). In this species the acrosomal membrane stains to demonstrate four distinctly different domains. Different regions of the acrosomal membrane are also demonstrable in other species (Holt, 1979a), perhaps indicating regions of analogous membrane structure or function in widely different species. It is of interest that epididymal transit brings about further modifications of the observed staining patterns in the acrosomal membrane, and it is worth remembering at this point that the freeze-fracture patterns evident in this membrane are also modified by passage through the epididymis.
V. Epididymal Contribution to Sperm Surface Heterogeneity The development of sperm fertility during epididymal transit involves, as discussed previously, a complex array of phenomena. An obvious question to ask, therefore, is how does the passage of a cell through a series of differing environments cause these changes to occur? It is of interest that fertility is not acquired simply by an aging process, the prevention of sperm migration through the duct successfully inhibiting the acquisition of fertility (Bedford, 1966). Conversely, Orgebin-Crist and Jahad (1979) were able to induce fertility in epididyma1 spermatozoa, in v i m , by the inclusion of epididymal secretory products in the culture medium. One aspect of epididymal function which has received considerable attention has been its possible involvement in the modification of the sperm surface; although this has been reviewed previously (for example, Holt, 1982; Moore and Bedford, 1983), some points of relevance to the development of membrane heterogeneity are worth mentioning here. Following earlier observations that spermatozoa placed in an electrical field
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would become oriented with the head toward the anode (Mudd and Mudd, 1929; Bangham, 1961; Nevo et al., 1961; Bedford, 1963b; Bey, 1965), a number of cytochemical investigations were performed to examine the chemistry of the cell surface in more detail. Studies using colloidal iron hydroxide as an electron-dense marker for negatively charged groups, in conjunction with spermatozoa from a number of species were reviewed by Bedford and Cooper (1978), and showed in essence that considerable species specificity existed in the nature of the binding patterns observed. There was good evidence, however, from species such as the rabbit and rhesus monkey (Bedford et al., 1972), that different membrane domains could be distinguished on the basis of their net negative charge. There was also evidence to show that the net negative charge increased, in some species, during epididymal transit, thus confirming earlier electrophoretic observations (Bedford, 1963b; Holt, 1980). In conjunction with the increase in net negative charge, other sperm surface characterisics have been observed to undergo modification as the cells pass through the epididymal duct. Taken together, these observations begin to point to mechanisms by which the sperm surface develops during maturation; additionally, these studies may provide some insight into the possible role of the epididymis in the maturation of sperm membranes. Nicolson et al. (1977) demonstrated a decrease in the concanavalin A-binding ability of rabbit spermatozoa as they passed through the epididymis. They also showed decreases in the ability of spermatozoa to bind two other lectins, wheat germ agglutinin (WGA) and Ricinus communis agglutinin (RCA), respectively, as sperm maturation progressed. As WGA and RCA bind to N-acetyl-Dglucosamine and galactose residues, respectively, both sugars are therefore present on the sperm surface prior to the completion of epididymal transit. These observations prompt the suggestion that decreases in lectin binding may be causally related to the progressive increase in electronegativity of the sperm surface which accompanies sperm maturation. The implication here is that this effect is mediated by the epididymal environment. Sialic acid, a negatively charged sugar acid, frequently occupies the terminal position of oligosaccharide side chains on glycoproteins and glycolipids, while acetyl-glucosamine and galactose are commonly found to occupy the penultimate position. It has been suggested (Nicolson et al., 1977) that the epididymal environment may participate in the completion of partially synthesized oligosaccharides on the sperm surface, with the result that penultimate groups would become unavailable for lectin binding. As a hypothe,sis, this has a number of attractive aspects. Studies of isolated sperm membrane glycoprotein fractions, though limited in number, have shown that glucosamine, galactose, and sialic acid are amongst the component carbohydrates (Nasir-ud-din et al., 1980; Hermann and Keil, 1981). It has also been shown (Olson and Hamilton, 1978) that removal of sialic acid groups from the
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rat sperm surface results in increased exposure of galactose residues. Comparative lectin-binding studies, reviewed by Koehler (198 I ) , lend some, but not overwhelming, support to this proposed mechanism for glycosylation of the sperm surface. Bedford and Millar (1978), in a study of the ascrotal hyrax, showed that there was a tendency for WGA receptors to disappear during sperm maturation, coupled with an increase in electronegativity. Significant reductions in the number of concanavalin A- (Fournier-Delpech and Courot, 1980) and RCA- (Hammarstedt et al., 1982) binding sites on the ram sperm surface have been demonstrated in conjunction with epididymal transit. The electronegativity of the ram sperm plasma membrane increases considerably during epididymal transit; cytochemical studies using the enzyme neuraminidase in conjunction with ferric colloid clearly demonstrated that the increase in negative charge was due almost entirely to the acquisition of sialic acid groups (Holt, 1980). Histochemical studies of the ram epididymis also showed that the epithelial cells of the corpus and cauda epididymides were coated by a neuraminidase-sensitive mucosubstance (Holt, 1979b). If the proposal that oligosaccharide side chains of the sperm surface are modified during epididymal transit is valid, the presence of glycosyltransferases in the epididymal fluid or on the sperm surface would be expected. Such transferase enzymes have actually been detected on the surface of intact mouse spermatozoa (sialyltranferase, Durr et al., 1977; N-acetylglucosamine:galactosyltransferase, Shur and Bennett, 1979), and similar enzymes have been found in the luminal fluids of the epididymis and vas deferens (Letts et al., 1974; Hamilton, 1980; Bernal et al., 1980). Further support for the possibility that sperm surface glycoproteins become modified during epididymal transit comes from studies which have shown that sialic acid is readily available in epididymal fluids (Bose et al., 1966, 1975; Rajalakshmi and Prasad, 1968; Fournier-Delpech et al., 1973; Gupta et al., 1974). It must be said, however, that while donor groups for use by glycosyltransferases are usually in the form of nucleoside sugars, rather than free molecules, the existence of such donor groups in epididymal fluids remains to be demonstrated. Olson and Hamilton (1978) performed a series of experiments to investigate modifications to the surface of rat spermatozoa as they passed through the epididymis. These authors showed that the passage of spermatozoa from the caput to the cauda epididymidis was associated with increases in the amount of sialic acid and galactose exposed to radiolabeling probes. Moreover, the labeled groups were associated with a particular membrane glycoprotein, originally regarded as having a molecular weight of 37,000, but later shown to be smaller, only 23,000 (D. W . Hamilton, personal communication). A membrane glycolipid, a ganglioside, was also shown to gain sialic acid groups during epididyma1 transit. Interpretation of this study by Olson and Hamilton (1978) is complicated in
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that a number of options are open for the origin of the newly exposed groups. A completely new glycoprotein might be added to the sperm surface, perhaps after secretion by epididymal cells. Epididymal protein secretion in the rat has been studied in some detail (see Brooks, 1981, and references quoted therein) and among the various segments of the epididymis pronounced regional differentiation in the profile of secreted proteins has been noted. This interpretation is supported by studies showing the appearance of antigens in epididymal cells, followed by their binding to spermatozoa (Lea et al., 1978; Moore, 1980; Faye et al., 1980; Lea and French, 1981; Feuchter et al., 1981; Vernon et al., 1982), and by other studies showing the adsorption of new proteins to the sperm surface (Voglmayr et al., 1980, 1982). Immunofluorescence studies have demonstrated clearly that the binding of such proteins takes place only over well-defined membrane domains of the sperm surface. Alternatively, in support of the arguments advanced previously, the newly exposed groups might represent minor modifications to the glycosidic moieties of incomplete oligosaccharides. The complex sequence of environments to which the spermatozoa are exposed during epididymal transit thus seems to modify the sperm surface, perhaps by several different mechanisms. However, it is difficult to envisage a mechanism by which discrete regions of the sperm surface could be selectively modified by exposure to enzymes or other proteins, in solution, unless regional differentiation of the cell surface already existed prior to entry of the spermatozoa into the epididymis. This view stresses once again the probable importance of spermiogenesis as the major process responsible for organization of the sperm plasma membrane.
VI. Functional Aspects of Membrane Heterogeneity in Spermatozoa A. SPERMSURFACE MODIFICATIONS Given that the sperm surface is modified during epididymal transit, what are the likely functional results of such a change? Clearly, surface changes would be reflected in the acquisition of new receptor sites, perhaps for the sperm-egg interaction. Evidence for this has been provided by Saling (1982) who showed that mouse spermatozoa recovered from the caput, corpus, and cauda epididymidis, respectively, showed an increasing ability to bind to the zona pellucida. As the occurrence of the acrosome reaction was not necessary for the observation of this effect, it would appear that this ability is conferred upon the plasma membrane by an epididymal process. Whether the cell surface modifications are related to more fundamental changes within the sperm plasma membrane, for example modulation of membrane fluidity, permeability or the mobility of intramembranous proteins, is an interesting question which can only be answered to a limited extent at present.
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Freeze-fracture studies, cited previously, demonstrated that while the sperm head plasma membranes from a number of species were reported to contain randomly dispersed intramembranous particles, this result was more commonly found in ejaculated cells. Geometric patterns in the sperm head plasma membrane occurred in epididymal spermatozoa, but were then disrupted at a time coincident with the removal of the glycocalyx (Suzuki and Nagano, 1980a; Friend, 1980, 1982b). Could the modification of the cell surface affect the disposition of intramembranous particles in this way? Theoretical arguments suggest that it might. For ideal membranes above the phase transition temperature of their component lipids, the entropy of mixing ensures that intramembranous proteins and lipids are randomly distributed throughout the available membrane area. In practice, mutual interactions between membrane components, and restraining influences imposed by cytoskeletal elements modulate the tendency toward randomness. Gingell (1976) and Barber (1982) in reviews of electrostatic interactions in membranes, both pointed out that van der Waal’s forces acting between intramembranous particles would induce particle aggregation unless a mechanism for its prevention also existed. Electrostatic repulsion between charged surface groups would be one preventative mechanism. Thus it could theoretically be argued that as rat spermatozoa acquire their negatively charged sialic acid residues, as suggested by Olson and Hamilton ( 1978), during the later stages of epididymal maturation, the intramembranous particles would randomize in accordance with the freeze-fracture observations. Such an argument has some interesting consequences; the randomization of particles would favor the prevention of premature membrane fusion, such as degenerative interactions with the acrosomal membrane. Removal of the surface charge, perhaps during capacitation (Johnson, 1975), would then induce particle clustering and the formation of protein-free areas of lipid, regarded by some as a prerequisite for the initiation of membrane fusion (Ahkong et al., 1973). Aggregation of membrane proteins would also favor the formation of ionophoric channels within the membrane (Gingell, 1976), and it is noteworthy that an influx of extracellular calcium is required to stimulate the acrosome reaction after capacitation (Yanagimachi and Usui, 1974). Clearly, this is an oversimplified argument, but one worth consideration. B . MEMBRANE HETEROGENEITY AND FUSION INTERACTIONS Upon exposure to the female reproductive tract environment the sperm plasma membrane becomes modified, mainly in terms of changes in receptor specificity, membrane permeability, and fluidity. Such changes, covered by the general term “capacitation,” are thought to prepare the spermatozoa for the initiation of the acrosome reaction and union of gametes. The mechanisms involved in capacitation have been explored in numerous studies (for reviews, see Barros, 1974;
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Austin, 1975; Gwatkin, 1976; Bedford and Cooper, 1978; Shapiro and Eddy, 1980; Shapiro et al., 1981; Yanagimachi, 1981; Moore and Bedford, 1983), however, some recent observations concerning changes to the local distribution of sperm membrane structures and domains are of sufficient significance to justify further consideration in this article. Particular attention will be paid here to the significance of these findings in terms of membrane fluidity, at a local level where possible, since this is an influential factor in controlling the occurrence of membrane fusion, fertilization being a prime, if specialized, example of this process. Despite differences between models of membrane fusion (see, Lucy, 1978; Papahadjopoulos, 1978) there is some agreement that for fusion to proceed, membranes must at some stage exist in a fluid state, either before or during fusion. The implications of this conclusion are of interest for their relevance to sperm function, the first question which presents itself being, are sperm membranes in a fluid state at any time during their development? Evidence from a number of different sources suggests that posttesticular sperm maturation is associated with an increase in membrane fluidity, the potential for membrane fusion being suppressed during this process, but finally being realized immediately before fertilization. Clearly, with such a highly controlled sequence of fusion events local differences in fluidity between the individual membrane domains of the spermatozoon have to be maintained. Subtle variations such as these are not amenable to study by gross biochemical methods, although these can provide useful guidelines and more informative techniques have to be used. Epididymal spermatozoa experience an increase in their unsaturated:saturated fatty acid ratio as they progress through the epididymis, this being combined with a decline in cholesterol concentration (Scott et al., 1967; Poulos et al., 1973). Reasoning from physical-chemical principles both of these factors would tend to suggest that increased fertility is associated with higher membrane fluidity. However, this evidence from the analysis of whole cells is insufficiently informative to justify this conclusion, as all the intricate intracellular variability in membrane properties would be represented in the overall average. The isolation and analysis of purified sperm membrane fractions are hardly more informative in this respect, although some workers are currently investigating the use of fluorescent probes to monitor membrane fluidity in these preparations (e.g., Vijayasarathy et al., 1982). Freeze-fracture images can be interpreted to some extent in terms of membrane fluidity and protein mobility (see Nicolson, 1979), the most useful criteria being that orderly distributions of intramembranous particles are usually immobile, held in place by exclusion from surrounding domains of crystalline lipid molecules, or by cytoskeletal elements. According to this view the highly ordered structure of the guinea pig epididymal sperm head plasma membrane would be indicative of low translational mobility. Similarly the regularly arranged particles of the rat and boar sperm head plasma membrane (Suzuki and
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Nagano, 1980a; Suzuki, 1981) could be interpreted in this way. This inference is upheld by the lectin-binding studies of Schwarz and Koehler (1979) where little or no capacity for temperature-induced receptor mobility was found on the guinea pig sperm surface. Koehler (1975b) had earlier shown the absence of translational mobility among receptors of the rabbit sperm surface, a finding essentially in agreement with studies by Nicolson and Yanagimachi (1974). O’Rand (1977), however, claimed that a rabbit sperm membrane glycoprotein antigen exhibited translational mobility, expressed as temperature-induced patching, in epididymal and ejaculated spermatozoa. Commenting upon this discrepancy, Schwarz and Koehler (1979) drew attention to the possible differences in mobility between receptors recognized by lectins, which may bind to numerous proteins, and those with an ability to bind specific antibodies. They also pointed out, however, that sperm membrane damage sustained during the experimental procedures used by O’Rand might misleadingly cause patching of label. Recent studies of the sperm surface in which monoclonal antibodies have been used (for example: Bechtol et al., 1979; Herr and Eddy, 1980; Feuchter et al., 1981; Myles et a l . , 1981; Gaunt, 1982; Schmell et al., 1982) have all demonstrated the binding of different antibodies to well-defined membrane domains. The implication of this work is that membrane surface components on epididyma1 and ejaculated spermatozoa, in which these studies have been performed, are strictly confined within their respective domains. Nicolson and Yanagimachi (1974) in a study employing lectins showed, as mentioned above, that no evidence of receptor mobility was apparent over the acrosomal region of the rabbit spermatozoon. These authors believed, however, that receptor clustering occurred over the postacrosomal region. This evidence needs to be considered with caution, since the shedding of membrane coat components, as seen by Koehler (1976) when rabbit spermatozoa were exposed to isotonic and hypertonic media, might lead erroneously to the production of electron micrographs showing clustered lectin receptors. Some success has been obtained in the assessment of sperm membrane properties and composition by the use of filipin and other membrane probes. Information gained in this way is particularly helpful in understanding some events associated with capacitation. Friend (1980, 1982a,b) showed that filipin, a polyene antibiotic with an affinity for membrane sterols, was bound avidly by the guinea pig sperm plasma membrane. The anterior acrosomal region displayed approximately four times as many filipin/sterol complexes as the postacrosomal region, a finding confirmed in the bull and ram (Bradley et al., 1979, 1980). The filipin/sterol complexes of the guinea pig sperm head were distributed between the plaques of quilt patterning, suggesting that such patterned plaques contain ordered arrays of membrane components capable of excluding the sterols: in contrast, filipin/sterol complexes in the bull and ram sperm plasma membrane were irregularly distributed.
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Removal of cholesterol from spermatozoa has been suggested as a function of albumin in the induction of the acrosome reaction (Davis, 1980; Davis et al., 1980), thereby helping to destabilize the plasma membrane. Furthermore good correlation has been noted in a number of species between capacitation time and sperm cholesterol content (Davis, 1981). Further elaboration of this proposal was provided by Langlais et al. (198 I), who studied the localization of cholesteryl sulfate in the plasma membranes of human spermatozoa. These authors noted that this ester was mainly associated with the plasma membrane overlying the acrosome, in keeping with the filipin studies, and suggested that subsequent removal of the membrane sterol would only occur after cleavage of the ester linkage. The action of sterol sulfatase would thus be an important mechanism for the control of capacitation. The considerable increase in anionic lipids in the outer leaflet of the plasma membrane during capacitation in the guinea pig (Bearer and Friend, 1981) is a provocative finding. Papahadjopoulos ( 1978) proposed that membranes containing a high proportion of anionic lipids would be especially susceptible to fusion, as calcium ions can induce them to undergo a liquid to gel phase transition. Thus, anionic lipid domains present at, or slightly above, their phase transition temperature would form liquid crystalline domains of high fusibility upon the addition of calcium ions. It was proposed that fusion occurred through the instability of such structures; packing faults, domain boundaries, or regions of transient hydrocarbon-water contact were regarded as contributing to the reduction of stability. Clearly, the obligatory role of calcium in promoting the acrosome reaction (Yanagimachi and Usui, 1974) can be explained in terms of this membrane fusion model. Lucy and his colleagues have proposed that membrane fusion is more likely to occur when lipids are in a fluid, rather than crystalline condition. The behavior of the sperm plasma membrane under capacitating conditions is consistent with this view. Evidence obtained using the guinea pig as a model showed that circular clearings, initially free of membrane sterols, but later of membrane proteins too, form over the acrosome (Friend, 1980) thus providing exactly the type of protein-free lipid domains envisaged by Ahkong et al. (1975) as an essential prelude to fusion. An increase in membrane fluidity over the acrosomal region has also been detected using rnerocyanine s540 (Bearer and Friend, 1981). In contrast membrane fluidity does not increase sufficiently over the postacrosomal region to be detectable with this reagent. Indirect evidence strengthens the view that increased membrane fluidity accompanies capacitation. Exposure of spermatozoa to membrane lipid perturbants such as lysolecithin (Gabara et al., 1973; Jones, 1976) or monoolein (Holt and Dott, 1980; Fleming and Yanagimachi, 198I), will promote membrane vesiculation similar to the acrosome reaction. Fusion of ram spermatozoa with hen erythrocytes was achieved using monoolein (Holt and Dott, 1980; Holt, 1981), and despite the artificiality of the system the sperm head membranes behaved as
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they would in normal fertilization (Fig. 7). Initially the membranes of the acrosoma1 region vesiculated, and this was followed by fusion of the postacrosomal plasma membrane with the erythrocyte membrane. These observations underline the intrinsic importance of membrane heterogeneity in the sequential organization of the fertilization reaction. Ahkong et al. (1980), in further investigations of erythrocyte fusion mechanisms, found that a calcium-activated thiol-proteinase was involved in the promotion of membrane protein aggregation. The proteolytic release of constraints upon particle aggregation is therefore likely to be an important aspect of membrane fusion in sperm interactions. This argument is supported by the observation that trypsin increases membrane fluidity over the anterior acrosomal portion of the guinea pig sperm plasma membrane (Bearer and Friend, 1981). It is evident therefore that an interpretation of the roles of calcium and anionic lipids in capacitation based too heavily upon the experimental models used by Papahadjopoulos ( 1978) would be excessively oversimplified. There is an interesting parallel between the use of filipin as a probe of membrane structure, as described above, and studies in which the physiological effects of filipin on sperm function are examined. Russell et al. (1979) noted that boar spermatozoa exposed to filipin in the presence of calcium exhibited plasma and acrosomal vesiculation in a manner resembling the acrosome reaction. Ultrastructural examination of spermatozoa treated in the absence of calcium merely demonstrated evidence of filipin binding to the acrosomal region of the plasma membrane. Corresponding results are found if ram spermatozoa are similarly treated (Holt, unpublished observations). Apparently the formation of filipin/sterol complexes facilitates the entry of calcium ions into the acrosome (Bradley et al., 1979), thereby bypassing a lengthy period of capacitation but causing membrane destabilization nevertheless. Such observations indicate that the complex sequence of membrane modifications which accompanies capacitation may result in the transport of calcium ions into the acrosome, where they might be required to interact with the inner leaflet of the plasma membrane or the outer leaflet of the acrosomal membrane, both of which would normally be inaccessible to calcium. Once inside the acrosome, calcium ions could interact with anionic lipids or function as enzyme activators as discussed above. Studies more specifically directed toward elucidating the role of calcium in capacitation have employed calcium ionophores (Summers et al., 1976; Talbot et al., 1976; Singh et al., 1978; Green, 1978; Shams-Borhan and Harrison, 1981; Byrd, 1981; Jamil and White, 1981; Jamil et al., 1982; for review, see Garbers and Kopf, 1980). In essence these studies confirm that entry of calcium into the acrosome effectively precipitates an acrosome reaction; in these instances entry is gained by the formation of ionophoric channels in the plasma, and possibly acrosomal, membranes. The morphological events accompanying and preceeding the artificially induced acrosome reaction resemble those seen normally;
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however, as membrane vesiculation is observed to occur over the equatorial segment in the bull (Byrd, 1981) and ram (Shams-Borhan and Harrison, 1981), a region which does not normally show this change, such treatment perhaps exaggerates the membrane permeability change. The loss of sperm motility associated with the use of ionophores would indicate the same thing. Jamil et al. (1982) studied the effects of ionophore treatment upon human spermatozoa by the use of freeze-fracture electron microscopy. Their findings indicate that considerable membrane disruption is caused by this treatment, extending to the inner and outer acrosomal membranes and the nuclear membrane. Considerable rearrangement of plasma membrane particles preceded the breakage and loss of this membrane. Such results are difficult to interpret usefully in terms of membrane heterogeneity; they do confirm, however, the likelihood that calcium entry into the cell is considerably increased after exposure to this reagent.
VII. Concluding Remarks The ideas discussed above have largely concerned the initiation of the acrosome reaction, since this event is easily studied in vitro. Fusion of the sperm head with the egg plasma membrane involves the postacrosomal region and equatorial segment (Bedford et al., 1979), areas which differ in properties both from one another and from the acrosomal region. The postacrosomal region apparently contains few sterols and anionic lipids, and failed to bind merocyanine s540 (Bearer and Friend, 1982), even after proteolytic treatment. The production of particle-free membrane domains was reported to occur in this region, however (Friend, 1980), indicating that sites of imminent membrane fusion are formed in response to capacitating conditions. The importance of factors other than changes in lipid composition should not be overlooked when considering mechanisms of fertilization. Space does not permit a discussion of the role of enzymes, cyclic nucleotides, surface receptors for the zona pellucida and vitelline membrane, changes in motility characteristics coupled with modifications to the flagellar membrane, however, these topics have received detailed treatment elsewhere (Garbers and Kopf, 1980; Shapiro and Eddy, 1980; Moore and Bedford, 1983).
ACKNOWLEDGMENTS I am most grateful to Dr. D. S . Friend who encouraged me to write this article. I would like to express my appreciation to Prof. J . A. Lucy and Dr. Q. F. Ahkong for the use of their freeze-fracture equipment, and for their assistance with the technique.
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INTERNATIONAL REVIEW OF CYTOLOGY. VOL. 87
Capping and the Cytoskeleton LILLYY. W. BOURGUIGNON AND GERARD J. BOURGUIGNON Department of Anatomy and Cell Biology, School of Medicine, University of Miami, Miami, Florida Introduction . . . . . . . . . . . . ............................. General Capping Phenomena . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Ligand-Dependent Processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B . Ligand-Independent Processes. . . . . . ........... 111. Microfilaments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1v. Microtubules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Intermediate Filaments. . . . . . . . . VI. Capping-Related Regulatory Mole ............. A. Ca2+-Calmodulin , . . B. Myosin Light Chain C. Cyclic AMP (CAMP) and Adenylate Cyclase . . . . . . . . . . . . . . . .................. VII. Mechanisms of Capping VIII. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I.
11.
I95 196 196 I97 198 203 207 208 209 210 213 213 217 220
I. Introduction In the 12 years since the aggregation or “capping” of cell surface receptors was first defined (Taylor et al., 1971), a great deal of research effort has been directed toward determining the molecular mechanisms responsible for this very interesting phenomena. In the past 5 or 6 years, significant progress has been made toward this goal as our understanding of the nature of the cytoskeleton and its relationship to the plasma membrane has increased. It is now generally agreed that the cytoskeleton is involved, either directly or indirectly, in the lateral redistribution of surface molecules into a cap structure. Recently, three reviews concerning capping have been published (Loor, 1981; Oliver and Berlin, 1982; Yahara, 1982) which propose different models as possible mechanisms for surface receptor capping. In this article we have summarized the most recent results obtained in capping/cytoskeleton research and then attempted to incorporate this new information, along with some of the previously proposed models, into what we now believe is the most likely mechanism for the capping process. Surface receptor redistribution appears to be very important physiologically in a variety of different cell types. For example, histamine release by mast cells is known to result from the clustering of Fc receptors of IgE on the cell surface 195 Copyright 0 19114 by Academic Press. Inc. All rights of reproduclion in any form reserved. ISBN 0-12-364487-9
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(Ishizaka et al., 1977); the receptors for insulin and epidermal growth factor immediately form aggregates on the cell surface after hormone binding (Schlessinger et al., 1978); and lymphocytes are stimulated to differentiate and proliferate by the binding of bivalent anti-immunoglobulin (Ig) or lectins (Cunningham et al., 1976) which aggregates certain membrane glycoproteins.
11. General Capping Phenomena A. LIGAND-DEPENDENT PROCESSES
Lymphocyte surface receptors are known to aggregate into small clusters upon exposure to externally added ligands (e.g., antibodies against surface antigens or lectins) at cold temperatures (0-4°C). Incubation at ambient temperature or 37°C causes these small clusters (so-called patches) to be further collected into a large aggregate at one pole of the cell (so-called caps) via an energy-dependent process (Taylor et al., 1971). Because of the fluid nature of the plasma membrane (Singer and Nicolson, 1972), it has been suggested that the ligand and its receptor (membrane components) are clustered due to a cross-linking event at the cell surface which is highly dependent on the valency of the ligand (de Petris and Raff, 1972; Taylor et al., 1971). For example, divalent antibodies against surface immunoglobulin (Ig) are required for the induction of Ig capping; monovalent Ig is unable to cause redistribution of the Ig receptor (Schreiner and Unanue, 1976). In the case of capping induced by the lectin, concanavalin A (Con A), the tetrameric form of Con A (at the proper concentration) is required whereas the dimer form (e.g., succinyl Con A) does not induce the surface Con A binding sites to aggregate (Edelman et al., 1973; Gunther et al., 1973). Recently, we have tested the ability of monoclonal antibody raised against T-200 (a major glycoprotein with MW 200,000 on the surface of murine Tlymphocytes) (Trowbridge, 1978) to induce capping of the lymphocyte surface T-200 molecules. This was of interest because one would predict that monoclonal antibodies. which recognize only a very limited number of antigen amino acid sequences, should cause much less cross-linking of the surface T-200 molecules than polyclonal antibodies which bind to a variety of different antigen amino acid sequences. Generally, T-200 molecules are found uniformly distributed over the entire plasma membrane (Fig. la). As shown in Fig. lb, we observe no cap formation but only small patchlike clusters on the surface of cells treated with monoclonal anti-T-200 antibody. This was true even when relatively high concentrations of monoclonal antibody were used. If the cells, however, were treated with two consecutive antibodies (the primary monoclonal antiT-200 antibody followed by a secondary polyclonal antibody such as anti-antiT200), apparently sufficient cross-linking of the receptors occurs in order to
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FIG. 1. lmmunofluorescent staining of murine lymphocyte surface T-200 molecules. (a) Cells were first fixed with 2% paraformaldehyde, then labeled with monoclonal rat-anti-T-200 antibody followed by rabbit anti-rat immunoglobulin and fluorescein-conjugated goat antibody against rabbit immunoglobulin ( X IOOO). (b) Unfixed cells treated with monoclonal rat anti-T-200 antibody at 37°C for 15 minutes, fixed with 2% paraformaldehyde, and then stained with rabbit anti-rat antibody plus fluorescein-conjugated goat antibody against rabbit immunoglobulin ( x 1OOO). (c) Unfixed cells treated with monoclonal rat anti-T-200 antibody at 37°C for 15 minutes followed by rabbit anti-rat immunoglobulin at 37°C for 15 minutes; after fixation with 2% paraformaldehyde, cells were labeled with fluorescein-conjugated goat antibody against rabbit immunoglobulin ( X IOOO). (Similar results were obtained with monoclonal rat anti-Thy-1 and gp 69/71 antibodies.)
induce about 35% of the cell population to form cap structures (Fig. lc and Table I). Thus, these data further support the notion that a certain threshold level of surface receptor cross-linking is required for the induction of cap formation.
PROCESSES B . LIGAND-INDEPENDENT A number of different reagents, such as colchicine and hypertonic media, can cause the formation of cap structures in the absence of any externally added ligand (Yahara and Kakimoto-Sameshima, 1977, 1978; Schreiner et al., 1977a; Bourguignon et al., 1981). In the case of colchicine, we have demonstrated that treatment with this microtubule-disrupting agent induces a variety of lymphocyte antigens, such as T-200, Thy- 1, and gp 69/7 1, to aggregate into a typical cap structure (Bourguignon et al., 1981). In addition, colchicine-induced capping was found to be both temperature dependent and cytochalasin D sensitive (Table I). Vinblastine, another microtubule-disrupting agent, also induces surface receptors to form cap structures via the same ligand-independent process (Table I).
198
LlLLY Y. W. BOURGUIGNON AND GERARD J. BOURGUIGNON TABLE I LIGAND-DEPENDENT AND LIGAND-INDEPENDENT RECEPTOR CAPPING"
Conditions
Reagents Ligand-dependent processes Monoclonal rat anti-T-200 antibody Monoclonal rat anti-T-200 antibody + polyclonal rabbit anti-rat antibody Ligand-independent process Colchicine (I x lop4 M ) Colchicine ( I X M) Vinblastine ( I x M) Colchicine or vinblastine (1 X M ) + cytochalasin D (20 kg/ml) Colchicine or vinblastine (1 X M ) + sodium azide (50 pg/ml)
37°C. 15 minutes 3 7 T , 15 minutes + 3 7 T , 15 minutes
Percentage capping
5 35
0°C. 60 minutes 3 7 T , 15 minutes 37°C 15 minutes 37°C. 15 minutes
40 35
37°C. 15 minutes
10
1
I
"The standard deviation for capping experiments is 25%
These findings support earlier studies which indicated that microtubules are intimately involved in the regulation of surface receptor movement (Edelman et al., 1973). Morphological studies, using both transmission and scanning electron microscopy, of cells forming caps in hypertonic medium have identified a strong association between cap structures and microvilli (Yahara and KakimotoSameshima, 1977). This appears to be a unique consequence of hypertonic treatment since no other capping studies have reported such an association with microvilli. A number of similarities, however, do exist between colchicine and hypertonic medium-induced capping. For example, both treatments induce the capping of the same lymphocyte surface molecules including Ig, H-2, Thy-1, T-200, Con A receptors, and both types of capping are inhibited by sodium azide and by the microfilament-disrupting agent, cytochalasin B . In addition, during the course of these two different treatments, the receptor cap structure is always associated with a dramatic cell shape change generally referred to as uropod formation (Bourguignon et al., 1981; Yahara and Kakimoto-Sameshima, 1977, Table 1). Further analysis of ligand-independent capping remains to be carried out. 111. Microfilaments
The involvement of contractile microfilaments in surface receptor capping was first suggested by Taylor et al. (197 1) who found that cytochalasin B partially
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inhibited the capping of surface Ig on mouse splenic lymphocytes. Since then numerous reports have confirmed this observation for a variety of different receptors on the surface of mouse and human lymphocytes (Schreiner and Unanue, 1976; de Petris, 1977; Loor, 1981). There are, however, apparently contradictory effects caused by cytochalasin B in other cell types. For example, both cytochalasin B and D have been found to induce capping in a number of transformed cell lines but not in normal fibroblastoid lines or lymphoblastoid lines (Sundqvist and Ehmst, 1976; Sundqvist et al., 1978; Mousa et al., 1978; Godman et al., 1980a,b). Recently, de Groot et al. (1981) have demonstrated that cytochalasin B and D stimulates the capping of surface Ig in rabbit lymphocytes in contrast to the inhibition of capping observed with mouse lymphocytes. Obviously, these results indicate that differences must exist in the way microfilaments are involved in the movement of surface components in different cell types. At the present time, the mechanism of action of the various types of cytochalasins (A, B, C, D, and E) is still not fully understood. There is good evidence for the binding of the drug to the growing end of an actin filament thereby blocking further formation of F-actin (Lin et al., 1980). It also appears that cytochalasin can cause the fragmentation of preformed actin filaments and can affect the actin-associated ATPase activity (Korn, 1982). In any case, all the data confirm the idea that actin-containing microfilaments are intimately involved in surface receptor capping (Loor, 1981). Further important evidence supporting this idea has been obtained using immunocytochemical procedures to localize intracellular actin and myosin in capped cells. A number of investigators have demonstrated that actin is accumulated directly beneath the surface cap formation in a variety of different cells (Sundqvist and Ehmst, 1976; Bourguignon and Singer, 1977; Toh and Hard, 1977; Gabbiani et al., 1977; Oliver et al., 1977; Bourguignon et al., 1978a; Singer et al., 1978; Butman et al., 1980; Bourguignon and Rozek, 1980). The same accumulation has been found for myosin (Bourguignon and Singer, 1977; Schreiner et u l . , 1977a,b; Bourguignon et al., 1978; Bourguignon, 1980). More conclusive evidence for a transmembrane association between intracellular microfilaments and surface receptors has been acquired from experiments in which capped cells are treated with nonionic detergents in order to isolate the actin-containing cytoskeleton. Flanagan and Koch ( 1978), Braun et al. (1982), and our own laboratory (Bourguignon et al., 1978a, 1983b; Butman et al., 1980; Bourguignon and Bourguignon, 1981a) have determined that lymphocyte surface receptors undergoing capping form a fairly stable association with the actin-containing cytoskeleton. Similar observations have been made following the capping of Con A receptors on the slime mold Dictyostelium discoidium (Condeelis, 1979) and on neutrophil leukocytes (Sheterline and Hopkins, 1981). The results of Braun et al. (1982) and our laboratory (Bourguignon and Singer, 1977; Bourguignon et al., 1983b) have also established that the
200
LlLLY Y. W. BOURGUIGNON AND GERARD J . BOURGUIGNON
attachment of the surface receptors to the cytoskeleton occurs at the patching stage before actual cap formation. The same conclusion was obtained earlier using immunocytochemical techniques (Bourguignon and Singer, 1977). Several years ago we proposed that a specific transmembrane interaction occurs between intracellular actomyosin filaments and surface receptors undergoing capping (Bourguignon and Singer, 1977; Bourguignon et a l . , 1978b). In particular, this hypothesis stated that membrane-associated actin and myosin are either directly or indirectly bound to an integral plasma membrane protein (or class of proteins) called X protein(s) following the binding of a multivalent ligand (e.g., antibodies against specific receptors or lectins) to the cells and the subsequent aggregation of the surface receptors into ‘‘patches. These receptor aggregates, which are linked to actin and myosin through the X protein(s), are then collected into a cap by a sliding filament mechanism analogous to that occurring during muscle contraction (Bourguignon and Singer, 1977). In recent years this hypothesis has attracted a good deal of attention; but to date, identification of the putative X protein(s) has not been accomplished. Since actin filaments play a very important role in receptor capping, it becomes necessary to determine whether any changes occur with actin during capping. One such change which has been observed is the polymerization of Gactin to F-actin during the process of capping. Several groups have reported that there are large pools of monomeric actin (G-actin) in nonmuscle cells which are polymerized into F-actin upon receiving an appropriate stimulus (Carlsson et al., 1979; Giltler et al., 1980). Using the newly developed DNase I assay to determine the ratio of G- to F-actin, Laub et al. (198 1) have found that a significant amount of actin polymerization occurs during antibody-induced Thy- 1 capping. This finding suggests one obvious control mechanism for the initiation of microfilament-mediated contraction during the capping process. Recently, a number of actin-associated proteins have been localized in the subcap region: a-actinin (Geiger and Singer, 1979; Hoessli et al., 1980), fodrin (a spectrin-like antigen) (Figs. 2 and 3 and Levine et al., 1981; Levine and Willard, 1983), and an ankyrin-like protein (Figs. 2 and 3 and Bourguignon et a l . , 1983b). In particular, our laboratory has utilized a monospecific antibody raised against brain fodrin to characterize lymphocyte fodrin (Fig. 4). We have determined that lymphocytes contain a fodrin-like antigen (molecular weight of 240,000) in both the plasma membrane and membrane-associated cytoskeleton fractions (Fig. 4) which increases significantly in the membrane-associated cytoskeleton fraction during patching and capping (Fig. 4). Nelson et al. (1983) have also recently reported that a 240,000-dalton protein from lymphocytes cross-reacts with erythrocyte a-spectrin and colocalizes with surface receptor aggregates. The possible role of these actin-associated proteins in capping will be discussed in Section VII. ”
uniform
patches
Con A
Fodrin
Con A
Ankyrin
FIG. 2. Double immunofluorescence staining of murine lymphocyte surface receptors and intracellular fodrin and ankyrin. Surface labeling with F1-Con A (a) and (g) of prefixed cells stained with intracellular fodrin (b) and intracellula ankyrin (h). Surface labeling with FI-Con A (c) and (i) of unfixed cells at patched state stained with intracellular fodrin (d) and intracellular ankyrin (j).Surface labeling with FI-Con A (e) and (k) of unfixed cells at capped condition stained with intracellular fodrin (f)and intracellular ankyrin (1). (Similar fodrin and ankyrin localization results were obtained when cell surface were labeled with antibodies raised against Thy-I or T-200.) lmmunoreagents used in these double label experiments are ( I ) rabbit anti-pig fodrin followed by rhodamine-labeled goat anti-rabbit immunoglobulin for labeling intracellular fodrin; and (2) rabbit anti-human erythrocyte ankyrin followed by rhodamine-labeled goat anti-rabbit immunoglobulin for staining intracellular ankyrin.
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LILLY Y. W . BOURGUIGNON A N D GERARD J. BOURGUIGNON
CAPPING AND THE CYTOSKELETON
203
Ankyrin 3e
FIG. 3. Analysis of intracellular fodrin and ankyrin in uncapped and patchedlcapped cells using immunoelectron microscopy on ultrathin frozen sections. Fodrin (a) and ankyrin (e) are found scattered over the cytoplasm (Cyj and close to the plasma membrane (Pm) in uncapped cells. Vi, Villus; Pm, plasma membrane; Cy, cytoplasm. ~60,000.(Arrowheads indicate the submembrane fodrin and ankyrin labeling.) Fodrin (b and c are identical pictures) and ankyrin (fl and (g) are preferentially aggregated at the plasma membrane (Pm) in patched and capped cells. Fodrin appears to be arranged in radial (possibly filamentous) arrays in the cytoplasm (Cy) close to the plasma membrane in patchedlcapped cells as depicted by the lines connecting the colloidal gold particles in (a) and (bj. (Both arrowheads and arrows indicate the submembrane fodrin and ankyrin labeling.) In the control sample (d), there is negligible binding between preabsorbed anti-fodrin or anti-ankyrin and protein Alcolloidal gold on the sections prepared from patched/capped or uncapped cells. Immunoreagents used in these experiments for locating intracellular fodrin and ankyrin are rabbit anti-pig fodrin and rabbit anti-human ankyrin, respectively, followed by colloidal gold-conjugated protein A.
IV. Microtubules The role of microtubules in surface receptor capping has remained a controversial topic for a number of years. The most common method used to investigate this question has been to examine the effect of mitotic inhibitors, such as colchicine and vinblastine, on the capping process. Such experiments have generally found that microtubule inhibitors either stimulate capping or permit it to occur under unfavorable conditions. For example, it has been shown using lymphocytes and fibroblasts that low concentrations of Con A (below 10 p,g/ml) induce patching and capping of the surface lectin receptors (at temperatures above 20°C), whereas high concentrations of Con A (above 50 p,g/ml) cause the immobilization of the receptors under the same incubation conditions (Unanue et al., 1972; Yahara and Edelman, 1973; Loor, 1974; Bourguignon and Rosek, 1980). Because the inhibition of receptor clustering by high concentrations of
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LlLLY Y.W. BOURGUIGNON AND GERARD I. BOURGUIGNON
FIG.4. SDS-PAGE analysis of the proteins in NP-40 solublelinsoluble fractions of total plasma -membrane from capped or uncapped cells. (a) Molecular weight markers used: myosin, 200,000; pgalactosidare, 130,000;lactoperoxidase, 78,000; bovine serum albumin, 68,000; actin, 42,000; Con A, 25,000. (b) Total plasma membrane from uncapped cells (in the absence of NP-40). (c) NP-40 soluble fraction of plasma membrane from uncapped cells. (d) NP-40 insoluble cytoskeleton fraction of plasma membrane from uncapped cells. (e) Total plasma membrane from patchedkapped cells (in the absence of NP-40). (f) NP-40 soluble fraction of plasma membrane from patchedlcapped cells. (8) NP-40 insoluble cytoskeleton fraction of plasma membrane from patched/capped cells. (h) TW 260/240. (i) Erythroid spectrin. fj) Pig brain fodrin. (k) Same as (8). (I.) Autoradiogram of NP-40 insoluble cytoskeleton fraction from either uncapped or capped cells following immunoblotting in the presence of anti-fodrin antibody plus '251-labeled protein A. (Similar results were obtained with total plasma membrane material from both uncapped and patchedlcapped cells.) Except for (I) all protein bands were detected by Coomassie blue staining.
Con A can be reversed by pretreatment of the cells in the cold (0-4°C) or with colchicine (but not with lumicolchicine, an analog of colchicine known not to interact with microtubules), it has been suggested that microtubules primarily retard the movement of cell surface receptors and must be disassembled, at least in the cell cortex, for capping to occur. There are, however, some studies which report that colchicine has either no effect or an inhibitory effect on receptor movement (for reviews see Nicolson, 1976, and Loor, 1981). These seemingly contradictory results could be due to one or more of a number of complications
CAPPING AND THE CYTOSKELETON
205
which arise in such drug-related studies. For instance, different cell types may respond differently, and certain drugs could have multiple effects on any particular type of cell. In this regard, it is known that colchicine can affect certain transport activities of the plasma membrane in addition to its effect on microtubules (Mizel and Wilson, 1972; Stadler and Franke, 1972, 1974). Using the more direct approach of localizing intracellular tubulin by immunofluorescence techniques, it has been found that microtubule organization is significantly altered during capping in lymphocytes (Yahara and KakimotoSameshima, 1978; Oliver et al., 1980). However, there does not appear to be a marked accumulation of tubulin directly underneath the surface cap structure as is found with actin and myosin (Bourguignon et al., 1978a). Recently, we have utilized the antitumor drug, taxol, to further examine the relationship between microtubule organization and the capping process in lymphocytes. This drug is known to promote both the assembly of microtubules in vitro (Schiff et al., 1979) and the aggregation of microtubule bundles in vivo at sites other than the centrosome (Schiff and Horwitz, 1980; De Brabander et al., 1981). In mouse T-lymphoma cells, our data show that taxol clearly inhibits Thy-1 capping (Table 11). This result is consistent with the proposed requirement for some microtubule disassembly in order for capping to occur. During taxol treatment, the drug also appears to trigger the extensive reorganization of microtubles into large bundles extending from the centrosome (Fig. 5, and Paatero and Brown, 1982). Possibly, the changes in the spacial organization of microtubules induced by taxol have uncoupled the intricate relationships between microtubules and microfilaments required for the activation of receptor capping. However, in the case of Ig capping, taxol has been reported to relieve the inhibition of Ig capping caused by Con A (Paatero and Brown, 1982). Most interestingly, it has been shown recently that the red blood cell cyto-
TABLE I1 EFFECTOF TAXOLON CAPPING"
Reagents Monoclonal rat anti-Thy-1 antibody + polyclonal rabbit anti-rat antibody (untreated samples) Pretreatment cells with taxol (20 Fglml + monoclonal rat anti-Thy-1 antibody + polyclonal rabbit anti-rat antibody (taxol-treated samples)
Condition
Percentage capping
37°C. 15 minutes + 37OC, 15 minutes
44
37°C. 30 minutes + 3 7 T , 15 minutes + 3 7 T , 15 minutes
10
OThe standard deviation for capping experiments is 25%.
206
LILLY Y. W. BOURGUIGNON AND GERARD J. BOURGUIGNON
FIG. 5 . Electron micrograph of mouse T-lymphoma cells treated with Taxol. Cells were treated with 20 p.g/ml taxol at 37°C for 30 minutes, followed by fixation with 2% glutataldehyde and 2% Os04; dehydration with a series of alcohol and embedding with Epon. Thin sections were cut and examined in a Philips 300 electron microscope. The electron micrograph illustrates bundle formation of microtubules (indicated by arrows) radiating from the centrosome (CT) in taxol-treated cell.
CAPPING AND THE CYTOSKELETON
207
skeletal protein, ankyrin, bears a marked resemblance antigenically to one of the microtubule-associated proteins (MAP 1) (Bennett and Davis, 1981). Moreover, ankyrin copurifies with microtubules during successive cycles of polymerization and depolymerization. In addition, another microtubule-associated protein (MAP 2) has been found to be immunologically related to the actin-binding protein spectrin (Davis and Bennett, 1982). It, therefore, seems quite possible that microtubules and microfilaments are closely interacting in the cell through their respective associated proteins. Furthermore, we have recently reported that during colchicine-induced capping the 20,000-dalton light chain of lymphocyte myosin is phosphorylated and preferentially accumulated in the plasma membrane fraction of mouse T-lymphoma cells (Bourguignon et al., 1981). These colchicine-induced effects of myosin light chain phosphorylation and aggregation of actomyosin underneath the capped structure (Bourguignon et al., 1981) may be one of the first pieces of direct biochemical evidence supporting the idea that microtubules are involved in the triggering of microfilament-mediated contractility during capping.
V. Intermediate Filaments Although intermediate filaments have received a great deal of attention in the past few years, it seems fair to say that very little is definitely known concerning the specific function(s) of this most recently discovered class of cytoskeletal filaments. Immunological and biochemical studies have revealed that there are at least five classes of intermediate filaments: vimentin, keratin, desmin, glial filaments, and neurofilaments (Lazarides, 1980, 1982; Steinert et al., 1982). Recently, Franke and his colleagues (Schmid et al., 1983) reported that in the presence of hormones such as hydrocortisone, insulin, and prolactin a bovine mammary epithelium cell line expresses keratin but not vimentin. Another bovine epithelial cell line which had been maintained in culture for a long period of time without hormone supplement was found to contain both keratin and vimentin suggesting that there are extracellular signals which can control the differential expression of intermediate filament proteins. At the present time, it is generally believed that intermediate filaments form the most stable component of the cytoskeleton and are possibly involved in the intracellular distribution of the cellular organelles+specially the nucleus (Lazarides, 1980; Wang and Choppin, 1980). There is now evidence that certain types of intermediate filaments, such as vimentin, interact with microtubules and possibly also with the microfilament network (Geiger and Singer, 1980; Weber and Osborn, 1981; Zackroff er al., 1981; Geuens et al., 1983). Very recently, it has been reported that disruption of the microfilaments (by cytochalasin D) and the microtubules (by colchicine) also induces the reorganization of keratin filaments in culture epithelial cells (Knapp et al., 1983). These data all support the
208
LILLY Y. W. BOURGUIGNON AND GERARD J. BOURGUIGNON
notion that important interactions occur between intermediate filaments and the other major components of the cytokeleton. The first report that intermediate filaments may be involved in the capping process was published by Zucker-Franklin et al. (1979). Electron microscopic studies on normal and chronic lymphocytic leukemic (CLL) lymphocytes showed that bundles of 10-nm filaments accumulated in the region of the cell underneath the cap formation on either normal or certain types of malignant lymphocytes (Zucker-Franklin, 1979). Two groups using double immunofluorescence techniques have recently confirmed that vimentin-type intermediate filaments cocap with different lymphocyte surface receptors such as Con A (Bourguignon and Bourguignon, 1981b), and µglobulin or Ig antigens (Dellagi and Brouet, 1982). Further important evidence that intermediate filaments are involved in cap formation was provided by Dellagi and Brouet (1982) who reported that vimentin is present in the detergent-insoluble cytoskeleton fraction isolated from capped lymphocytes. In addition, vimentin has been recently identified as forming a complex with a 140,000-dalton plasma membrane glycoprotein in cultured human fibroblasts (Lehto, 1983) and has been found attached to the plasma membrane-derived “boundary lamina” of Triton X- 100 treated Ehlich ascites cells (Nelson and Traub, 1981). In this regard, it is also very interesting that two groups have reported evidence suggesting that the cell nucleus is required for capping (Berke and Fishelson, 1976; Otteskoog et al., 1981). In both cases it was shown that enucleated cells lose the ability to cap surface receptors while the karyoplast (nucleus plus a residual thin rim of cytoplasm and plasma membrane) is able to cap normally. In addition, Otteskoog et al. have found that surface receptor capping is accompanied by the aggregation of certain nuclear membrane antigens into a region of the nuclear membrane which is directly opposed to the surface cap structure. Furthermore, intermediate filaments have been found to interact with both the nuclear membrane (Woodcock, 1980) and the plasma membrane (Nelson and Traub, 1981; Lehto, 1983). It is conceivable, therefore, that intermediate filaments could form a physical linkage (possibly via microfilaments) between the cell surface and the nucleus which functions to transmit signals, particles, and/or vesicles directly from the cell surface to the nucleus.
VI. Capping-Related Regulatory Molecules and Enzymes The set of proteins which have been found to be closely associated with surface cap structures now include several important enzymatic activities such as myosin light chain kinase (Bourguignon et al., 1982), 5’-nucleotidase, Na+ -K -ATPase (Raz and Bucana, 1980), and adenylate cyclgse (Bourguig+
CAPPING AND THE CYTOSKELETON
209
non and Hsing, 1983). In addition, several other ions and molecules, known to be involved in muscle contraction and cellular regulation, such as Ca2+, calmodulin, and cyclic AMP (CAMP) have also been implicated in the receptor redistribution process (Bourguignon and Balazovich, 1980; Salisbury et al., 1981; Nelson et al., 1982; Bourguignon and Kerrick, 1983). Our current knowledge in this area is summarized as follows: A. Ca2 -CALMODULIN +
It is well known that Ca2 plays an important role in the regulation of a large number of cellular activities, some of which are related to surface receptor redistribution. Calmodulin is one of several Ca2+ binding proteins found in a variety of eukaryotes which have been shown to be involved in some of these Ca2 regulated processes (Cheung, 1979, 1980). Several antipsychotic drugs, including chloropromazine (thorazine) and trifluoperazine (stelazine), are known to impair the function of calmodulin (Weiss and Levine, 1978; Weiss and Wallace, 1980) and also inhibit surface receptor capping (Bourguignon and Balazovich, 1980; Salisbury et al., 1981; Nelson et al., 1982). Using a double immunofluorescence technique, intracellular calmodulin was found to be accumulated underneath both Con A and Ig cap structures (Salisbury et al., 1980; Nelson et al., 1982). Other studies have indicated that Ca2+ efflux from lymphocytes takes place subsequent to anti-Ig binding to Ig receptors (Braun et al., 1979). Thus, the involvement of Ca2+ and calmodulin during receptor capping is strongly implicated. There are, however, some experimental observations which appear to argue against Ca2 being involved in anti-Ig induced Ig capping. For example, in the presence of a Ca2+ ionophore, Ca2+ does not facilitate Ig capping (Poste and Nicolson, 1976); Ig capping is reversed by a Ca2 ionophore such as A23 187 in the presence of Ca2+ (Schreiner and Unanue, 1976); Ca2+ transients precede Ig capping and reach low levels before capping begins (Pozzan et al., 1982); and Ig capping can take place in the absence of external Ca2 (Schreiner and Unanue, 1976); The inhibition and reversal of capping by the Ca2+ ionophore, A23187 (dose range between l o p 5 and lo-’ M),in the presence of Ca2+ may be due, however, to nonspecific detergency properties of the ionophore rather than a rise in intracellular Ca2 activity (Bourguignon and Pressman, 1983). In smooth muscle, Ca2 reaches high levels prior to contraction and decreases to low levels before contraction begins (Fay et al., 1979). Therefore, some important similaries with regard to internal Ca2 accumulation patterns appear to exist between smooth muscle contraction and receptor capping. Recently, it has been found that low doses (10-7-10-8M) of monensin, a Na ionophore, stimulates lymphocyte receptor capping (Bourguignon and Pressman, 1983). Low doses of monensin are known to increase the intracellular +
+
+
+
+
+
+
+
+
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LILLY Y. W. BOURGUIGNON AND GERARD J . BOURGUIGNON
Na level (Pressman and Fahim, 1982) which subsequently raises intracellular Ca2 activity, either by releasing intraceffufarfysequestered Ca2 or exchanging with extraceflufarCa2 (van Breemen er af., 1979). It is quite likely that the intracellular Ca2 level (not necessarily only the external Ca2 concentration) plays a crucial role in receptor capping process. The apparent discrepancies which argue against Ca2+ involvement during receptor capping may all be explained by the differences in experimental approaches and in the interpretation of experimental results by different laboratories (Schreiner and Unanue, 1976; Poste and Nicolson, 1976; Pozzan et af., 1982). What is needed to definitely decide this issue is a direct demonstration of a Ca2 requirement during receptor capping. In order to allow Ca2 and other small molecules to freely penetrate across the lymphocyte membrane, we have made use of an EGTA-buffered solution to render the lymphocyte membrane reversibly permeable to ions and small proteins (no larger than 80,000 daltons) (Hoar et af., 1979; Bourguignon and Kerrick, 1983). Our data show that increasing concentrations of both Ca2+ (10-5-10-3 M ) and calmodulin (3.9 X 10V8-6.3 X lo-’ M ) cause a graded increase in capping. Most interestingly, the reversal of capping readily occurs upon the removal of Ca2+ (Bourguignon and Kerrick, 1983). These findings strongly support the notion that a Ca2+calmodulin-regulated mechanism is involved in the lymphocyte receptor capping event. These Ca2 -calmodulin-mediated activies may include (1) the association of the acto-myosin containing cytoskeleton with receptors as proposed in the red cell membrane-spectrin system (Nicolson et af., 1971); (2) the activation of myosin light chain kinase to carry out acto-myosin mediated contraction as described in muscle cells (Adelstein and Klee, 1980; Dedman et af., 1979); (3) the regulation of microtubule assembly-disassembly process (Marcum et af., 1978); (4) the control of Ca2 -Mg2 -ATPases of cell membranes and subsequent regulation of the intracellular ionic state of the cell (Kobayashi et af., 1979; Niggli et al., 1979); and (5) the modulation of various cellular enzymes such as adenylate cyclase, guanylate cyclase, Ca2 -dependent protein kinase, phosphorylase kinase, phospholipase, etc. (Cheung, 1980). In this regard, we have recently observed that capping in mouse leukemic cells occurs in a cell cycle-specific manner-primarily in S phase (Bourguignon et af., 1983a). It is noteworthy that calmodulin levels have also been found to increase specifically in late G,/early S phase (Chafouleas et af., 1982) which is consistent with its proposed role in the regulation of the capping process. +
+
+
+
+
+
+
+
+
+
+
+
B. MYOSINLIGHTCHAINKINASE(MLCK) As mentioned earlier, an actomyosin sliding filament mechanism has been suggested as a possible mechanism for aggregating receptors into cap structures (Bourguignon and Singer, 1977). During such an event, bipolar myosin filaments would bridge F-actin filaments from adjacent receptor patches (clusters)
21 1
CAPPING AND THE CYTOSKELETON
enabling the collection of receptor patches into a cap by the cyclic interaction of myosin cross-bridges with actin resulting in the sliding of actomyosin filaments. Recently, using an EGTA-buffered solution we have been able to introduce ATP into lymphocytes and examine directly the role that ATP plays during the capping process. Our results show that ATP (but not other ATP analogs such as ITP, GTP, CTP, and UTP) is definitely required for cap formation (Table 111, Bourguignon and Kerrick, 1983). In muscle, ATP is required for the cyclic interaction of myosin and actin which is the basis for the contraction mechanism (Huxley, 1972). This strong correlation between capping and the specific requirement of ATP is consistent with the hypothesis that capping is the result of a cyclic actomyosin interaction. Studies on both smooth muscle and nonmuscle cells indicate that actomyosinmediated contraction is regulated by myosin light chain kinase which in turn is regulated by Ca2 -calmodulin (Adelstein and Klee, 1980; Dedman et al., 1979). Specifically, the myosin light chain kinase-mediated phosphorylation of myosin light chain (MW 20,000) has been shown (1) to control the change in conformation of myosin molecules from a folded form to an extended monomeric form which is able to assemble into filaments (Craig e t a f . ,1983); ( 2 ) to be directly correlated with the enhancement of the actin-activated myosin ATPase activity (Adelstein and Eisenberg, 1980; Chacko, 1981; Lebowitz and Cooke, 1979; Trotter and Adelstein, 1979); and (3) to be associated with force production in both smooth muscle cells (Aksoy et a f . , 1982; Barron et a f . , 1979; Cassidy et al., 1979; De Lanerolle and Stuil, 1980; Dillon and Murphy, 1982) and actomyosin threads spun from purified proteins (Lebowitz and Cooke, 1978). Phosphorylation of myosin light chains has been found to take place in a +
TABLE I11 EFFECTOF ATP ANALOGSON CAPPING"
Nucleotideb
Conditions"
Percentage capping
ATP ATP ITP GTP CTP UTP
pCA = 8.0 pCa = 4.0 pCa = 4.0 pCa = 4.0 pCa = 4.0 pCa = 4.0
9.0 30.0 9.0 6.0 5.0 3.0
"The standard deviation for capping experiments is 2 5 % . bATP, Adenosine triphosphate; ITP, inosine triphosphate; GTP, guanodine triphosphate; CTP, cytosine triphosphate; UTP, uridine triphosphate. CpCa = -loglo 1 0 - 8 (M)= 8.0; pCa = -loglo 1 0 - 4
(M)= 4.0.
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LILLY Y . W. BOURGUIGNON AND GERARD J . BOURGUIGNON
variety of cell types, including skeletal muscle cells (Barany et af., 1979; Pires and Perry, 1977; Stull and High, 1977), smooth muscle cells (Chacko et af., 1977; Gorecka et af., 1976; Sobieszek, 1977), and nonmuscle cells (Adelstein and Conti, 1975; Daniel and Adelstein, 1976; Scordilis and Adelstein, 1977; Yerna et af.,1978; Trotter and Adelstein, 1979; Bourguignon et af., 1981; Fechheimer and Cebra, 1982; Keller and Mooseker, 1982; Craig et af., 1983) and has also been shown to be involved in a number of cellular motility and secretion-related events (Bourguignon et af., 1981, 1982; Fechheimer and Cebra, 1982; Fox and Phillips, 1982; Keller and Mooseker, 1982). During receptor capping, it has been found that the 20,000-dalton light chain of lymphocyte myosin is both phosphorylated and preferentially accumulated in the plasma membrane of T-lymphoma cells (Bourguignon et af., 1981). As mentioned above, the capping process (1) can be inhibited by the drug trifluorperazine (stelazine) (Bourguignon and Balazovich, 1980; Nelson et af., 1982) which binds to calmodulin in a Ca2 dependent manner; (2) is directly regulated in vitro by micromolar concentrations of Ca2 and calmodulin (Bourguignon and Kerrick, 1983); and (3) involves the phosphorylation of 20,000-dalton myosin light chain. Together, these observations strongly support the contention that capping is a myosin light chain kinase-dependent nonmuscle cell contractile process (Adelstein and Klee, 1980). Recently, a specific antibody raised against chick gizzard myosin light chain kinase (Guerriero et al., 1981) has been utilized to identify the presence of a Ca2 -calmodulin activated myosin light chain kinase in mouse T-lymphoma cells. The lymphocyte myosin light chain kinase is found to be a 130,000-dalton protein which is present in the nonionic detergent insoluble plasma membranecytoskeleton fraction and is preferentially accumulated under the surface Con A cap structure. It has been shown previously that phosphorylation of the kinase significantly lowers the activity of the enzyme (Conti and Adelstein, 1981). During metabolically labeling of cells with inorganic 32Pi,this enzyme has also been found to be phosphorylated in uncapped lymphoma cells. Consequently, under these conditions the light chain of myosin does not appear to be phosphorylated. However, the lymphocyte myosin light chain kinase is able to carry out the phosphorylation of both endogenous lymphocyte myosin light chains and those from smooth and skeletal muscle in a Ca2+-calmodulin dependent and trifuoperazine-sensive manner (Bourguignon et af., 1982). We have also recently demonstrated the functional involvement of myosin light chain kinase during cap formation using a permeablizing EGTA-buffered solution (Kerrick and Bourguignon, 1982). For example, Ca2 -activated capping has been found to be inhibited by the catalytic subunit of cyclic AMP (CAMP)-dependent protein kinase (Kerrick and Bourguignon, 1982) which is known to decrease the activity of myosin light chain kinase (Conti and Adelstein, 1980). Furthermore, we have utilized the ATP analog, ATPyS, which is known +
+
+
+
CAPPING AND THE CYTOSKELETON
213
to serve as a substrate for the myosin light chain kinase. The thiophosphorylated myosin light chains become resistant to the phosphatase resulting in the irreversible activation of actomyosin ATPase activity (Sherry et al., 1978). Our data show that ATPyS in the presence of Ca2+ is able to significantly stimulate cap formation. Other ATP analogs, such as CTP, GTP, and GTPyS, which are not used as a substrate by myosin light chain kinase, do not allow Ca2+-activated receptor capping to occur (Kerrick and Bourguignon, 1982). These data further demonstrate that receptor capping is regulated by a Ca2 -activated myosin light chain kinase. +
CYCLASE C. CYCLICAMP (CAMP) AND ADENYLATE Cyclic adenosine monophosphate (CAMP)has been implicated as an important regulatory molecule in a variety of cellular events (Greengard, 1978; Cheung, 1980). Previously, it has been reported that agents known to elevate intracellular levels of CAMP, such as N6, 02-dibutyryl adenosine 3’,5’-cyclic monophosphoric acid (dibutyryl CAMP) and theophylline, cause either stimulation of T-lymphocyte receptor capping (Butman et al., 1980, 1981) or have no effect on anti-Ig-induced B-lymphocyte capping (Unanue et al., 1973; Schreiner and Unanue, 1975). The discrepancy between these results may be due to different drug response in T and B lymphocytes. The fact that cAMP has been shown to be preferentially accumulated underneath anti-Ig-induced B-lymphocyte patches and caps (Earp et al., 1977; Curtain, 1979) suggests that cAMP may be intimately involved in the Ig-capping of B-lymphocytes. Using a sensitive radioimmunoassay we have found that the level of cAMP increases at least twofold during receptor capping (Butman et al., 1981; Bourguignon and Hsing, 1983). Most recently, it has been reported that membrane-bound adenylate cyclase is induced to cocap with independent membrane molecules such as Thy-1 antigens (Bourguignon and Hsing, 1983). The redistribution of adenylate cyclase, which takes place during receptor capping, suggests that the membrane-bound enzyme may be activated by ligand binding to the surface receptor. Subsequently, the elevated levels of cAMP may then cause the activation of a CAMP-dependent protein kinase (Trotter and Adelstein, 1979). It is speculated that this CAMPdependent kinase could phosphorylate the appropriate cytoskeleton proteins and/or regulatory molecules necessary for the force generating mechanisms to function during the receptor capping process.
VII. Mechanisms of Capping Conflicting points of the view exist with regard to the mechanism of capping, in general, and the transmembrane interaction hypothesis, in particular. Earlier
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data obtained primarily by Unanue and colleagues indicated that there may be two distinct mechanisms of capping for different lymphocyte surface receptors (Schreiner and Unanue, 1977; Braun et al., 1978a,b). One is a rapid, active mechanism involving a transmembrane linkage between the surface receptors and the submembrane microfilament network. The second mechanism is characterized by a relatively slow, passive redistribution of surface receptors in which there is no aggregation of myosin beneath the cap structure. The latter results have not been substantiated in subsequent experiments by several different laboratories. Specifically, our laboratory has consistently observed aggregation of both actin and myosin in the subcap region during capping of all surface receptors studied including those previously examined by Unanue and co-workers (Bourguignon and Singer, 1977; Bourguignon et a l . , 1978a). In addition, no significant differences in the rate of capping have been found by Hoessli et af. (1980) and by Corps et al. (1982) for the two classes of lymphocyte surface receptors distinguished by Unanue et al. Data obtained in our laboratory also agree with the conclusion of Corps et al. (1982) that the rate of capping is highly dependent both on the density of surface receptors and the concentration of crosslinking ligands added to the cells. Corps et al. (1982) have demonstrated that a number of different lymphocyte receptors all cap at about the same optimal rate and are all inhibited to about the same extent by cytochalasin B. Recently, similar results have been reported for three different receptors on the surface of human (Kammer et al., 1983) and rat (Woda and Gelman, 1983) lymphocytes. Therefore, we believe it is very likely that only one mechanism exists for liganddependent capping. There are, however, reports claiming that the capping kinetics for lymphocyte Ig receptors varies with different strains of mice (Fram et af., 1976) or with lymphocytes from different species such as rabbit (de Groot and Wormmeester, 1981) or guinea pig (Rosenthal et al., 1973). Recently, Oliver and Berlin (1982) have proposed a mechanism for capping which does not require the attachment of surface receptors to the submembrane cytoskeletal network. This mechanism (termed a “surfboard” mechanism) involves initially a contraction of the submembrane microfilament network toward one pole of the cell. This contraction sets up waves on the cell surface which sweep only those surface receptors aggregated into clusters or patches toward the region of microfilament accumulation. Although it is possible that this mechanism may occur in certain types of cells or under certain special conditions, the results of our experiments plus those of other investigators appear to argue strongly against such a “surfboard” mechanism applying to the usual capping situation in lymphocytes. For example, we have consistently observed that microfilament aggregation occurs beneath the patched structures which precede the actual capping events; and direct in vivo evidence for the formation of microfilament-surface receptor complex during capping has been recently obtained by Heath (1983). In addition, we and others have recently shown that lymphocyte
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surface receptors become specifically attached to the cytoskeleton at the early patching stage (Braun et al., 1982; Bourguignon et al., 1983b). All other capping models of which we are aware propose that surface receptors become either directly or indirectly linked to the cytoskeleton. Loor (1981), for example, has recently presented a capping mechanism which entails the existence of three different domains of membrane receptors: one controlled by microfilaments, one by microtubules, and one domain of receptors free to diffuse in the fluid membrane. He proposes that the clustering of the freely diffusing surface receptors by the binding of multivalent ligand traps some of the microfilament attached surface components which then form a cap structure via a contractile ring mechanism. The microfilament-attached surface components are analogous to the “X-protein” proposed by Bourguignon and Singer (1977) to be the linker molecules bridging the surface receptors and the intracellular cytoskeleton. Karnovsky and colleagues have presented a model which focuses on lipid/ protein interactions and the role of Ca2 in the capping mechanism (Klausner et al., 1980). They suggest that a transmembrane protein exists which is both attached to the cytoskeleton and reversibly binds Ca2 ion. The cross-linking of surface receptors by multivalent ligands to form patches is thought to induce the association of the aggregated receptors with the transmembrane linker protein and the subsequent release of bound Ca2 required for the cytoskeletal contraction to form a cap structure. Further evidence in favor of a direct transmembrane interaction between surface receptors and the cytoskeleton has been presented by Young-Karlan and Ashman (1981) who have studied the sequential effects of a series of reversible inhibitors of capping. Our current concept of the capping mechanism is basically similar to that proposed earlier (Bourguignon and Singer, 1977). We now propose that there is a minimum, threshold size required for the cluster of patch of surface receptors in order to bind to the putative X-protein. Patch formation may then activate a number of membrane-associated enzymes such as adenylate cyclase (Bourguignon and Hsing, 1983), a membrane-associated proteolytic enzyme and/or a methylation enzyme as is found during the clustering of IgE on mast cells (Ishiyaka, 1982). We believe that the X-protein and its attachment to the cytoskeleton may be very analogous to the cytoskeletal/membrane organization which exists in erythrocytes. Our most recent data indicate that lymphocytes contain a transmembrane glycoprotein (possibly analogous to glycophorin in red blood cells) (Fig. 6), an ankyrin-like (Figs. 2h, i, and 1, and 3e-g) and a fodrin-like (Figs. 2b,d, and f, and 3a-d) molecule all of which cocap with all the surface receptors tested. It has been shown previously that a number of different membrane proteins are associated with the cytoskeleton network in murine cells (Mescher et al., 1981). +
+
+
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LILLY Y. W. BOURGUIGNON AND GERARD J. BOURGUIGNON
-3
x10
B
A
C
D
E
F
G
H
I
200
BO k-
130
78
68
42
25-
?5ka
b
c i a
b cia b c 'a b c
T
FIG. 6 . SDS-PAGE analysis of membrane proteins from NP-40 solublelinsoluble fractions and from total plasma membranes of capped and uncapped lymphocytes. (A-D) Autoradiograms of lactoperoxidase-catalyzed12JI-labeledproteins from mouse-T-lymphoma cells. (A) Uncapped cells. (B) Con A-treated patchedlcapped cells. (C) Thy-I antibody-treated patchedlcapped cells (similar results were obtained from T-200 or viral glycoproteins 69/7 1 antibody-treated cells). (D) Uncapped cells treated with cytochalasin D: (a) 12sI-labeled membrane proteins in NP-40 insoluble fraction of plasma membrane; (b) i2sI-labeled membrane proteins in NP-40 soluble fraction of plasma membrane; (c) '2sI-labeled membrane proteins in total plasma membranes in the absence of NP-40. (E-J) SDS-PAGE and autoradiographic analysis of anti-T-200 ( I80.000 dalton) protein-mediated immunoprecipitates. (E) Coomassie blue staining of 1251-labeledmembrane proteins isolated from uncapped cells. (F-G) Coomassie blue staining (F) and autoradiograms (G) of 12sI-labeled membrane proteins isolated from uncapped cells and imrnunoprecipitated by rabbit anti-T-200 ( 180,000 dalton) protein. (H)Autoradiogram of [3H]glucosamine-labeled membrane proteins isolated from uncapped cells and immunoprecipitated by rabbit anti-T-200 (180,000 dalton) protein. (I-J) Control samples were carried out using preabsorbed antibody (T-200 free antibody)-mediated nonspecific immunoprecipitation from uncapped cells labeled with 12sI (I) autoradiogram; (J) Coomassie blue staining.
J
217
CAPPING AND THE CYTOSKELETON
Recently, we have found that a major glycoprotein with a molecular weight of about 180,000 is preferentially attached to the lymphocyte cytoskeleton during patching and capping. In addition, immunoprecipation of the 180,000-dalton protein by a specific antibody causes selective coprecipitation of fodrin-like polypeptides (Fig. 6). Therefore, we propose that the 180,000-dalton protein and fodrin (spectrin-like) protein are closely associated in vivo. Our preliminary data indicate that the 180,000-dalton protein may be a glycophorin-like transmembrane molecule (unpublished observation). Direct visualization of the lymphocyte membrane/cytoskeleton organization in uncapped cells using freeze-fracture and deep-etching techniques (Heuser and Salpeter, 1979) is provided in Fig. 7. In addition to the submembranous microfilaments (MF) and intermediate filaments (IF), one can readily observe in these electron micrographs a class of very thin filaments (2-3 nm in diameter) which appear to link microfilaments to the plasma membrane. It is quite possible that these filaments contain fodrin-like molecules since fodrin (a spectrin-like protein) is known to form filaments of this diameter (Glenney et al., 1982). We also propose that the increased concentration of intracellular Ca2 upon binding of the patch to the cytoskeleton is responsible for inducing the local disassembly of microtubules and is required for the contraction of the actin/myosin network formation during the capping event. The specific source and mechanism of Ca2+ action remains to be determined. One possible source of Ca2 ion could be the plasma membrane or plasma membrane-associated Ca2 binding proteins as previously suggested by Klausner et al. (1980). Our current model for receptor capping is presented in Fig. 8. +
+
+
VIII. Concluding Remarks Although our knowledge concerning the capping process has increased greatly in the last few years, it is obvious that much more work is required to attain a complete understanding. We now seem to be close to having a good picture of the basic mechanism, especially with regard to the cytoskeleton. Much less clear is our understanding of the mechanisms involved with Ca2+ and cyclic AMP regulation, the protein phosphorylation events and the possible role of phospholipid metabolism in capping. In addition, further information on the interactions which probably occur between microfilaments, microtubules, and intermediate filaments should prove to be very important. In particular, we feel that intermediate filaments may function as anchoring sites toward which the microfilament network contracts during capping. Because intermediate filaments can potentially form a physical link between the plasma membrane and the nuclear membrane, it is also possible that they are responsible for transmitting important
FIG. 7. Organization of lymphocyte membranekytoskeleton visualized by freeze-fracture and deep-etching. Mouse T-lymphoma cells were quick-frozen with a liquid helium cooled machine and freeze-fractured in a Balzers apparatus as described previously (Heuser and Salpeter, 1979). Deepetching was carried out at -95°C for 5 minutes. Replicas were made by rotary shadowing with a mixture of platinum and carbon after the sample had been cooled to either - 120 or - 196°C. Samples were examined with a JOEL IOOCX electron microscope. The electron micrographs show bundles of microfilaments (MF) accumulated immediately underneath the plasma membrane (Pm). Some intermediate filaments (IF) were noted to be closely associated with MF and vesicles (V) in the cytoplasm. A class of thin filaments (arrowhead), possibly fodrin-like molecules, form a link between plasma membrane and microfilaments as well as cross-link the microfilament network. (Micrographs were obtained as a collaborative effort between J. Heuser and our laboratory.)
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CAPPING AND THE CYTOSKELETON
A
C
0
C
FIG. 8. Model for ligand-dependent surface receptor capping. I , Surface receptor; 2. other surface proteins; 3, transmembrane cross-linking proteins; 4, fodrin-like molecule either directly or indirectly attaching microfilaments (MF) to X protein(s); 5, G-actin; 6, F-actin chains (microfilaments or MF); 7, myosin filaments; 8, multivalent ligand. (A) Uniform distribution of unbound surface receptors. (B) Patches of ligand-bound surface receptors; they occur spontaneously at 0-4°C and cause an increase in intracellular Ca2+ concentration. (C) Cap formation; it occurs via an energy and temperature-dependent contractile process analogous to the sliding filament mechanism in muscle cells. It is important to keep in mind that this is a highly schematic diagram which undoubtedly will require future modification. For clarity, microtubules, intermediate filaments, calmodulin, myosin light chain kinase, and adenylate cyclase have not been included in the diagram. Their possible roles in the capping mechanism are described in the text.
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signals, or even membrane vesicles, from the cell surface to the nucleus during or following cap formation. Finally, it will be necessary to define more precisely the functional role of capping in both lymphocytes and other cell types. In this regard, we believe it is likely that external ligand (antibody, lectin, or some hormones)-mediated receptor patching and capping may be responsible for the following important immune-related responses: ( 1) proliferation and differentiation of the cells into antibody-secreting plasma cells (Koros et al., 1968; Tannenberg and Malaviya, 1968; Nossal, 1962); (2) the development of an increased number of precursor cells (Nossal, 1962; Gowans and Uhr, 1966; Sercarz and Coons, 1962) which are responsible for immunological memory; and (3) activation and proliferation of T cells which carry out cell-mediated cytotoxic killing (Shearer and Schmitt-Verhulst, 1977; Snell, 1978; Zinkernagel and Doherty, 1979). In nonimmune cells, capping may also be involved in such important cellular processes as endocytosis, chemotaxis, mitogenesis, and general cell-cell recognition.
ACKNOWLEDGMENT This work was supported by U.S. Public Health Grant A1 191 18.
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INTERNATIONAL REVIEW OF CYTOLOGY. VOL. 87
The Muscle Satellite Cell: A Review DENNISR. CAMPION USDA-SE-ARS, Animal Physiology Unit? Richard B . Russell Agricultural Research Center, Athens, Georgia, and Department of Foods and Nutrition, University of Georgia, Arhens, Georgia I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Cell Location . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Skeletal Muscle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Cardiac Muscle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Gross Morphology.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Fine Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Situations Affecting Satellite Cell Content A. Normal Growth . . . . . . . . . . . . . . . . . B. Nutrition.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Muscle Regeneration and Compensatory Hypertrophy . . . . . . . . VI. Activation Stimulus.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Summary .................... . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
225 226 226 231 231 233
243 244 245 246 247
I. Introduction During normal growth of skeletal muscle the total amount of DNA increases as muscle mass increases. Because of the constancy of the DNA content per nucleus within a species, it is reasonable to conclude that the number of myonuclei increases during growth. The fact notwithstanding that approximately 25% of the nuclei in human muscle (Cheek er al., 1971), 29-40% of the nuclei in rat muscle (Enesco and Puddy, 1964; Ontell, 1974; Schmalbmch and Hellhammer, 1977) and 41% of the nuclei in androgen-sensitive muscle (Venable, 1966) are extraplasmalemmal in location, considerable microscopic evidence is available to verify that the nuclear content of individual myofibers increases during normal growth (e.g., Enesco and Puddy, 1964; MacConnachie et a l ., 1964; Moss, 1968). Embryologically, mononucleated myoblasts fuse, forming a syncytium or myotube. The myotube ultimately develops into the mature myofiber. A point germane to the topic of this article is that investigations using a variety of techniques to study myogenesis in siru (e.g., Przybylski and Blumberg, 1966; Shafiq er al., 1968; Kelly and Zacks, 1969; Stockdale, 1970; Moss and Leblond, 1971; Allbrook et al., 1971; Cardasis and Cooper, 1975), and in virro (e.g., 225 ISBN 0-12-3644117-9
226
DENNIS R. CAMPION
Stockdale and Holtzer, 1961; Okazaki and Holtzer, 1966; Bischoff and Holtzer, 1969; Richler and Yaffe, 1970; Bischoff, 1974, 1975; Konigsberg et a l . , 1975; Yeoh and Holtzer, 1977; Pullman and Yeoh, 1978; Yeoh et a l . , 1978), have demonstrated that the nuclei of the myotube do not divide. Thus, nuclei contained within the myofiber, at least in the normal situation, are not capable of mitosis. In situations wherein there is a biological need to generate additional nuclei for normally growing myofibers, to generate new myofibers, or to repair damaged or diseased myofibers, the primary source of these nuclei is thought to be the satellite cell. This cell was first identified by name by Katz (1961) and Mauro (1961). Mauro (1961) originally suggested that satellite cells may be the source of nuclei added to myofibers during regeneration. In this article, the topic of the satellite cell in relation to muscle regeneration is limited as this area has been the subject of several recent reviews (Carlson, 1973; Reznik, 1976; Allbrook, 1981; Klishov and Danilov, 1981) and conferences (Mauro et al., 1970; Mauro, 1979). In the recent review of myogenic cell proliferation by Allen et al. (1979), particular attention was focused upon the importance of satellite cell and presumptive myoblast proliferation to meat animal production. Therefore, this latter relation is only minimally stressed in the present article.
11. Cell Location
A. SKELETAL MUSCLE The satellite cell is a mononucleated cell that lies under or embedded in the basal lamina of the myofiber. Frequently, the satellite cell lies within a groove that is parallel to the long axis of the myofiber. Some variation has been noted, however, as satellite cells were observed by scanning electron microscopy and by special lead staining techniques and light microscopy to be occasionally oriented obliquely or transversely to the long axis of myofibers of the frog sartorius muscle (Franzini-Armstrong, 1979; Larocque et al., 1980; Mazanet et al., 1982). A gap of 15-60 nm separates the opposing cell walls of the satellite cell and the myofiber. The wider spacings are more commonly found in immature muscle (Schultz, 1976). Generally, basal lamina material is not seen in the intervening gap. A notable exception, however, is Rana pipiens. Maruenda and FranziniArmstrong (1978) reported that in 21% of the satellite cell profiles in the sartorius muscle, basal lamina had penetrated a variable distance into the gap region. In addition, 6% of the satellite cell profiles was completely encapsulated by the basal lamina of the attendant myofiber. Other, less extensive exceptions have also been noted. Protrusion of basal lamina material into the gap region has
THE MUSCLE SATELLITE CELL
227
been observed in the lumbrical muscle of growing mice (Schultz, 1976) and in the soleus muscle of aging mice (Snow, 1978). Kelly (1978a) reported insinuation of basal lamina between the opposed plasma membranes of perisynaptic satellite cells and myofibers in the soleus muscle of the mature rat. In the tail muscles of lizards, the basal lamina did not protrude into the gap region (Kahn and Simpson, 1974). Several lines of evidence exist to demonstrate that satellite cells are independent of the adjacent myofibers. Muir el al. (1965) observed that cut, web-muscle fibers that had been immersed in 2.5 M sucrose became distended. The density of the satellite cells, on the other hand, was increased, presumably due to extraction of water from the satellite cells. These authors concluded, therefore, that there was no continuity of satellite cell cytoplasm with myofiber cytoplasm. A similar conclusion was reached for the satellite cells of frog muscle wherein it was found that horseradish peroxidase injected into the myofiber never appeared in the cytoplasm of the satellite cell (Cull-Candy et al., 1980). In addition, Schmalbruch (1978), in the only freeze-fracture study of satellite cells, saw no membrane specializations between opposing cell walls of satellite cells and myofibers of the adult rat soleus muscle. However, desmosome-like specializations were reported in the craniovelar muscle of the Atlantic hagfish (Sandset and Korneliussen, 1978). These specializations were not noted in the parietal muscle of this species. Hess and Rosner (1970) observed thickening in the membranes of satellite cells and myofibers of the extraocular muscles of the guinea pig. But the two membranes were not joined at these thickenings. The significance of these membrane specializations is not known. Satellite cell nuclei often appear near myofiber nuclei when muscle cross sections are examined (Allbrook et al., 1971; Sandset and Korneliussen, 1978; Schmalbruch and Hellhammer, 1977; Ontell, 1974, 1977). In the sartorius muscle of the fetal pig (Campion et al., 1978) and in the soleus muscle of the adult mouse (Snow, 198l), satellite cells are uniformly distributed throughout the length of the muscle. Maruenda and Franzini-Armstrong (1978) reported a significantly higher incidence of satellite cells in the distal portion of the adult frog sartorius muscle. However, when the data of these two authors were recalculated by Snow (1981), it became evident that the mean percentage of satellite cells associated with the distal, belly, and proximal locations was similar. Satellite cells are frequently seen in close association with cross-sectional profiles of myoneural junctions of the soleus (Kelly, 1978a; Cardasis and Padykula, 1981; Snow, 1981; Gibson and Schultz, 1982) and diaphragm muscle of the rat (Cardasis, 1979), and with the nerve terminals on intrafusal fibers of frog sartorius muscle (Katz, 1961). In the adult rat soleus muscle, there is a nearly 20-fold higher incidence of satellite cell nuclei in the synaptic than in the nonsynaptic area (Kelly, 1978a). A 14-fold higher incidence of satellite cells was reported in the synaptic than in the nonsynaptic area of the adult mouse soleus
228
DENNIS R. CAMPION
muscle (Snow, 1981). But, because the number of perisynaptic satellite cells constitutes a relatively small proportion of the total number of satellite cells, and because the frequency of sectioning through a nerve terminal is low, the effect of perisynaptic satellite cells on experimental quantitation of the percentage of nuclei within the basal lamina that are satellite cell nuclei is minimal (Snow, 1981). Interestingly, perisynaptic satellite cell clusters were not observed in the rat sternomastoid muscle (Mazanet, 1981). And their incidence was relatively low in the extensor digitorum longus muscle of the rat when compared to the soleus muscle (Kelly, 1978a; Gibson and Schultz, 1982). Thus, not all skeletal muscles within a species exhibit similar characteristics with respect to linear distribution of satellite cells. In anuran gastrocnemius muscle (Trupin, 1976), there is no obvious association between the satellite cell and the motor-end plate or vasculature. More than half of the satellite cells in rat muscle are associated with a capillary (Schmalbruch and Hellhammer, 1977). No difference in the incidence
INFLUENCE OF FIBERTYPEO N OF
Muscle Axolotl, trunk muscle "Young" Red Intermediate White Shark, axial muscle Red fibers White fibers Atlantic hagfish, parietal muscle Red fibers White fibers Atlantic hagfish, craniovelar muscle Red
TABLE I INCIDENCE OF NUCLEIIN TRANSVERSE SECTIONS MUSCLEFROM THE ADULT THE
Incidence of muscle fiber nuclei/fiber transected
Incidence of satellite cell nuclei/fiber transected
(a)
(%)
47 35 59 I00
0 (58)a 0 (135p 11.8 (l53)<' 0 (220)l'
Percentage nuclei within basal lamina that are satellite cell nuclei (70)
Reference Flood ( I97 I )
Kryvi and Eide (1977) 79 62
6.0 4.0
7.1 6. I
Sandset and Korneliussen (1978) 78 200
27 27
11.1
2.8
Sandset and Korneliussen (1978) 43
30
uPercentags based on incidence of cytoplasmic profiles
22.6
THE MUSCLE SATELLITE CELL
229
of satellite cells was noted in the myotendinous or myoneural junction of web muscles in the fruit bat (Muir et al., 1965). Satellite cells, in addition to their presence on extrafusal muscle fibers, are also present on intrafusal muscle fibers. This latter relationship was reported in frog (Katz, 1961; Karlsson et al., 1966; Karlsson and Anderson-Cedergren, 1971), mouse (Rumpelt and Schmalbruch, 1969; Snow, 1977a), rat (Landon, 1966; Rumpelt and Schmalbruch, 1969; Maynard and Cooper, 1973; Anastasi et a / . , 1979), cat (Adal, 1969), man (Rumpelt and Schmalbruch, 1969), dog (Banker and Girvin, 1971), and snake (Fukami, 1982) muscle. Bird and Allbrook (1980) calculated that the ratio of satellite cell nuclei to myofiber nuclei was 1:9 in chain fibers and 1:5 in bag fibers in the juxta-equatorial region of rat lumbrical muscle. In the less complex spindle of amphibian muscle, satellite cells are commonly present in the sensory compact zones. They are seldom seen in the reticular or motor compact zones (Karlsson et al., 1966). Satellite cells exist in association with the various fiber types present in skeletal muscle (Tables I, 11, and 111; Takahama, 1983). Schmalbruch and Hellhammer (1976) reported that satellite cells were associated with the various fiber types present in human skeletal muscle. They did not attempt to segregate satellite cell content by fiber type. However, from the fiber type data given in Table I, no unique relation is evident between fiber type and the incidence of satellite cell nuclei. Gibson and Schultz (1982) examined in detail the relation between the incidence of satellite cells and fiber type to determine if differences in fiber type
INFLUENCEOF FIBERTYPEON OF
Muscle Rat muscles Soleus EDL
Rat muscles Soleus Diaphragm Tibialis anterior
TABLE I1 INCIDENCEOF NUCLEI I N TRANSVERSE SECTIONS MUSCLEFROM THE ADULT THE
Incidence of muscle fiber nuclei/fiber transected (%)
Incidence of satellite cell nucleilfiber transected (%j
5-6.4 I .7-2.9
2.7 1.o
(4.6 x 1 0 4 ~ (6.4 x 1 0 4 ) ~ (2.3 x lo4)"
"Number of nuclei per mm3 muscle
Percentage nuclei within basal lamina that are satellite cell nuclei (%j
(4920)" (5310)" (920)"
10.7 8.5 4.0
Reference Aloisi et a / . (1973) Kelly (1978b); Gibson and Schultz (1982) Schmalbruch and Hellhammer (1977)
230
DENNIS R . CAMPION
TABLE I11 PERCENTAGE FIBER TYPECOMPOSITION IN THE GENERAL FIBERPOPULATION AND SATELLITE CELL FIBER POPULATION OF ADULTRAT MUSCLESU
IN THE
Satellite cell-fiber population
Satellite cell/general
Muscle
Fiber type
General fiber population
EDL
Type I1 A Type 1 Type I1 B
14.1 41.5 38.4
23.8 43.8 32.5
1.68 0.92 0.84
Soleus
Type I1 A Type I
14.9 85.1
4.0 96.0
0.26 1.12
“Adapted from Table 3, Gibson and Schultz (1982).
frequency could account for the higher satellite cell content of the rat soleus relative to the extensor digitorum longus muscle. The relative frequency of fiber types in the general population and in the population of fibers that contained satellite cell nuclei in muscle sections is given in Table 111 for the adult rat. The fiber type composition was significantly different between the general population and satellite cell-fiber population for both muscles. The satellite cell-fiber is the ratio of frequency of a given satellite cell-fiber type to the frequency of the corresponding fiber type in the general population. A ratio greater than 1 indicates that the particular fiber type has a relatively greater number of satellite cells per unit length. Since in the extensor digitorum longus, Type I1 B (glycolytic) fibers exhibited a satellite cell population which approximated that of Type I (red) fibers, it was concluded that the higher incidence of satellite cells in soleus muscle was not due to the higher proportion of red fibers present in this muscle when compared to the extensor digitorum longus muscle. A meaningful relation is established when the satellite cell content is compared with the myonuclear-cytoplasmic volume ratio (Table 11). Schmalbruch and Hellhammer (1977) observed that the concentration of satellite cells/mm3 muscle was positively associated with the concentration of myonuclei/mm3 muscle. Thus, a muscle (or fiber type) that possesses a high concentration of myonuclei will have a correspondingly high concentration of satellite cells. Expressing the data on a per unit volume basis eliminates the bias inherent in determining the incidence of satellite cell nuclei per fiber transected that results from variability in fiber diameter of the different fiber types. For example, in shark axial muscle (Table I), the incidence of satellite cell nuclei per fiber transected was 6% in the red fibers and 4% in the white fibers. But, because the red fibers were smaller in diameter and had a higher myonuclear-cytoplasmic volume ratio than the white fibers (Kryvi and Eide, 1977), the concentration of satellite cell/mm3 muscle must have been
THE MUSCLE SATELLITE CELL
23 1
greater in the red portion of the muscle. A similar association can be drawn between red and white muscle portions of the Atlantic hagfish as the red fibers of the parietal and craniovelar muscle were smaller in diameter than the white fibers (Korneliussen and Nicolaysen, 1973) of the parietal muscle. B. CARDIAC MUSCLE Satellite cells are present in the striated, skeletal muscle of almost all species of vertebrates (for representative listings see Muir, 1970; Midsukami, 1981). Possibly one exception to this rule exists as satellite cells are not evident in uninjured adult newt limb muscle (Hay, 1979; Popiela, 1976). Interestingly, satellite cells have not been found in vertebrate cardiac muscle (Mauro, 1961; Stenger and Spiro, 1961; Shafiq et al., 1968). But satellite cells have been identified in the cardiac muscle of a number of species in the order Decapoda (Midsukami, 1964, 1981; Aizu, 1973). In species of this order the cardiac satellite cells show close structural similarity to those cells described in other striated muscles. The cardiac satellite cells frequently extend long, thin processes into the cardiac fibers. These cells possess neither lipid droplets nor glycogen. Presumably their origin and function are similar to their skeletal muscle counterparts (Midsukami, 1981).
111. Gross Morphology
Muir (1970) described the satellite cell as fusiform in shape. However, considerable variation exists. For example, satellite cells on intrafusal muscle fibers of the frog were reported to ramify into smaller cell processes (Karlsson et al., 1966). When studied by freeze-fracture (Schmalbruch, 1978) lateral projections from the satellite cells of rat soleus muscle were seen to extend over the attendant myofiber. These projections resided in grooves on the surface of the myofibers as did the satellite cell proper. With scanning electron microscopy, tails were seen to eminate from central, longitudinally oriented fusiform bodies of satellite cells in frog sartorius muscle (Mazanet et al., 1982) and in rat sternomastoid muscle (Mazanet, 1981). The tails were generally paired and originated from either end of the cell body. When a third tail was evident, it originated either as a branch from an existing tail or from the cell body. In the frog sartorius muscle (Mazanet et al., 1982), the fusiform cell bodies were 7-15 pm long and 3-5 km wide. Tails extended up to 40 pm in length from either end of the cell bodies. While the satellite cell surface generally appeared smooth in the freeze-fracture study (Schmalbruch, 1978), use of scanning electron microscopy revealed that the cell body and tails of a few satellite cells were serrated. Serration, when evident, was more extensive in the tail than in the cell body. When serrations were present on
TABLE IV IN NORMAL MUSCLE SATELLITECELLDIMENSIONS Nuclear dimensions (pm)O Species
Muscle
Fruit bat Human, cat, and dog Mouse and rat Frog Fruit bat
Web Gastrocnemius Levator ani EDL (spindle) Web
Guinea pig Dog Axolotl Lizard Mouse Shark R . clamitans R . pipiens Rat Rat
Eye Gastrocnemius Trunk Tail Lumbrical Axial Gastrocnemius Gastrocnemius Hindlimb Soleus Tibialis anterior Diaphragm sartorius
R . pipiens Hagfish
R . pipiens
Parietal and craniovelar Sartorius and peroneus longus sartorius
Length
Width
Depth
Similar (8-12) 13 (16) 20 5.5-10.0 ( 10.0- 12.5)
(3) 4.5
(7)
Satellite cell length (pm) 25 30
2.8 (2.5) 50
25 20 10
2.8-3.5
600 <800 18.56 100
<2 (2-3)
Similar (12.6) 12 12.4 (23.1) 12.8 (24.0) 12.1 (12.6) 9.6 (13.4) 9.6 (16.0) 9.9 (11.5) 20.4 (22)
4.7 (5.1) 4.4 (4.9) 4.3 (5.9)
1.1 (2.6) 1.3 (2.4) 2.4 (2.8)
4.7 (4.6)
1.5 (1.2)
10-15
5
Similar (12.2)
OValues for myofiber nuclei are given in parentheses. Walculated from data in Tables 1 and 2 of Schultz (1974). CCell bodies 7-15 pm, tail extensions up to 40 pm long according to Mazanet er a / . (1982). dCalculated from data of Campion et nl. (1978, 1981a) for belly of muscle.
7-55c
Reference Muir et al. (1965) Ishikawa (1966) Venable ( 1966) Karlsson et al. (1966) Church ( 1970) Hess and Rosner (1970) Banker and Girvin (1971) Flood (1971) Kahn and Simpson (1974) Schultz (1974) Kryvi (1975) Trupin (1976) Trupin (1976) Snow (1977a) Schmalbruch and Hellhammer (1977)
30-50
Maruenda and FranziniArmstrong (1978) Sandset and Komeliussen (1978)
24.4d
Campion er a/. (1979)
40-90
Larocque er al. (1980)
THE MUSCLE SATELLITE CELL
233
the cell body, they were also present on the tails. This was not always the case in the converse situation. Kryvi (1975) also proposed that the satellite cells of G . melastomus axial muscle possessed bifurcating extensions. Extensions of satellite cell cytoplasm into the myofiber have been observed. In the case of the shark, these extensions can penetrate up to 7 pm into the myofiber (Kryvi, 1975). While the distinction may be a qualitative one, cytoplasmic projections of the myofiber into the satellite cell have only been reported in the muscle of the Atlantic hagfish (Sandset and Komeliussen, 1978). The length of the satellite cell generally ranges between 18 and 50 pm (Table IV). Lizard tail muscle (Kahn and Simpson, 1974) and axolotl trunk muscle (Flood, 1971) possess satellite cells of unusual length. In axolotl trunk muscle, satellite cell nuclei are rarely transected, but cytoplasmic profiles are numerous. The incidence of satellite cells based on cytoplasmic processes may be overestimated if these processes bifurcate or branch. The linear dimensions of the satellite cell nucleus are similar to or slightly smaller than comparable measurements of the myofiber nucleus. Typically, satellite cell nuclei measure 10-15 pm in length, 2-5 pm in width, and 1.1-2.8 pm in depth.
IV. Fine Structure With few exceptions (Flood, 1964; Laguens, 1963; Midsukami, 1964; Muir et al., 1965), the satellite cell nucleus has been described as more heterochromatic than the myofiber nucleus (Fig. 1). The distinction, however, is not always apparent. Occasionally, a myofiber nucleus may exhibit a relatively high degree of heterochromaticity. Moss and Leblond (1971) suggested that such myonuclei were recently incorporated from satellite cells. An oval shape is characteristic of satellite cell nuclei transected in a plane perpendicular to the long axis. Some variation in shape exists, however, as indentations (Maruenda and Franzini-Armstrong, 1978; Takahama, 1983) and an irregular outline (Karlsson et al., 1966) of satellite cell nuclei were reported in frog skeletal muscle. Occasional in-foldings were seen in satellite cell nuclei in human skeletal muscle (Wakayama, 1976) and in the muscle of malnourished or recovering children (Hansen-Smith et al., 1979). However, this situation was not observed in other studies of human muscle (Laguens, 1963; Ishikawa, 1966; Reger and Craig, 1968) which suggests that the satellite cell nucleus is more characteristically oval in shape. Lipofusin is present in the cytoplasm of satellite cells of more mature animals. These inclusions were reported in frog (Trupin, 1976), Atlantic hagfish (Sandset and Korneliussen, 1978), shark (Kryvi, 1975), mouse (Schultz, 1976), and human (Conen and Bell, 1970) satellite cells. The significance of the presence of
234
DENNIS R. CAMPION
FIG. 1. Satellite cell in the sartorius muscle of an oblob mouse at 2 weeks of age.The satellite cell lies beneath the basal lamina (arrowheads) of the myofiber. The chromatin material is slightly more heterochromatic in the satellite cell nucleus than in the myofiber nucleus. The high nuclearlcytoplasmic volume and paucity of cytoplasmic organelles is indicative of a relatively inactive satellite cell. ~ 2 1 , 0 0 0 .
THE MUSCLE SATELLITE CELL
235
lipofusion is not known. Specific mention of the presence of lysosomes has been made for satellite cells of the frog (Franzini-Armstrong, 1979), Atlantic hagfish (Sandset and Korneliussen, 1978), shark (Kryvi, 1975), and pig (Campion et al., 1978). The diversity of species within which lipofusin and lysosomes have been described suggests that they are normally occurring structures in satellite cells. The presence of glycogen, on the other hand, is restricted to the satellite cells in the axial muscles of G . melastomus (Kryvi, 1975). Using differential staining techniques to distinguish between small glycogen particles and free ribosomes, Schiaffino and Hanzlikova (1972) and Galavazi (1971) demonstrated the absence of glycogen particles in the satellite cells of young and adult rat skeletal muscle, respectively. The absence of glycogen particles in satellite cell cytoplasm has been reported in several other species, namely, human (Ishikawa, 1966; HansenSmith et al., 1979), cat, dog (Ishikawa, 1966), shark (Flood, 1964), mice and rats (Snow, 1977a), and pig (Campion et al., 1978). Glycogen particles are also not evident in satellite cells of Japanese quail (Coturnix coturnix japonica) and ob/ob mice (Campion, unpublished). Myofilaments are characteristically absent in satellite cells while the presence of microtubules and microfilaments is often observed. The mitochondria of satellite cells are few in number and smaller in size and exhibit fewer internal cristae when compared to myofiber mitochondria. Centrioles have been observed in the satellite cells of normal skeletal muscle of human, cat, dog (Ishikawa, 1966), pig (Campion et al., 1978), rat (Snow, 1977a), mouse (Muir et al., 1965; Shultz, 1976), fruit bat (Muir et al., 1965), frog (Franzini-Armstrong, 1979), and Atlantic hagfish (Sandset and Kornelieussen, 1978). Another characteristic observation is that pinocytotic vesicles are present at the satellite cell wall exposed to basal lamina as well as that exposed to the myofiber. Lamellar bodies were reported within endoplasmic reticulum cisternae and the nuclear envelope of satellite cells in the muscle of children and adults (Wakayama et al., 1981). The structures were most frequently observed in the space between the satellite cell and myofiber. Wakayama et al. (1981) proposed that lamellar bodies were part of a mechanism to transfer or exchange phospholipid material between satellite cell and fiber. As yet, there is no proof of this hypothesis. Bearing in mind that a spectrum of fine structural detail exists, satellite cells can be classified at the extremes as being either active or inactive based on fine structural analysis. The variation in ultrastructural detail was most eloquently described by Schultz (1976) for the mouse lumbrical muscle, by Snow (1977a) for the soleus and gastrocnemius muscles of the mouse and rat, and by HansenSmith et al. (1979) for the vastus lateralis muscle of humans. The physiological state of the muscle is basically reflected in the proportion of active to inactive satellite cells, e.g., in young, growing animals a high propor-
236
DENNIS R. CAMPION
tion of metabolically active cells is seen while the opposite is apparent at older ages. The ultrastructural characteristics described above are typical of satellite cells in general. But, in inactive cells the nuclear-cytoplasmic volume ratio is relatively greater, and the nucleus may be slightly more heterochromatic and nucleoli are very infrequent when compared to the activated state. If rough or granular endoplasmic reticulum is present, segments are short and fragmented. Kahn and Simpson (1974) stated that rough endoplasmic reticulum was not present in the satellite cells of lizard tail muscle. This latter situation is certainly unusual and represents the extreme. A variable number of free ribosomes are located in the cytoplasm. When observed, the Golgi apparatus possesses few folds and is otherwise poorly developed. Cilia are found in the satellite cells of healthy muscle from the fruit bat (Muir et al., 1965) and the mouse (Schultz, 1976) but their function is not known. The more dynamic state of the satellite cell is characterized by a lower nuclear-cytoplasmic volume ratio which is a result of the elaboration of cyto-
FIG. 2. Satellite cell in peroneus longus muscle of the pig at 95 days of gestation. The elaborate Golgi apparatus (arrowheads), and the presence of a centriole (arrow) in this satellite cell is indicative of a relatively more active cell. X 10,200.
FIG. 3. Satellite cell in the sartorius muscle of an oblob mouse at 2 weeks of age. Many pinocytotic vesicles (mows), elaborate channels of rough endoplasmic reticulum (arrowheads), and the presence of a relatively greater number of mitochondria indicate an active cell. Note that the nuclear chromatin is relatively similar to the inactive satellite cell depicted in Fig. 1 . The pronounced heterochromaticity of this active satellite cell is in contrast to the more nearly euchromatic appearance of the active satellite cell of Fig. 2. X21,OOO.
238
DENNIS R. CAMPION
plasmic organelles (Figs. 2 and 3). Nuclear chromatin may become more euchromatic and the nucleoli may become more prominent (Fig. 2). But a heterochromatic appearance of the nuclear chromatin is also commonly found (Fig. 3). The Golgi is well developed and more extensive. The rough endoplasmic reticulum is more elaborate and the channels appear longer and more torturous. Schultz (1976) suggested that basal lamina material might be produced by the satellite cell; but this hypothesis has not been tested. Satellite cells of mouse lumbrical muscle (Schultz, 1976) have polysomes of 5 to 6 units, while in the pig strands of 10 to 12 units are apparent (Campion et al., 1978). Based on the work of Heywood and Rich (1968) strands this length should be capable of producing proteins of 16,000 to about 35,000 daltons, respectively.
V. Situations Affecting Satellite Cell Content A. NORMALGROWTH The addition of myonuclei to growing fibers is brought about through mitotic activity (MacConnachie et al., 1964). That satellite cells undergo mitosis in vivo during normal growth and development is well documented. For example, satellite cells have been observed in metaphase after arrest with colchicine (Shafiq et al., 1968; Allbrook et al., 1971; Hellmuth and Allbrook, 1973). DNA synthesis and mitotic activity have also been demonstrated by the use of [3H]thymidine (Moss and Leblond, 1970, 1971; Allbrook el al., 1971; Hellmuth and Allbrook, 1973; Snow, 1977c; Kelly, 1978b). In addition, proliferation of satellite cells in cultures of isolated fiber preparations has been observed in mammalian (Bischoff, 1975) and in avian (Konigsberg et al., 1975) species. No evidence of mitosis in myofiber nuclei was found in any of these studies. From the original studies of Moss and Leblond (1970, 1971), the appearance of [3H]thymidinelabeled myonuclei was considered to be the result of incorporation of daughter satellite cell nuclei that were labeled during mitosis of the mother cell. Thus, nuclei added to muscle fibers during normal growth and development originate from satellite cell nuclei. Presumably, the daughter nuclei can be added to the growing myofibers without regard to location on the fiber (Snow, 1979). Satellite cells are first distinguishable morphologically when the basal lamina begins to envelope the individual muscle fibers (Church, 1969; Dorn, 1969; Kelly and Zacks, 1969; Conen and Bell, 1970; Ontell, 1974; Cardasis and Cooper, 1975; Ontell and Dunn, 1978). Usually by birth to 1-2 weeks postnatally in mammals and by the time of hatching in fowl, the basal lamina fully encompasses the individual myofibers. As the basal lamina forms it “entraps” myoblastic cells. These myoblastic cells, or satellite cells, become situ-
THE MUSCLE SATELLITE CELL
239
ated between the cell wall of the myofiber and the basement membrane. These cells remain throughout life. For example, Schmalbruch and Hellhammer (1976) observed a satellite cell in the extensor digitorum muscle of a 73-year-old man. Their presence has also been verified in the levator ani muscle of 3-year-old rats (Gutmann and Hanzlfkovi, 1972) and in the muscles of 29- to 30-month-old mice (Snow, 1977a; Allen et al., 1982) and rats (Schultz and Lipton, 1982). In the mouse, deposition of basal lamina happens about the nineteenth day of gestation (Cardasis and Cooper, 1975). However, newly deposited basal lamina material encompasses clusters of fetal myoblasts, myotubes, myofibers, satellite fibers, and other cell types (Kelly and Zacks, 1969; Ontell, 1977; Ontell and Dunn, 1978). Unless serial section is used at these early developmental stages to differentiate among the various cell types, identification of cells that contain no nucleus or myofibrils in cross section cannot be conclusively labeled as satellite cells or as early formed myotubes. In the early stages of development, when the incidence of satellite cells is high, many of these cells appear activated by the ultrastructural criterion outlined in Section IV. This morphology is qualitatively similar to that of the presumptive myoblast (Lipton, 1977; Przybylski, 1971). It is not known, however, if the postnatal satellite cell and the mononucleated myogenic cells of embryonic origin are unequivocally identical. On the one hand, Jones (1982) demonstrated that satellite cells isolated from regenerating muscle in adult rats and embryonic presumptive myoblasts from rats responded similarly in culture, i.e., each proliferated and ultimately fused to form myotubes. These cells did not differ in growth rate or fusion characteristics. The same increase in creatine kinase activity and shift in isozyme profile developed after fusion. Based on temporal and spatial changes in acetylcholinesterase activity in fetal rabbit muscle, Tennyson et al. (1973) concluded that the satellite cell was a remnant from embryonic development. Young ef al. (1979) cultured myogenic cells from mice less than 24 hours old and from 3-week and 7-week-old mice and measured protein synthesis in the myotubes. In this study, age did not affect quantitatively or qualitatively the ability of the cells to differentiate and to synthesize and assemble the myofibriller proteins in culture. On the other hand, Allen ef al. (1982) reported that myotubes which differentiated from neonatal muscle cells of the rat accumulated more than three times as much a-actin per myotube nucleus as myotubes differentiated from the satellite cells of older rats. Furthermore, Schultz and Lipton (1982) found that, as the age of normal donor rats increased from 6 days to 30 months of age, the satellite cells required a longer time to initiate proliferative activity in culture. Replicative capacity also decreased with age. Studies have not been conducted to determine if the satellite cells and embryonic myogenic cells isolated and cultured from the same species under the same conditions respond similarly to hormonal and other growth factors. Quantitative data (Table V) exist for several species to confirm that the satel-
AGE-ASSOCIATED CHANGES IN
THE
TABLE V RELATIVE AND ABSOLUTENUMBER OF SATELLITE CELLS Postnatal
Days gestation Species
Muscle
Rat
Subclavius
Mouse
Lumbrical
Mouse
pig
Trait %SCN/TN" Number ( X lO5)d
%SCNe %SCN/TN Numberg Gastrocnemius %SCN/TN Numberh sartorius
Peroneus Longus
%SCN %SCN/TN Numbern %SCN %SCN/TN Numbern
95
110
Days 1
7
31.7 1.06
25.36 1.496
32 50
18 28 23 27 55
14
21 17.4c 1.04'-
11
16 15 -20 48
10 15 15
28
Months
35
42 49 63
11.4 9.4 0.99 1.10
70
6
32
4.6 4.3 0.95 1.05
64
Reference Hellmuth and Allbrook (1973) Schultz (1974)
4f ( i f
7f 10 28
5 15
7 7 6 22 25 21
5 3 3-4 18 11 12-13 122 95 100-130 12 7 7-7 31 20 18-18 23 183 199-220
O%SCN/TN, percentage nuclei within basal lamina that are satellite cell nuclei. bTen day values. cFifteen day values. dLTsed N = PL/d, P = number of nuclei per cross section of muscle; L = muscle length, d = nuclear length. <%SCN, percentage fibers in cross section that exhibit satellite cell nuclei. mhirty day values. KCalculations of number of satellite cells by formula, N = PL/d, where P = %SCN, L = muscle length, d = nuclear length. hCalculations based on observations of isolated fibers.
4 6 450 5 8 800
Cardasis and Cooper (1975) I Campion er al. 1 (1979, 140 1981) 8 4 720
THE MUSCLE SATELLITE CELL
24 1
lite cell population is not constant throughout life. As more daughter nuclei are added to the growing muscle, the number of myonuclei increases out of proportion to any changes in the satellite cell population. Thus, the percentage of nuclei within the basal lamina that are satellite cell nuclei decreases until the mature muscle weight is attained. At very old ages this percentage will once again begin to decrease. Snow (1977a) suggested that this latter decrease might be due to passage of some satellite cells into the intestitial space during normal aging. In the mouse (Schultz, 1974; Cardasis and Cooper, 1975; Young et al., 1978), the apparent number of satellite cells decreased as age increased. This is the only species, however, wherein an inverse relation has been consistently shown. In the rat (Hellmuth and Allbrook, 1973), the apparent number of satellite cells in the subclavius muscle changed little between 1 day and 6 months, while apparent satellite cell number increased dramatically from 1 day to 32 weeks of age in two pig muscles (Campion et af., 1979, 1981b). Apparent satellite cell number also increased in the sartorius muscle of Japanese quail between 4 days and 4 weeks of age (Campion er af., 1982a). Between 1 month and 12 months of age, the number of satellite cells per unit length of muscle increased in the soleus muscle but decreased in the extensor digitorum longus muscle of the rat (Gibson and Schultz, 1982). Although muscle length was not determined in this study, it can be concluded that the apparent number of satellite cells increased in the soleus muscle over the age period studied because the satellite cell number per unit of muscle length increased. The variation seen in apparent satellite cell number suggests that each satellite cell mitosis does not necessarily result in the incorporation of one daughter nucleus into the myofiber and in the other daughter nucleus becoming a satellite cell. This is particularly shown by the increase in apparent satellite cell number in pig muscles between 1 and 32 weeks of age (Table IV) and in Japanese quail muscle between 4 days and 4 weeks of age. Conversely, the near twofold reduction in apparent satellite cell number between 14 and 28-30 days of age in mouse muscle indicates that either satellite cell nuclei can be incorporated directly into myofibers, or that both daughter nuclei can be incorporated. The higher incidence of satellite cells that characterizes the soleus muscle when compared to the extensor digitorum longus muscle of the rat is a distinction that appears early in myogenesis, even before histochemical differentiation is evident (Kelly, 1978b). In addition, the normal age-associated changes in fiber type composition in these muscles was not related to age changes in the distribution of the satellite cell-fiber population (Gibson and Schultz, 1982). In the extensor digitorum longus muscle, the concentration and rate of proliferation of myonuclei is lower than in the soleus muscle. This correlates with the finding of a relatively smaller satellite cell population and a more rapid decline with age in the satellite cell labeling index in the extensor digitorum longus muscle than in the soleus muscle (Kelly, 1978b).
242
DENNIS R. CAMPION
Satellite cells are present in anuran tadpole tail muscles during metamorphosis. From the hind-limb-bud stage to climax, the percentage of nuclei within the basal lamina that are satellite cell nuclei decreases from 19.2 to 2.1% in red fibers and from 11.9 to 0.4% in white fibers (Takahama, 1983). Based on morphological evidence, it seems that satellite cells migrate into the interstitium during the late stage of metamorphosis. A similar migration was suggested to occur during metamorphosis of several species of urodeles (Popiela, 1976). Popiela (1976) proposed that these cells were the progenitors of the “pericyte” found in adult salamander muscle. Satellite cells are not seen in adult salamander muscle (Hay, 1979). Whether or not the pericytes in salamanders are indeed myogenic stem cells that differ only in anatomical location has not been clearly established (Hay, 1979). At some stage of adulthood, the satellite cells become mitotically quiescent. This happens at least by 4 months of age in mouse tibialis anterior muscle (Schultz et al., 1978). At 30 months of age, mouse and rat muscle satellite cells were found in situ that contained centrioles, but no ultrastructural evidence of mitosis was seen (Snow, 1977a). Myonuclear proliferation has been investigated in animal models that vary in muscle mass and body composition. These models were employed to ascertain if the concentration of satellite cells and/or if the rate of mitosis and nuclear incorporation were related to differences in rate of muscle growth and in attainment of total muscle mass. In a study of muscle from Japanese quail selected for high body weight, the satellite cells in the muscle of growth strain quail when compared to the control strain (Campion et al., 1982a) exhibited a slightly higher mitotic rate. The muscles of the growth strain Japanese quail also possessed more muscle fibers (Fowler et al., 1980; Campion et al., 1982b). Since the incubation period for eggs is similar for the control and growth strain birds and mature myofiber number is established about the time of hatch, mitotic activity must have also been greater in the embryonic presumptive myoblasts. Therefore, selection for high body weight in this model appeared to result in a higher mitotic rate in the myogenic cells whether they be termed presumptive myoblasts or satellite cells. Penney et al. (1983) studied two strains of mice that differed in myofiber number and concluded that the primary difference in muscling between the two strains was due to a difference in the rate of proliferation of presumptive myoblasts before fusion. This conclusion is in general agreement with the results of the studies with Japanese quail. A question of considerable importance is whether the rate of mitotic activity and incorporation of nuclei into the myofiber can account for the increase in myofiber nuclei seen during normal growth and development. Moss and Leblond ( 197l), using autoradiographic and electron microscopic techniques, calculated the actual and relative incidence of labeled satellite cell nuclei and myofiber nuclei over a 72-hour period in 14- to 17-day-old rats. It was concluded that the
THE MUSCLE SATELLITE CELL
243
mitotic rate and incorporation rate was consistent with the hypothesis that satellite cells can account for all nuclei added to normally growing myofibers of the rat.
B. NUTRITION Skeletal muscle is responsive to the nutritional status of the animal (Winick and Noble, 1966). While the mass and DNA content of muscle are both responsive to nutritional manipulation, there is limited knowledge on how the satellite cell might be affected. Hansen-Smith et al. (1978a) examined the effect of 10 weeks of protein restriction or protein-energy restriction on the fine structure of the quadriceps muscle of rats. Most of the myofibers of the treated groups were similar to age matched controls. In each of the dietary groups, a small percentage of the myofibers possessed satellite cells. Morphologically, dietary treatment did not influence satellite cell fine structure. Nuclei were not quantitated in this study. Nutritional restriction of rats during gestation and lactation had a transient effect on the incidence of satellite cells in the skeletal muscles of the progeny (Beermann et al., 1981; Beermann, 1983). In 1-day-old rats from restricted- and control-fed dams, counts of satellite cells and myonuclei were similar. Counts were markedly decreased in control rats at 21 days of age but remained unchanged or elevated in restricted rats. Ad libitum feeding of restricted and control rats from weaning to 175 days of age resulted in similar counts in the two groups of offspring at 175 days. It might be inferred from this study that the satellite cell population changes in concert with the weight of the muscle rather than with the age of the animal. Seerley er al. (1974) first suggested the possibility of feeding high levels of dietary fat to the sow immediately before farrowing to improve the energy status of the piglets at birth. Work in this area was recently reviewed by Pettigrew (1981). We examined the effect of feeding diets containing 0, 2, 10, or 30% fat to sows during late gestation on the cellularity of the fetal sartorius muscle (Campion et al., 1983). Maternal diet had no effect on muscle weight, on myofiber nuclear content, or on satellite cell nuclear content. In the only studies conducted on humans, Hansen-Smith et al. (1978b, 1979) examined the myonuclear content of the vastus lateralis muscle of malnourished children. The percentage nuclei within the basal lamina that were satellite cell nuclei was lower in the muscle of malnourished children than in the muscle of either their clinically recovered or well-nourished counterparts. From the limited work in this important area, it can be suggested that only when muscle weight is affected by nutritional manipulation is the population of satellite cells affected.
244
DENNIS R. CAMPION
C. MUSCLEREGENERATION AND COMPENSATORY HYPERTROPHY Muscle regeneration has long been studied (Bottcher, 1858; Waldeyer, 1865; Volkmann, 1893). From recent reviews of skeletal muscle regeneration (Carlson, 1973; Reznik, 1976; Allbrook, 1981), it has been concluded that the essential process of regeneration was similar regardless of the cause of the injury or disease. In addition, muscle regeneration appears to capitulate embryonic myogenesis-mononucleated myogenic cells (embryologically, presumptive myoblasts) proliferate, line up usually within the confines of the residual basal lamina (embryologically basal lamina is formed after initial myotube formation), and fuse to form myotubes. These myotubes mature into fibers if innervated and if the blood supply is adequate. The satellite cell is thought to be a source of these myogenic cells which impart to skeletal muscle the ability to regenerate. Several lines of evidence exist to support this hypothesis. Satellite cells have been harvested and cultured (where they proliferate and fuse to form myotubes) from the skeletal muscle of mice (Young et al., 1978) and rats (Allen et al., 1980; Jones, 1982) at various ages, up to 30 months of age in the rat (Allen et al., 1982), and from adult humans (Nag and Foster, 1981). In addition, explants of muscle tissue or fibers from human (Mendell et al., 1972; Askanas and Engel, 1975), rat (Bischoff, 1974, 1975), avian (Konigsberg et al., 1975), and other species yield mononucleated cells in culture that also proliferate and differentiate into myotubes. Bischoff and Konigsberg et al. concluded from their studies that satellite cells were a source of the proliferative myoblasts observed in culture. The autoradiographic-electron microscopic studies of Snow (1 977c, 1978), Lipton and Schultz (1979), and Trupin et al. (1982) lend further evidence to implicate the satellite cell as a source of the myogenic cells involved in regeneration. The effect of a lesion in the central nervous system on the muscle satellite cell population was reported by Campion et al. (1978). Removal of the influence of the brain by spinal cauterization of the fetal pig in early gestation resulted in a reduced incidence of satellite cells in the sartorius muscle of the near-term fetus. Decapitation of' the fetal pig, however, had no influence on the content of myofiber nuclei or satellite cells in the peroneus longus muscle (Campion et al., 1981a). A gross endocrine imbalance existed in the decapitated fetus (Martin et al., 1983). Whether or not the effect of the endocrine imbalance counteracted the loss of the influence of the brain is not known. Since different muscles were examined in the two fetal pig studies, the differences in treatment effects may alternatively have been the result of variation in muscle specific characteristics. In a variety of situations, the satellite cell has shown considerable motility. For example, neofiber formation has been observed in denervated muscle (Miledi and Slater, 1969; Ontell, 1975; McGeachie and Allbrook, 1978; Schultz, 1978), in minced muscle implants (Snow, 1977b), in muscle with experimentally in-
THE MUSCLE SATELLITE CELL
245
duced fibrillation (Salleo et al., 1979), in muscle grafts (Hansen-Smith and Carlson, 1979; Ontell et al., 1982), and in muscle undergoing compensatory hypertrophy (McGeachie and Allbrook, 1978; Salleo et al., 1980). Under these various conditions, the satellite cells migrate out into the interstitium, line up, and fuse to form new fibers. The evidence to support migration, however, is largely circumstantial. Many cell types are labeled when tracer studies using [3H]thymidine are used, making it impossible to accurately follow movement of the satellite cell in situ. But, the work of Bischoff (1974, 1975), Konigsberg et al. (1975), and Lipton and Schultz (1979) revealed that satellite cells could migrate through injured or intact basal lamina. Miledi and Slater (1969) suggested that satellite cells may have a role in fiber splitting. There is still no direct evidence to support this hypothesis. In fact, Schiaffino et al. (1979) argued that the term “fiber splitting” was misleading, as “splitting” fibers can correspond to satellite fibers or to branched fibers. Recent studies of muscle hypertrophy in rats (James and Cabric, 1981) and in mice (Atherton et al., 1981) suggest that fiber splitting does not happen in skeletal muscle undergoing hypertrophy as a result of overload. Lipton and Schultz (1979) demonstrated that satellite cells, labeled with [3H]thymidine in culture, underwent extensive migration away from the implant site when returned to the muscle of the original donor. In both rats and quail, most of the labeled cells penetrated the basal lamina of intact myofibers while a few appeared in satellite fibers. It was suggested that, after an injury, satellite cells from undamaged fibers may migrate to the site of the lesion and participate in the regenerative process. Schultz (1978) also postulated this mechanism in denervated mouse and rat muscle. In addition, Hall-Craggs and Lawrence (1970) reported seeing satellite cells forming a bridge between two myofibers in the soleus muscle of the rat 10 days after crushing. Apparent migration of satellite cells between myofibers was described in normal mouse lumbrical muscle (Schultz, 1976), in the gastrocnemius muscle of the human fetus (Ishikawa, 1966), and in shark muscle (Kryvi, 1975). In situations evaluated solely by morphological observation, however, the possibility that the bridging cell was an invasive cell rather than a satellite cell cannot be entirely ruled out (Trupin, 1979; Trupin and Hsu, 1979; Maruenda and Franzini-Armstrong, 1978).
VI. Activation Stimulus Certain hormones provide a mitogenic stimulus to myogenic cells (for recent reviews, see Allen et al., 1975; Allen, 1979). Little information is available, however, on the effect of hormones on the satellite cell population in situ. After 12 months of streptozotocin-induced diabetes mellitus, the incidence of satellite cells appeared to be increased in the red fibers of the diaphragm muscle when compared to control rats (Bestetti et al., 1981). Injection of glucocorticoids into
246
DENNIS R. CAMPION
rabbits from 1 to 9 days after birth caused retardation of muscle differentiation and satellite cell degeneration (Jirmanova et al., 1982). It is not known if the effects observed on the satellite cells in these studies were directly or indirectly related to the particular hormones. In culture, synthesis of muscle specific actin by fused rat satellite cells was not influenced by addition of growth hormone or testosterone (Allen er al., 1983). Myotube cultures from neonatal rat, embryonic chick, and Yaffe’s L-6 myogenic cell line did not alter rates of incorporation of a-amino isobutyric acid in the presence of near physiological concentrations of growth hormone (Ewton and Florini, 1980). While more careful comparisons are needed, the results from these two studies suggest that myotubes derived from presumptive myoblasts and satellite cells do not respond to growth hormone. Passive stretch of chicken wing muscles results in an increase in total muscle DNA content (Bamett et al., 1980; Holly er al., 1980). In this situation, neuromuscular junction activity is not altered. Although fiber damage (and subsequent release of mitogens) cannot be ruled out, it seems possible that mechanical stretch of the satellite cell, per se, may initiate activation of the satellite cell. Satellite cells were activated in the bulbi rectus superior muscle of the rat by light compression (Teravainen, 1970). That no discernible injury occurred further suggests that activation can result by a purely physical mechanism. Furthermore, denervation atrophy was inhibited when denervation was combined with synergistic incapacitation (Schiaffino and Hanzlikovi, 1970). Murray and Robbins (1982a,b) suggested that denervation of muscle causes tissue disruption that results in release of mitogen(s) which enhance proliferation of connective tissue cells and satellite cells. They cited altered surface membrane proteins, altered ionic permeabilities, increased release of enzymes or degradation products, and appearance of proteolytic enzyme activity as potential mitogenic signals. These “signals” exist in denervated mammalian muscle for several weeks. Selective stimulation of satellite cell proliferation may result from release of a muscle-cell specific mitogen during injury (Bischoff, 1981). Salleo et al. (1979) postulated that fibrillation may be the common link whereby denervated muscle and muscle undergoing compensatory hypertrophy exhibit similar characteristics in terms of satellite cell activation and neofiber formation. And Anastasi et al. (1979) hypothesized that satellite cell division could be stimulated by motor activity. As yet there is no unified hypothesis to explain activation of satellite cells and how the activation process is regulated.
VII. Summary Since the first reports of satellite cells in 1961, considerable knowledge has accumulated concerning their phylogenetic distribution and their location, mor-
THE MUSCLE SATELLITE CELL
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phology, and function. There is no doubt that satellite cells are capable of undergoing mitosis and that they have considerable motility. These cells function as the progenitors of the myofiber nuclei that must be added during normal (postnatal) growth of muscle. In muscle undergoing or attempting to undergo regeneration, the satellite cell functions as a myogenic stem cell to produce myoblasts that line up and fuse within the scaffolding of the remnant basal lamina or migrate into the interstitium to produce neofibers. A number of problems remain to be solved concerning the regulation of satellite cell function. At this time it is equivocable whether or not the presumptive myoblast and the satellite cell are functionally identical and at the same stage of myogenic differentiation. Apparently there is species variation in terms of the ability of myotubes from embryonic myogenic cells and satellite cells to synthesize protein. The mechanism(s) by which a wide variety of stimuli activate satellite cells is not known, nor is the mechanism(s) by which satellite cells become inactive during the latter stages of growth and adulthood known. Mitogenic factors are present in damaged muscle; but the specific characteristics of these factors and their mechanism of activation are also unknown. Hormones are certainly involved in the regulation of proliferation and differentiation of myogenic cells, but whether presumptive myoblasts and satellite cells or their myotubes respond similarly to hormones in culture has not been adequately examined. Greater understanding of these mechanisms will increase the possibility of total muscle recovery from severe injury or disease. Such knowledge would also have particular application to the production of meat animals and to a greater understanding of the growth process in general.
REFERENCES Adal, M . N. (1969). J . Ultrasrruct. Res. 26, 332-354. A i m , S . (1973). Tohoku J . Exp. Med. 111, 101-117. Allbrook, D . (1981). Muscle Nerve 4, 234-245. Allbrook, D . B . , Han, M . F., and Helmuth, A . E. (1971). Pathology 3, 233-243. Allen, R. E. (1979). Proc. Ann. Recip. Meat Conf., 32nd, Chicago pp. 99-107. Allen, R. E . , Young, R. B . , Strorner, M. H . , and Goll, D . E. (1975). Proc. Ann. Recip. Meat Corf., 28th, Chicago pp. 182-201. Allen, R . E . , Merkel. R. A , , and Young, R. B . (1979). J . Anim. Sci. 49, 115-127. Allen, R. E . , McAllister, P. K . , and Masak, K. C . (1980). Mech. Ageing Dev. 13, 105-109. Allen. R. E., McAllister, P. K . , Masak, K. C . , and Anderson, G. R. (1982). Mech. Ageing Dev. 18, 89-95. Allen, R. E., McAllister, P. K . . and Merkel, R. A . (1983). J . Anim. Sci. 56, 833-837. Aloisi, M . . Mussini, I . , and Schiaffino, S. (1973). In “Basic Research in Myology” (B. A . Kakulas, e d . ) , pp. 338-342. Excerpta Medica, Amsterdam. Anastasi, G . , Salleo, A . , Falzea, G . , Denaro, M. G . , La Spada, G . , and Magaudda, L. (1979). J . Submicrosc. Cytol. 11, 463-472. Askanas, V . , and Engel, W . K . (1975). Neurdogy 25, 58-67.
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Atherton, G . W . , James, N. T., and Mahon, M. (1981). Experientia 37, 308-310. Banker, B. Q., and Girvin, T. P. (1971). J . Neuroparhol. Exp. Neurol. 30, 155-195. Barnett, I . G., Holly, R. G., and Ashmore, C. R. (1980). Am. J . Physiol. 293, C39-C46. Beermann, D. H. (1983). J. Anim. Sci. 57, 328-337. Beermann, D. H., Hood, L. F., and Liboff, M. (1981). J. Anim. Sci. 53 (Suppl. I ) , 206. Bestetti, G., Zemp, C., Probst, D., and Rossi, G. L. (1981). Acra Neuroparhol. 55, 11-20. Bird, D. O., and Allbrook, D. B. (1980). J. Anat. 130, 202. Bischoff, R. (1974). Anat. Rec. 180, 645-661. Bischoff, R. (1975). Anat. Rec. 182, 215-236. Bischoff, R. (1981). J. Cell Biol. 91, 342a. Bischoff, R., and Holtzer, H. (1969). J. Cell Biol. 41, 188-200. Bottcher, A. (1858). Arch. Pathol. Anar. Physiol. Klin. Med. 13, 227-392. Campion, D. R., Richardson, R. L., Kraeling, R. R., and Reagan, J. 0. (1978). Growth 42, 189-204. Campion, D. R., Richardson, R. L., Kraeling, R. R., and Reagan, J. 0. (1979). J. Anim. Sci. 48, 1 109- 1115. Campion, D. R., Hausman, G. J., and Richardson, R. L. (1981a). Biol. Neonore 39, 253-259. Campion, D. R., Richardson, R. L., Reagan, J. 0.. and Kraeling, R. R. (1981b). J. Anim. Sci. 52, 1014- 1018. Campion, D. R., Marks, H. L., and Richardson, R. L. (1982a). Acta Anat. 112, 9-13. Campion, D. R., Marks, H. L., Reagan, 1. O., and Barrett, J. B. (1982b). Poultry Sci. 61,212-217. Campion, D. R., Kveragas, C. L., and Seerley, R. W. (1983). J. Anim. Sci. 57 (Suppl. I ) , 239. Cardasis, C. A. (1979). In “Muscle Regeneration” (A. Mauro, ed.), pp. 155-166. Raven, New York. Cardasis, C. A., and Cooper, G. W. (1975). J. Enp. Zoo/. 191, 347-358. Cardasis, C. A,, and Padykula, H. A. (1981). Anat. Rec. 200, 41-60. Carlson, B. M. (1973). Am. J . Anor. 137, 119-150. Cheek, D. B., Holt, A. B., Hill, D. E., and Talbert, J . L. (1971). Pediarr. Res. 5, 312-328. Church, J. C. T. (1969). J . Anar. 105,419-438. Church, J. C. T. (1970). J. Anat. 32, 531-537. Conen, P. E., and Bell, C. D. (1970). I n “Regeneration of Striated Muscle, and Myogenesis” (A. Mauro, S. A. Shafiq, and A. T. Milhorat, eds.), pp. 194-211. Excerpta Medica, Amsterdam. Cull-Candy, S. G., Miledi, R., Nakajima, Y.,and Uchitel, 0. D. (1980). Proc. R . Soc. London Ser. B 209, 563-568. Dom, A. (1969). Anar. Anz. 124, 513-550. Enesco, M., and Puddy, D. (1964). Am. J. Anat. 114, 235-244. Ewton, D. Z., and Florini, J. R. (1980). Endocrinology 106, 577-583. Flood, P. R. (1964). Proc. Eur. Conj. Electron Microsc., 3rd 575-576. Flood, P. R. (1971). J. Ulrrasfruct. Res. 36, 523-524. Fowler, S. P., Campion, D. R., Marks, H. L., and Reagan, J. 0. (1980). Growth 44,235-252. Franzini-Armstrong, C. (1979). In “Muscle Regeneration” (A. Mauro, ed.), pp. 233-238. Raven, New York. Fukami, Y. (1982). J. Neurophysiol. 47, 810-826. Galavazi, G. (1971). Z. Zellforsch. 121, 531-547. Gibson, M. C., and Shultz, E. (1982). Anar. Rec. 202, 329-337. Gutmann, E., and Hanzlikovi, V. (1972). “Age Changes in the Neuromuscular System.” Scientechnica, Bristol. Hall-Craggs, E. C. B., and Lawrence, C. A. (1970). Z. Zellforsch. 109, 481-494. Hansen-Smith, F. M., and Carlson, B. M. (1979). J. Neurol. Sci. 41, 149-173. Hansen-Smith, F. M., Van Horn, D. L., and Maksud, M. G. (1978a). J. Nurr. 108, 248-255. Hansen-Smith, F. M., Picou, D., and Golden, M. N. H. (1978b). Pediat. Res. 12, 167-170.
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Hansen-Smith, F. M., Picou, D., and Golden, M. N. H. (1979). .I. Neurol. Sci. 41, 207-221. Hay, E. D. (1979). In “Muscle Regeneration” (A. Mauro, ed.), pp. 73-81. Raven, New York.. Hellmuth, A. E., and Allbrook, D. (1973). Proc. Inr. Congr. Muscle Dis., 2nd pp. 343-345. Hess, A., and Rosner, S. (1970). Am. J . Anar. 129, 21-40. Heywood, S. M., and Rich, A. (1968). Proc. Nail. Acad. Sci. U.S.A. 59, 590-597. Holly, R. G., Barnett, J. G., Ashmore, C. R., Taylor, R. G., and Mole, P. A. (1980). Am. J. Phvsiol. 238, C62-C7 1. Ishikawa, H. (1966). Z . Anar. Enhvicklungssch. 125, 43-63. James, N. T., and Cabric, M. (1981). E r . J. Exp. Parhol. 62, 600-605. Jirmanova, I., Soukup, T., and Zelena, J . (1982). Virchows Arch. E . Cell Parhol. 38, 323-336. Jones, P. H. (1982). Exp. Cell Res. 139, 401-404. Kahn, E. B . , and Simpson, S. B., Jr. (1974). Dev. Biol. 37, 219-233. Karlsson, U.,and Anderson-Cedergren, E. (1971). J . Ulrrasrruct. Res. 34, 426-438. Karlsson, U., Anderson-Cedergren, E., and Ottoson, D. (1966). J . Ultrasrruct. Res. 14, 1-35. Katz, B. (1961). Philos. Trans. R. Soc. London Ser. E 243, 221-240. Kelly, A. M. (1978a). Anar. Rec. 190, 891-904. Kelly, A. M. (1978b). Dev. Eiol. 65, 1-10. Kelly, A. M., andZacks, S. I. (1969). J . CeflEiol. 42, 135-152. Klishov, A. A., and Danilov, R. K. (1981). Ark. Anar. Cisrol. Embriol. Leningrad 80, 95-107. Konigsberg, U . R., Lipton, B. H., and Konigsberg, I. R. (1975). Dev. Eiol. 45, 260-275. Korneliussen, H., and Nicolaysen, K. (1973). Z . Zellforsch. 143, 273-290. Kryvi, H. (1975). Anar. Embryol. 147, 35-44. Kryvi, H., and Eide, A. (1977). Anar. Embryol. 151, 17-28. Laguens, R. (1963). Virchows Arch. Parhol. Anar. 336, 564-569. Landon, D. N. (1966). I n “Control and Innervation of Skeletal Muscle” (B. L. Andrews and S . Livingston, eds.), pp. 96-1 10. Livingstone, London. Larocque, A. A., Politoff, A. L., and Peters, A. (1980). Anar. Rec. 196, 373-385. Lipton, B. H. (1977). Dev. Eiol. 60, 26-47. Lipton, B. H., and Schultz, E. (1979). Science 205, 1292-1294. MacConnachie, H. F., Enesco, H. F., and Leblond, C. P. (1964). Am. J. Anar. 114, 245-253. Martin, R. J . , Campion, D. R., Hausman, G . J., and Gahagan, J. H. (1983). Submitted. Maruenda, E. H., de, and Franzini-Armstrong, C. (1978). Tissue Cell 10, 749-772. Mauro, A. (1961). J. Biophys. Eiochem. Cyrol. 9, 493-495. Mauro, A. (1979). “Muscle Regeneration.” Raven, New York. Mauro, A., Shafiq, S. A., and Milhorat, A. T. (1970). “Regeneration of Striated Muscle, and Myogenesis.” Exerpta Medica, Amsterdam. Maynard, J. A., and Cooper, R. R. (1973). Z . Anar. Enrwicklungsgesch. 140, 1-9. Mazanet, R. (1981). Satellite Cells and Pericytes: Their Roles in Skeletal Muscle. PhD dissertation, University of Pennsylvania. Mazanet, R., Reese, B. F . , Franzini-Armstrong, C., and Reese, T. S. (1982). Dev. Eiol. 93,22-27. McGeachie, J . , and Allbrook, D. (1978). Cell Tissue Res. 193, 259-267. Mendell, J. R., Roelofs, R. I., and Engel, W. K. (1972). Exp. Neurol. 31, 433-446. Midsukami, M. (1964). Okajimas Foliz Anat. Jpn. 40, 173-185. Midsukami, M. (1981). Cell Tissue Res. 219, 69-83. Miledi, R., and Slater, C. R. (1969). Proc. R. SOC. London Ser. E 174, 253-269. Moss, F. P. (1968). Am. J . Anar. 122, 555-563. Moss, F. P., and Leblond, C. P. (1970). J. Cell Eiol. 44, 459-462. Moss, F. P., and Leblond, C. P. (1971). Anar. Rec. 170, 421-436. Muir, A. R. (1965). J . Anar. 99, 27-46. Muir, A. (1970). In “Regeneration of Striated Muscle, and Myogenesis” (A. Mauro, S. Shafiq, and A. T. Mihorat, eds.), pp. 91-100. Exerpta Medica, Amsterdam.
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Muir, A. R.. Kanji, A. H. M., and Allbrook, D. (1965). J . Anut. 99, 435-444. Murray, M. A,, and Robbins, N. (1982a). Neuroscience 7, 1817-1822. Murray, M. A., and Robbins, N. (1982b). Neuroscience 7, 1823-1833. Nag, A. C., and Foster, J. D. (1981). J . Anut. 132, 1-18. Okazaki, K., and Holtzer, H. (1966). Proc. Nutl. Acud. Sci. (I.S.A. 56, 1484-1490. Ontell, M. (1974). Anat. Rec. 178, 21 1-228. Ontell, M. (1975). Cell Tissue Res. 160, 345-353. Ontell, M. (1977). Anat. Rec. 189, 669-690. Ontell, M., and Dunn, R. F. (1978). Am. J . Anut. 152, 539-556. Ontell, M., Hughes, D., and Bourke, D. (1982). Anut. Res. 204, 199-207. Penney, R. K . , Prentis. P. F., Marshall, P. A., and Goldspink, G. (1983). Cell Tissue Res. 228, 375-388. Pettigrew, J. E. (1981). J . Anim. Sci. 53, 107-1 17. Popiela, H. (1976). J . Exp. Zool. 198, 57-64. Przybylski, R. J . (1971). J . Cell B i d . 48, 214-221. Przybylski, R. J . , and Blumberg, J. M. (1966). Lab. Invest. 15, 836. Pullman, W. E., and Yeoh, G. C. T. (1978). J . Cell. Physiol. 96, 245-252. Reger, J. F., and Craig, A. S . (1968). Anat. Rec. 162, 483-500. Reznik, M. (1976). Differentiurion 7, 65-73. Richler, C., and Yaffe, D. (1970). Dev. B i d . 23, 1-22. Rumpelt, H.-J., and Schmalbruch, H. (1969). Z . Zellforsch. 102, 601-630. Salleo, A., Anastasi, G., Spada, G . , la, Denaro, M. G . , Falzea, G . , and Magaudda, L. (1979). Dixerentiation 15, 119-125. Salleo, A , , Anastasi, G., Spada, G . , la, Falzea, G., and Denaro, M. G. (1980). Med. Sci. Sports Exercise 12, 268-273. Sandset, P. M., and Korneliussen, H. (1978). Cell Tissue Res. 195, 17-27. Schiaffino, S . , and Hanzlikovii, V. (1970). Experientia 26, 152-153. Schiaffino, S . , and Hanzlikovii, V. (1972). J . Cell B i d . 52, 41-51. Schiaffino, S . , Bormioli, S. P., and Aloisi, M. (1979). In “Muscle Regeneration” (A. Mauro, ed.), pp. 177-188. Raven, New York. Schmalbruch, H. (1978). Anat. Rer. 191, 371-376. Schmalbruch, H., and Hellhammer, U. (1976). Anat. Rec. 185, 279-288. Schmalbruch, H., and Hellhammer, U . (1977). Anat. Rec. 189, 169-176. Schultz, E. (1974). Anut. Rec. 180, 589-596. Schultz, E. (1976). Am. J . Anat. 147, 49-70. Schultz, E. (1978). Anat. Rec. 190, 299-312. Schultz, E., and Lipton, B. H. (1982). Mech. Ageing Dev. 20, 377-383. Schultz, E., Gibson, M. C., and Champion, T. (1978). J . Exp. Zool. 206, 451-455. Seerley, R. W., Pace, T. A , , Foley, C. W., and Scarth, R. D. (1974). J . Anim. Sci. 38, 64-70. Shafiq, S. A., Gorycki, M. A , , and Mauro, A. (1968). J . Anut. 103, 135-141. Snow, M. H. (1977a). Cell Tissue Res. 185, 399-408. Snow, M. H. (1977b). Anar. Rec. 188, 181-200. Rec. 188, 201-218. Snow, M. H. ( 1 9 7 7 ~ )Anar. . Snow, M. H. (1978). Cell Tissue Res. 186, 535-540. Snow, M. H. (1979). In ‘‘Muscle Regeneration” (A. Mauro, ed.), pp. 91-100. Raven, New York. Snow, M. H. (1981). Anaf. Rec. 201, 463-469. Stenger, R. T., and Spiro, D. (1961). J . Biophys. Biochem. Cyrol. 9, 325-353. Stockdale, F. E. (1970). Dev. Eiol. 21, 462. Stockdale, F. E., and Holtzer, H. (1961). Exp. Cell Res. 24, 508. Takahama, H. (1983). Cell Tissue Res. 228, 573-585.
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INTERNATIONAL REVIEW OF CYTOLOGY. VOL. 87
Cytology of the Secretion in Mammalian Sweat Glands KAZUMASAKUROSUMI ,* SUSUMUSHIBASAKI,? AND TOSHIOITOI *Department of Morphology, Institute of Endocrinology, Gunma University, Maebashi, Japan, ?Department of Anatomy, Gunma University School of Medicine, Maebashi, Japan, and $Department of Anatomy, Teikyo University School of Medicine, Tokyo, Japan I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Historical Survey of Light Microscopic Studies of Sweat Gland Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. General Histology of the Sweat Glands.. . . . . . . . . . . . . . . . . . . B. Light Microscopic Cytology of the Apocrine Sweat Glands.. . . C. Light Microscopic Cytology of the Eccrine Sweat Glands . . . . . 111. Ultrastructures and Their Functional Significance of the Human Sweat Glands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. The Human Apocrine Sweat Gland . . . . . . . . . . . . . . . . . . . . . . . B. The Human Eccrine Sweat Gland.. ....................... C. Excretory Ducts of the Human Sweat Glands . . . . . . . . . . . . . . . IV. Ultrastructural Cytology of the Secretory Activity in the Sweat Glands of Nonhuman Mammals.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Animal Apocrine Sweat Glands . . . . . . . . . . . . . . . . . . . . . . . . . . B. Animal Eccrine Sweat Glands. . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References .............
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I. Introduction The mammalian skin glands have been repeatedly used as material for the study of cytology of secretion; for example, Schiefferdecker (1917) studied the mode of secretion release using the skin glands. Among the different kinds of skin glands, the sweat glands are widely distributed through almost all the species of mammals, and vary remarkably in fine structure according to species. The human sweat glands are best differentiated as compared with those of nonhuman mammals. A great number of histological and cytological studies on the human sweat glands, especially those distributed in the axillary skin, were performed by Japanese researchers. For this reason it may be noticed that many Japanese hate the axillary odor, that is dermatologically called osmidrosis. That is not a disease 253 Copyright Q 1984 by Academic Press, lnc. All rights of reproduction in any form reserved. ISBN 0-12-364487-9
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but a kind of hereditary constitution, since persons having axillary odor are in the minority among the Japanese population, and such persons often receive a surgical operation to remove a part of the skin at the axillary region. Therefore, normal fresh specimens of axillary sweat glands of both apocrine and eccrine types could be easily taken for research of histology and cytology in Japan. This custom, however, has been changed recently and such operations have been rather infrequent, because other methods for deodorant techniques became popular. Still morphological research on the human sweat glands continues in some laboratories in .lapan using various advanced techniques for microscopy. There are some difficulties in research of cell biology concerning the secretion of human sweat glands, because no hazardous experiments can be applied to the human skin such as autoradiography. Therefore, the progress of research on the secretion mechanism in the human sweat glands has been rather slow, as compared with some other glands of experimental animals such as the pancreas of some rodents. However, comparative studies on the ultrastructure of sweat gland cells using human and animal glands may lead to certain progress in understanding the secretory mechanism of human apocrine and eccrine sweat glands. It is worthwhile to review studies on the sweat glands of both human and nonhuman mammals to promote further studies which will bring a final resolution of certain problems concerning the secretory mechanism of these complicated glands in man, and which may contribute to some extent to investigative dermatology, especially the pathology of skin gland tumors.
11. Historical Survey of Light Microscopic Studies of Sweat Gland
Morphology A. GENERAL HISTOLOGYOF THE SWEATGLANDS Purkinje (1833) has been honored as the discoverer of the sweat glands, but other researchers had already found the sweat pores on the human skin and the tubules extending from the pores downward into the dermis, before Purkinje observed the secretory portion of the sweat glands. Schiefferdecker (1917, 1922) has been the most famous in the field of research of skin glands including the sweat glands, because he classified the skin glands into several types according to the mode of secretion discharge by these gland cells. At first he divided the skin glands into the holocrine gland and merocrine gland, the former corresponding to the sebaceous gland and the latter to the sweat gland. In the holocrine gland the gland cells change entirely into a secretory product, while the merocrine gland is the gland in which only a part of the gland cell cytoplasm becomes the secretion. Schiefferdecker divided the merocrine glands into the apocrine gland and eccrine gland. Apocrine secretion is characterized by the fact that the cytoplasm extends a process, which is pinched-off into
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the gland lumen and becomes the secretion; in the eccrine gland, the secretory material is leaked out penetrating through the surface membrane on the luminal side of the cell. The latter mechanism is erroneously called merocrine secretion in some textbooks, but the merocrine must contain both apocrine and eccrine mechanisms. Schiefferdecker (1922) classified the sweat glands of the human skin into two groups, the apocrine and eccrine sweat glands, based on their mechanism of secretion release as described above. In the human body, the apocrine sweat glands are restricted to certain regions, such as the axillary fossa, external auditory meatus, eyelids, wings of the nostril, nipple and areola of the breast, perianal region, and some parts of the external genitalia, while the eccrine sweat glands are distributed over almost all the body surface except the lips, external auditory meatus, prepuce, glans penis, labia minora, and clitoris. The apocrine gland generally opens into the pilary canal (inside the hair follicle), but the eccrine glands pours the sweat onto the surface of the epidermis, and the secretory portion of the apocrine sweat gland is larger than that of the eccrine sweat glands. Therefore the former is called a large sweat gland, while the latter is called a small sweat gland. The distribution of sweat glands is quite different in most mammals from those in man and primates; the eccrine sweat glands in most mammalian species are limited to the palms and soles, including the volar surface of fingers and the plantar surface of toes. On the general body surface of these mammals, apocrine sweat glands are distributed, though most of these apocrine sweat glands are not effective for regulation of body temperature as in the human body; the only exception occurs in the horse. Concerning the secretory mechanism, the difference between the eccrine and apocrine sweat glands becomes vague, since the fact that the eccrine sweat glands also release the secretion by the mechanism of apocrine secretion was found (Minamitani, 1941a; Ito and Iwashige, 1951). However, the presence of these two different types of sweat glands is evident both morphologically and physiologically. Therefore, terms “eccrine” and “apocrine” sweat glands are to be understood not to indicate their mode of secretion release, but to be only names. Both types of sweat glands have common morphological characteristics, that is, both are simple tubular glands. In most cases the secretory portion forms a glomerulum. Therefore, the secretory tubule runs in a tortuous course, and is often called the secretory coil. The cross diameter of secretory tubules of the apocrine sweat glands is always larger than that of the eccrine sweat glands, and the lumen of the secretory tubule is also wider in the apocrine glands than in the eccrine glands. Sometimes the branching of secretory tubules is observed both in the apocrine (Talke, 1903; Sperling, 1935; Kato and Nagata, 1938) and eccrine types of sweat glands (Backmund, 1901; Clausen and Alexanderson, 1929). The excretory ducts of the eccrine sweat glands are rather simple tubules and
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are divided into two parts: the dermal duct and epidermal duct. The initial part of the dermal duct participates in the formation of secretory glomerulum and is called the coiled duct, but the remainder of the dermal duct is straight and goes upward and approaches the skin surface, entering the epidermis at the bottom of the epidermal rete peg. The epidermal ducts are also called terminal ducts and wind spirally like a cork screw and open onto the skin surface at the summit of skin ridges. As a special part of the gland tubule of the human sweat gland, the transitional portion is found between the secretory coil and coiled duct (Ito and Enjo, 1949). The transitional portion of the eccrine sweat gland is conspicuous, but that of the apocrine gland is known to be very short or invisible. Because the duct of the apocrine gland opens to the hair follicle at the level of the isthmus, the epidermal duct is absent in the apocrine sweat gland. Some of the apocrine sweat glands in the external auditory canal (ceruminous glands), however, open onto the surface of the epidermis. Therefore, the epidermal duct similar to that of the eccrine sweat glands is observed in the ceruminous glands. It is known that the ducts of most apocrine sweat glands do not contribute to the formation of the glomerulum, and hence the coiled ducts are absent in these glands.
B. LIGHTMICROSCOPIC CYTOLOGY
OF THE
AP~CRINE SWEAT GLANDS
The secretory portion of the apocrine sweat glands is a glomerulum composed of secretory tubules whose cross diameters are different from place to place and often form strong dilatations. The secretory tubules sometimes send out a branch which may end blindly or anastomose to the main tubule to form a ring (Sperling, 1935). In the human skin, the apocrine sweat glands in the axillary region are best developed, and have the most complicated glomerulum. The wall of the secretory tubule of the apocrine sweat glands consists of two different kinds of cells, the glandular secretory cells and the contractile myoepithelial cells. The glandular cells comprise a simple epithelium and the myoepithelial cells cover the former from the outside. The heights of the glandular cells are variable; in most parts they are tall and form a simple columnar epithelium, but in other parts they are short and comprise a simple cuboidal or flat epithelium (Fig. la and b). No intercellular canaliculi were observed (Ito, 1949). The myoepithelial cells are very long rodlike or threadlike cells whose longitudinal axes are oriented almost parallel or slightly oblique to the long axis of the gland tubule. They are arranged parallel to one another and form a kind of a bundle or a lattice around the tubular gland (Fig. lb). They are often spirally arranged because each myoepithelial cell is set slightly oblique to the long axis of the tubule. It has often been reported that the myoepithelial cells are better developed in the apocrine sweat glands than in the eccrine sweat glands. It has been argued that the myoepithelial cells are tightly packed and no gaps are left
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FIG. I . Photomicrographs of human axillary apocrine sweat glands. (a) Apical processes of cytoplasm protruding into the lumen (arrows), suggestive of apocrine secretion; (b) a grazed section through the periphery of the secretory tubule shows parallel-arranged myoepithelial cells (ME).
between the neighboring cells in the human apocrine gland (Talke, 1903; Schiefferdecker, 1922), but it was found that there is a gap between the adjacent myoepithelial cells as observed by light microscopy (Kan, 1941) and electron microscopy (Kawabata and Kurosumi, 1976). Even in a single section of a glomerulum of an apocrine sweat gland seen in a histological preparation, a wide variety of morphology of the secretory tubule is observed: some are high columnar epithelium whereas others are flat or cuboidal epithelium. These differences in the shape and height of glandular cells were thought to be related to the functional state of the gland cells. It is the general view that the tall cells are more active as compared with the flat cells. Some authors argued that the loss of cytoplasm due to repeating release of parts of the cytoplasm by the apocrine secretion may result in a short cuboidal or flat shape of gland cells (Talke, 1903; Brinkmann, 1908, 191 1; Mislawsky, 1909; Schiefferdecker, 1922; Hoepke, 1927). On the other hand Schaffer (1927) observed that the contraction of myoepithelial cells may cause the flattening of the secretory epithelium. Talke (1903) described the presence of two types of glandular cells, clear cells and dark cells, in the human apocrine gland, but they are transitional to each other (Mislawsky, 1909) and are fundamentally of the same cell type. Today all investigators of sweat glands believe that the glandular secretory cells of the apocrine sweat glands are only one type, being different from the eccrine sweat
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glands which possess two different types of secretory cells. In most cases the glandular cells are tall and cylindrical and often extend apocrine processes into the lumen. In contrast to the irregular distribution of nuclei in the secretory epithelium of the human eccrine sweat gland, the nuclei of the apocrine gland cells are found almost in the same level, that is, slightly lower than the middle of the cell height or a little more basal. There are many dark stained large granules in the cytoplasm above the nucleus (Fig. la). Almost all light microscopists thought that these large granules are nothing but the secretory granules, but recent electron microscopic studies demonstrated that there is no evidence of extrusion of these large dense granules. Therefore, these granules are not called “secretory granules” in this article, though almost all studies of light microscopy did so designate these granules. We call them large dense granules in order to coincide with the description by electron microscopy. The real secretory granules found by electron microscopy (Kurosumi and Kurosumi, 1982) cannot be observed with the light microscope due to their small size (about 150 nm in diameter), so they did not appear in the description of light microscopic research. Even if the large dense granules are abundantly accumulated in the supranuclear cytoplasm, they are absent in the cytoplasmic zone just beneath the luminal surface of the cell. This homogeneous cytoplasmic zone devoid of dark granules was called the “cuticle” by earlier authors (Tadokoro, 1909; Tachibana, 1936) but Minamitani (1941a,b) and It0 (1949) called this zone the “crust.” Minamitani (1941b) also observed a structure like the brush border on the very surface of this zone, by very careful light microscopic observation of microvilli which were later easily discerned by electron microscopy. Brinkmann (1908) was the first to find mitochondria in animal apocrine sweat glands, and Nicolas et al. (1914) studied them in the human glands. Both of them argued that the secretory granules might be derived from the mitochondria. Minamitani (1941b) also observed the mitochondria in the human axillary apocrine sweat glands, and reached the same conclusion as the previous authors, because he found an inverse proportion between the numbers of mitochondria and secretion granules. Electron microscopy demonstrated an enormous hypertrophy of mitochondria in the secretory cells of the human apocrine sweat glands (Kurosumi et al., 1959; Kawabata, 1964; Kurosumi and Kawabata, 1976; Kurosumi and Kurosumi, 1982), and in early reports electron microscopists also described these large mitochondria as light secretory granules (Kurosumi et al., 1959), which were undoubtedly derived from the normal sized mitochondria. However, there has been no evidence of release of these mitochondria1 “secretory” granules into the gland lumen (Yasuda et al., 1962; Kurosumi and Kurosumi, 1982). Therefore, it may be better not to call these granules “secretory granules. The distinction between large dense granules and hypertrophied mitochondria is almost impossible with the light microscope, because they are almost the same size, as described in a later section of this article. The conclu”
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sions of Nicolas et al. (1914) as well as Minamitani (1941b) were not correct, but their observation was very accurate. The so-called secretory granules of these light microscopists were known to contain iron and lipid (Homma, 1925; Richter, 1933). The iron was not observed, however, in the granules of axillary apocrine glands of nonosmidrotic individuals, who occupy the majority (80-90%) of the Japanese population (Iwashige, 1951). The lipid in the granules of apocrine gland cells is colored and hence is called lipochrome or lipofuscin. The Golgi apparatus is situated immediately above the nucleus and is shaped like a basket. Its size is comparable to that of the nucleus. Minamitani (1941b) argued that the so-called secretory granules (large dense granules) are not related to the Golgi apparatus, as far as the results of light microscopic morphology is concerned. He suggested that the granules originate from the mitochondria below the Golgi apparatus, and move upward through the Golgi area, where the maturation of the granules takes place, namely, some granules acquire lipid, pigment, and iron. The mechanism of secretion release from the apocrine gland cells was believed to be primarily the so-called apocrine secretion which is also called decapitation. From the luminal surface of the gland cell, tonguelike or balloonlike processes extend into the lumen (Fig. la). Most of these processes contain no formed elements and appear homogeneous (Mislawsky, 1909; Minamitani, 1941b), but some authors argued that they contain vacuoles which were formed through the liquefaction of secretory granules (Brinkmann, 191 1 ; Schaffer, 1927; Krompecher and Rudinai-Molnar, 1935). There are two mechanisms of detachment of the apocrine secretory processes: the pinching-off through the constriction of the base of the process and the formation of a demarcation membrane at the base which is formed from the fusion of hematoxylin-stainable grains (Minamitani, 1941b). Other secretory mechanisms were also advocated for the secretory cells of the human apocrine sweat glands. Holocrine secretion was assumed by Holmgren (1922). The degeneration and death of gland cells may occur, and these dead cells may be desquamated into the gland lumen, but it is questionable whether this event may be one of the physiological processes of secretion or not. The eccrine mechanism of secretion release was also applied to some gland cells of the apocrine sweat glands (Kan, 1941), but direct evidence of eccrine secretion (that may be comparable with exocytosis in electron microscopy) was not discernible by light microscopy; however, electron microscopic research has now demonstrated evidence of this mechanism in the apocrine gland cells, as will be discussed in detail in a later section. C. LIGHTMICROSCOPIC CYTOLOGY OF
THE
ECCRINESWEATGLANDS
The formation of the glomerulum is more conspicuous in the eccrine sweat glands. The outside diameter of secretory tubules of this gland is always smaller
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than that of the apocrine gland, but the lumen is very narrow and hence the thickness of the wall is rather larger than that of the apocrine gland. Myoepithelial cells are arranged on the outer surface of the secretory coil, being oriented longitudinally or spirally and aligned parallel to one another. The secretory epithelium has a much more complicated structure than that of the apocrine gland, and consists of two different types of glandular cells, as first described by Ito (1943). He named these cell types “superficial cells” and “basal cells,” however, they are not arranged in two layers but in a pseudostratified epithelium (Fig. 2a). The distribution of nuclei is irregular and arranged roughly in two lines. Such an irregular distribution of secretory cells is one of the most prominent characteristics of the human eccrine sweat gland. The superficial cells are situated centrally and their free surface faces the gland lumen but their basal parts extend to the basement membrane, while the basal cells are arranged peripherally abutting on the myoepithelial cells or the basement.membrane; most of them do not reach the gland lumen. There are intercellular secretory canaliculi between the adjacent basal cells, and their secretion may be transported through the canaliculi toward the main lumen of the gland tubule. Because the superficial cells contain dark granules well stained with iron
FIG. 2. Photomicrographs of human eccrine sweat glands in the axillary region. (a) Two sections of secretory coil containing dark cells (D), clear cells ( C ) ,and myoepithelial cells (ME). (b) Transitional portion of the eccrine sweat gland; most of the epithelial cells are cylindrical and are arranged in a single layer. Myoepithelial cells are also present.
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26 1
hematoxylin and the cytoplasm is also darkly stained, these cells were called “dark cells” by Montagna et al. (1953). On the other hand, they called the basal cells of Ito (1943) “clear cells,” since these cells have a pale stain (Fig. 2a). The secretory granules of the superficial (dark) cells are intensely stained with PAS reaction and therefore they may contain acid mucopolysaccharides (Montagna et al., 1953; Formisano and Lobitz, 1957). Lee (1960) proposed the name “mucin cells” for the superficial cells and “chief cells” for the basal cells, but the latter nomenclature was not widely adopted. The first differentiation of these two kinds of cells in the eccrine sweat gland by Ito (1943), that is into the “basal cells” and “superficial cells,” was appropriate for the human eccrine sweat glands, but they are not suitable for animal glands, because the two types of glandular cells are mostly arranged in a single layer. Even in the human eccrine sweat glands, clear (basal) cells sometimes directly face the gland lumen, and these cells are not basal as in such a case. Therefore, the nomenclature of Montagna has been more frequently adopted by cytologists working on the sweat glands. According to It0 (1943), the Golgi apparatus of the superficial cells is quite different from that of the basal cells in structure. Therefore, a possibility of transition between these two types has been completely denied, and no intermediate forms of the gland cells could be observed. Minamitani (1941a) indicated that the secretory granules of the eccrine sweat gland are smaller than those of the apocrine gland, and they are distributed in the very superficial part of the cells abutting the lumen, because the eccrine sweat gland cells have no crust which is characteristic of the apocrine sweat gland. Ito and Iwashige (195 1) argued that the secretory granules of superficial (dark) cells are also formed from the mitochondria. Secretory granules are stained dark with iron hematoxylin, but there were no signs indicating the presence of either iron, lipid, or pigment (Ito, 1949). As already described, they are PAS-positive and hence it is clear that the granules contain mucopolysaccharide. At times among secretory granules clear vesicles of the same size as granules are mixed. They are secretory vacuoles which are probably derived from the secretory granules through the liquefaction of granular substance. Ito (1949) and Ito and Iwashige (195 1) indicated that the secretory granules of the basal (clear) cells are larger than those of superficial (dark) cells, and they are also derived from mitochondria. By electron microscopy the presence of secretory granules in the dark cells was proved, but no secretory granules were observed in the clear cells (Kurosumi and Kurosumi, 1982). It is not clear to what the socalled secretory granules of the basal cells correspond, though the lysosomes, mitochondria, and lipid droplets of various sizes were observed in this type of cell by electron microscopy. Intercellular secretory canaliculi are always seen between the clear (basal) cells, but not between the dark (superficial) cells. Some earlier authors described the intracellular canaliculi (Zimmermann, 1898; Holmgren, 1922; Klaar, 1924;
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Hoepke, 1927; Kan, 1941), but they have not been observed by both light and electron microscopy. The Golgi apparatus is characteristic of both types of glandular cells. The morphological difference of the Golgi apparatus in the superficial and basal cells was the clue to discovering the presence of these two different types of secretory cells in the human eccrine sweat gland (Ito, 1943). The Golgi apparatus of the superficial (dark) cell is a circumscribed network situated above the nucleus, while that of the basal (clear) cell consists of scattered Golgi elements, each of which is either semilunar, semicircular, or rodlike (Fig. 3a). According to the classification of Hirschler (1927), the Golgi apparatus of the superficial cell corresponds to the complex type, while that of the basal cell is the diffuse type. The separate elements of the Golgi apparatus of the basal cell are often observed along the intercellular canaliculi. Lipid droplets are often seen in both cell types, and are especially abundant in the Golgi field. They are either monovesicular or polyvesicular (Fig. 3b). There is a tendency for the lipid droplets to increase in size and number, as the age of the individual possessing the sweat gland under histological examination increases. Ito et al. (1946) studied the distribution of glycogen in the human sweat glands, and showed that glycogen is not present in the apocrine glands, but is present in the eccrine glands. Furthermore, glycogen was found only in the basal
b*
i
‘
I
FIG.3 . Drawings of the Golgi apparatus of the human eccrine sweat gland. (a) Different natures of Golgi apparatus between the dark (superficial) cells and clear (basal) cells; the former is the complex form and the latter is the diffuse form. (b) Lipid droplets of mono- and polyvesicular types are associated with the Golgi apparatus. (From Ito, 1943.)
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(clear) cells and not in the superficial (dark) cells (lto, 1949; Montagna et al., 1952). In animal eccrine sweat glands, however, Tsukagoshi (1951) could not obtain agreement with the human case, concerning the distribution of glycogen. Minamitani (1941a) as well as Ito and Iwashige (1951) found the occurrence of the apocrine mechanism of secretion release from the superficial (dark) cells of the human eccrine sweat gland. This finding indicated that the classification of sweat glands by Schiefferdecker (1922), namely, the apocrine and eccrine glands, did not show the secretion mechanism accurately but only showed the names of sweat gland types. Although the apocrine mechanism of secretion release occurs in both the apocrine and eccrine glands, the apocrine secretion is slightly less frequent in the eccrine glands than in the apocrine glands.
111. Ultrastructures and Their Functional Significance of the Human
Sweat Glands A. THEHUMANAPOCRINE SWEATGLAND Many electron microscopists studied the ultrastructure of the human apocrine sweat glands using material from the axillary skin (Kurosumi er al., 1959; Charles, 1959; Yamada, 1960; Hibbs, 1962; Biempica and Montes, 1965; Munger, 1965b; Hashimoto et a l . , 1966c; Bell, 1974; Schaumburg-Lever and Lever, 1975; Kurosumi and Kurosumi, 1982), while a few reports from the first author’s laboratory (Kawabata, 1964; Kurosumi and Kawabata, 1976; Kawabata and Kurosumi, 1976) concerned the apocrine gland of the external auditory meatus which is also called the ceruminous gland. 1. The Secretory Cell
The secretory portion of the apocrine sweat gland consists of coiled tubules whose lumen is very wide. The wall is variable in thickness, but in most cases it is a simple columnar epithelium made up of tall secretory cells, and underlying myoepithelial cells are interposed between the secretory epithelium and the basement membrane. Cylindrical or cuboidal secretory cells contain spherical nuclei situated in the basal half of the cell (Fig. 4). If the cells are flattened, the nuclei are also slightly flattened and their vertical sections are elliptical with their long axes oriented parallel to the base of the epithelium. The nucleolus is usually well developed and roughly spherical in shape. The nuclear envelope is provided with numerous pores. Four kinds of granules are observed in the cytoplasm: (1) large dense granules, (2) cored vacuoles, (3) large less dense granules, and (4) small secretory granules. a. Large Dense Granules. Large dense granules are very unique and easily
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FIG. 4. Electron micrograph of the secretory portion of the human axillary apocrine sweat gland. Cylindrical cells contain large round mitochondria (M), large dense granules (D) in the middle part of the cell, and small secretory granules (S) along the apical surface membrane. Myoepithelial cells (ME) and folding of the basal plasma membrane (BF) are seen at the bottom. (From Kurosumi and Kurosumi, 1982.)
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observable even by light microscopy due to their enormous size (0.8-1.0 pm), and are often erroneously called secretory granules by light microscopists (Minamitani, 1941a,b). No evidence for releasing of these granules is observed (Kurosumi and Kurosumi, 1982), but authors of early reports by electron microscopy, including us (Kurosumi et al., 1959; Charles, 1959; Hashimoto et al., 1966c), thought that the substance of these granules might be secreted either by way of liquefaction forming vacuoles and diffusing away in the apical cytoplasm (Kurosumi et al., 1959). or budding of many vacuoles and vesicles from the large dense granules which might be discharged by exocytosis (Fig. 5 ) (Kurosumi and Kawabata, 1976). Light microscopists (Nicolas et al., 1914; Minamitani, 1941b) thought that these granules might originate from mitochondria, but there is no relation between mitochondria and large dense granules as observed by electron microscopy. From the earliest study on this gland to now, we thought that the large dense granules may arise from the cored vacuoles derived from the Golgi apparatus or smooth endoplasmic reticulum (Kurosumi et al., 1959; It0 and Shibasaki, 1966b; Kurosumi and Kurosumi, 1982). The large dense granules can be further classified into two types: A and B. Type A has a rather smooth contour and is mostly spherical or oval in shape, though there are some with an elongate shape. The granules of this type contain
FIG. 5 . Large dense granules of the secretory cell of the human ceruminous gland. Clear vacuoles are seen protruding from the surface of the dense granules. (From Kurosumi and Kawabata, 1976.)
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KAZUMASA KUROSUMI ET AL.
some small vesicles, which often protrude from the surface of the granule (Figs. 5, 6b, and 7a). These less dense vesicles may be shed off from the granule and move to the apical surface of the cell (Kurosumi and Kawabata, 1976). The internal substance of this type of dense granules is very dense and relatively homogeneous, as observed in low magnification, but very small particles of three different orders of diameter can be detected in high magnification electron micrographs (Fig. 6a). The smallest is about 50 A, the middle is about 75 A, and the largest is about 140 A. They are not evenly distributed, but are arranged in a pattern with a fingerprint or speckled appearance (Fig. 6b). Though Munger (1965b) stated that the large dense granules might contain keratin filaments, no filamentous substance was observed by us (Kurosumi and Kurosumi, 1982). Type B is a large heterogeneous granule and often looks like an aggregate of many small dense granules (Fig. 7b). The matrix of the granule of this type is slightly lower in electron density than that of the Type A granule, and often contains many clear vesicles. This type is similar in morphology to the so-called lipofuscin granules of other tissues. Ito and Shibasaki (1966b) observed a complex of dense granules corresponding to the Type B granules in this description, and conjectured that such heterogeneous granules are the breakdown step of the Type A large dense granules which they called secretory granules. After break-
FIG.6. Large dense granules of the axillary apocrine gland. They contain clear vesicles and much denser particles either scattixed randomly or arranged lineally forming a fingerprint-like pattern. (From Kurosumi and Kurosumi, 1982.)
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down, the material of the dense secretory granules might be scattered in the cytoplasm, and released to the gland lumen by either apocrine or eccrine secretory mechanisms. Biempica and Montes (1965), Hashimoto et ul. (1966c), and Kurosumi and Kawabata (1976) demonstrated the activity of acid phosphatase in the large dense granules (Fig. 8a). Therefore, these granules were thought to be one of the lysosomes. Most of these papers did not divide the large dense granules into two types, so that it is not clear which type contains acid phosphatase, but it is probable that both types are positive in acid phosphatase reaction. Kurosumi and Kawabata (1976) showed the positive reaction not only on the dense granules but also on the vesicles which have probably been released from the dense granules and move about in the cytoplasm. We thought that these vesicles containing lysosomal enzymes may be secreted into the lumen by exocytosis (Fig. 8b). Later we found most of the secretory granules and vesicles arranged beneath the apical surface and extruded by exocytosis may be directly derived from the Golgi apparatus (Kurosumi and Kurosumi, 1982), but the former mechanism (enzyme secretion) is also possible. As we demonstrated the reaction products of acid phosphatase in the peripheral part of the gland lumen, we conjectured that the lysosomal hydrolases released into the lumen may act to digest the cytoplasmic
FIG.7. Granules of the human apocrine sweat glands. (a) Type A large dense granules and cored vacuoles (arrows) of the axillary gland; (b) Type B large dense granule of the ceruminous gland. (From Kurosumi and Kawabata, 1976.)
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FIG. 8. Results of cytochemical detection of acid phosphatase activity. Reaction products are present on the large dense granules and clear vesicles in the apical cytoplasm as well as in the gland lumen. The human ceruminous gland. (From Kurosumi and Kawabata, 1976.)
debris which has been detached from the main cell body by the mechanism of apocrine secretion. Bell (1974) classified the dense granules into three types: the Type I granule is the same as Type A, and Type I1 corresponds to our Type B. The Type I11 granules of Bell are those we called cored vacuoles. Bell thought that the Type I granules are secretory granules like our first report (Kurosumi et af., 1959) and light microscopic reports (Minamitani, 1941b). She observed 250 8, particles contained in Type I granules and reported that these particles were dispersed after stimulation by epinephrine injection (Bell, 1974). She considered that Type 1 granules might originate from Type I11 granules. This idea is the same as ours proposed as early as 1959 (Kurosumi et al., 1959). In this report we called the cored vacuoles ‘‘encapsulated granules” and presumed that these vacuoles may grow larger and become the large dense granules (dark secretory granules). In a recent paper we again assumed the conversion from the cored vacuole into the large dense granules (Kurosumi and Kurosumi, 1982). b. Cored Vacuoles. The cored vacuoles are variable in size from 170 to 550 nm in diameter. Each vacuole is limited by a single membrane and contains less dense or medium dense substance. A dark core is always observed in the vacuole in an eccentric position and is often attached to the inner aspect of the limiting
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FIG. 9. Cored vacuoles (arrows) and large less dense granules derived from mitochondria in the secretory cells of the axillary apocrine gland. (From Kurosumi and Kurosumi, 1982.)
membrane at one side of the vacuole (Figs. 7a and 9). The outline of the core is not sharp but fades away gradually into the less dense matrix surrounding the core. The density as well as the positive reaction to methenamine silver are similar to the dense substance of large dense granules, and therefore the hypothesis that the advancing accumulation of dense core substance within the vacuole may develop into the large dense granules was suggested (Kurosumi et al., 1959; Bell, 1974; Kurosumi and Kurosumi, 1982). The origin of the cored vacuole is not clear, but it is possible that the accumulation of a dark substance within the Golgi vacuole may form this organelle. The cored vacuoles, however, often appear in the basal cytoplasm far below the Golgi apparatus, where only mitochondria and endoplasmic reticulum exist. Therefore, the origin of cored vacuoles from components other than the Golgi apparatus cannot be excluded. The endoplasmic reticulum was often assumed to be the origin of these vacuoles, but the surface of the vacuole is always smooth without attached ribosomes. Therefore, Kurosumi et al. (1959) presumed that the cored vacuoles (encapsulated granules) might occur from the Golgi apparatus or smooth surfaced endoplasmic reticulum. But Bell (1974) wrote that the Type 111 granules (corresponding to the cored vacuoles) might arise from vesicles associated with the Golgi zone or with the granular endoplasmic reticulum. The authors cannot accept the direct relation of rough endo-
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plasmic reticulum to the cored vacuoles, though the final solution of this problem has not been achieved. c. Large Less Dense Granules. Occurrence of very large mitochondria is one of the peculiar characteristics of the secretory cells of the human apocrine sweat gland. They are sometimes larger than the large dense granules and reach a size as large as 3 pm in diameter, and are also called large less dense granules. They are usually round and limited by a double membrane indicating mitochondrial origin. The internal matrix is moderate in electron density and almost homogeneous. They contain remnants of cristae mitochondriales, which are mostly curved in an archlike shape, being attached at both ends on the same wall, although some cristae are straight (Fig. 9). Such a strong hypertrophy of mitochondria probably caused light microscopic cytologists and some electron microscopists in the early stage of the electron microscopic cytology of the sweat glands to consider that mitochondria may turn into the secretory granules of this gland (Minamitani, 1941b; Ito, 1949; Kurosumi et al., 1959). Kurosumi et al. ( 1959) called these hypertrophied mitochondria light secretory granules which was opposite to the dark secretory granules (large dense granules). Charles (1959) also found the presence of two types of secretory granules and called them rough and smooth secretory granules, but he denied the mitochondria1 origin of secretory granules. At first Kurosumi et al. (1 959) conjectured that light granules of enormous size might be secreted into the lumen, but this possibility was negated by many authors (Hibbs, 1962; Yasuda et al., 1962; Kawabata, 1964; Kurosumi and Kawabata, 1976; Kurosumi and Kurosumi, 1982). Such an enormous hypertrophy of mitochondria is observed only in the human apocrine Sweat glands of both axillary and external auditory regions; it is not observed in the human eccrine sweat glands and animal sweat glands of both apocrine and eccrine types. The significance of this hypertrophy and the chemical nature of the substance accumulated in the mitochondria have not been elucidated. d. Small Secretory Granules. In both the axillary gland and the ceruminous gland, small granules and vesicles about 100-200 nm in diameter are scattered in the cytoplasm immediately beneath the luminal surface membrane (Fig. 10a and b). Each granule is covered with a smooth limiting membrane. The intefnal substance varies markedly from very dense to watery clear (Fig. 1Oc). The latter case is called vesicle instead of granule. But if methenamine silver staining was applied, almost all the granules and vesicles arranged in the apical cytoplasm are darkly stained. This result indicates that the granules and vesicles are only varieties of the same entity and they contain mucopolysaccharide. Opening of these granules and vesicles onto the surface of the gland cell is frequently observed and suggestive of exocytosis leading the internal substance to be released into the lumen (Fig. lob). Such a finding strongly indicated that
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FIG. 10. Small secretory granules arranged along the cell surface. Sometimes they appear as empty clear vesicles. Arrow indicates the exocytotic release of the granule. (From Kurosumi and Kurosumi, 1982.)
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these granules are the true secretory granules. Sometimes balloonlike processes containing these vesicles are observed. The small secretory granules or vesicles may be dropped out with the cytoplasmic matrix into the lumen by the mechanism of apocrine secretion (Kurosumi and Kurosumi, 1982). These small secretory granules were first described by Yamada (1960). He classified the granules of the apocrine gland cell into three types, G I , G2, and G3. According to him, G 1 granules are the young secretory granules produced at the Golgi apparatus, and grow into G2 granules which correspond to the large dense granules of Kurosumi and Kurosumi (1982). G3 granules are small secretory granules arranged along the luminal surface membrane. G2 granules contain less dense vesicles and they often protrude from the surface of the granule. They may be detached from G2 granules and become G3 granules which are finally released into the lumen. This idea was later supported by Kawabata (1964) and Kurosumi and Kawabata (1976), and therefore the large dense granules were called prosecretory granules. But the later study on the axillary gland (Kurosumi and Kurosumi, 1982) did not show strong evidence of small secretory granules or vesicles arising from the large dense granules. The clear spots in the large dense granules were not positive to methenamine silver reaction; though both the small secretory granules (vesicles) and the dark part of the large dense granules strongly reacted with methenamine silver, the clear spots in the large granules might be unrelated to the secretory granules. Ito and Shibasaki (1966b) found clear vesicles just beneath the luminal surface membrane, and thought that these are the secretory vesicles containing material other than the large dense granule, and probably released by exocytosis (eccrine mechanism). Bell (1974) described in detail the large dense granules, but she was unaware of small secretory granules. Munger (1965b) described both large dense granules and small secretory granules (vesicles). He argued that the large dense granules contain keratin, but this assumption was denied by Bell (1974). Munger (1965b) did not correlate the secretoiy granules (vesicles) arranged in the apical cytoplasm with the large dense granules which he assumed to be keratin. He concluded that these secretory vesicles (vacuoles) containing mucopolysaccharide are formed in association with the Golgi apparatus and are liberated from the cytoplasm by a merocrine mechanism. In his terminology “merocrine” means exocytosis. Munger’s conclusion is in complete agreement with our findings using methenamine silver staining (Fig. 11) (Kurosumi and Kurosumi, 1982), though we did not agree with Munger (1965b) as far as the nomenclature of merocrine instead of exocytosis and the assumption that the large dense granules might contain keratin. There are some small granules in the Golgi apparatus and these granules are positively reacted with methenamine silver; the small secretory granules may arise at the Golgi apparatus, but not be related to the large dense granules (Kurosumi and Kurosumi, 1982).
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FIG. 1 I .
273
Result of methenamine-silver staining for polysaccharides. Both large dense granules
(D)and small secretory granules (S) reacted positively. Small granules in the Golgi area (arrows) are equal in size to the apical secretory granules. (From Kurosumi and Kurosumi, 1982.)
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e. Apocrine Secretion Mechanism. Pinching-off of the cytoplasmic projection extended from the cell body is known as “apocrine secretion” or “decapitation,” and the former term became the origin of the name of this kind of sweat gland. When we observe the apocrine sweat glands with the light microscope, we can observe tonguelike processes projecting into the lumen very frequently, and none of the light microscopic researchers doubted this mechanism of secretion discharge. Transmission electron microscopy of the thin sections, however, seldom demonstrates the cytoplasmic processes suggestive of apocrine secretion, and many researchers denied this phenomenon as a physiological mechanism of discharge of secretory substance and they ascribed the ballooning of apical cytoplasm to an artifact which occurred during specimen preparation (Yamada, 1960; Hibbs, 1962; Biempica and Montes, 1965; Munger, 1965b; Bell, 1974). The electron microscopic images, however, suggesting the apocrine secretion were obtained not only with the transmission electron microscope (Fig. 12a and b) (Kurosumi et al., 1959; Kawabata, 1964; Hashimoto et a l . , 1966c; Schaumburg-Lever and Lever, 1975; Kurosumi and Kurosumi, 1982), but also with the scanning electron microscope (Fig. 13a and b) (Kurosumi and Kawabata, 1976). In the mammary gland of a lactating rat, pinching-off of cytoplasmic projections was frequently observed (Kurosumi et a l . , 1968). In this case the secretory
FIG. 12. Large cytoplasmic processes protruding into the lumen suggestive of apocrine secretion. The process shown in b contains secretory vesicles. The human axillary apocrine gland. (From Kurosurni and Kurosumi, 1982.)
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process contains a large fat droplet and a few vacuoles containing granules of milk protein (casein), but in most cases of the apocrine sweat glands, the interior of the secretory process is homogeneous. In our recent observation, however, we observed small vesicles aligned along the surface plasma membrane of an apocrine secretory process (Kurosumi and Kurosumi, 1982). Like the casein granules in the mammary gland, mucopolysaccharide-containingsecretory granules (vesicles) of the human apocrine gland may be released in two ways, by the apocrine mechanism and exocytosis. Kurosumi et al. (1959), Ito and Shibasaki (1966b), and Hashimoto et ul. (1966~)speculated that destruction of the large dense granules might result in the dispersion of the material of large granules in the apical cytoplasm which would be detached and drop into the gland lumen, but the features of breakdown of large granules were very rarely observed. Schaumburg-Lever and Lever ( 1975) proposed three mechanisms: merocrine (exocytosis), apocrine, and holocrine secretion. As the original meaning of the term ‘‘merocrine” included apocrine and eccrine secretory mechanisms (Schiefferdecker, 1917, 1922), the use of this term for indicating exocytosis is erroneous, but the evident demonstration of exocytosis of the secretory vesicles, which originated from the Golgi apparatus as they assumed, was convincing.
FIG. 13. Scanning electron micrographs of the luminal surface of the human ceruminous gland. (a) Many small projections for microapocrine secretion; (b) a large projection for macroapocrine secretion. (From Kurosumi and Kawabata, 1976.)
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They showed three steps of apocrine secretion: at the beginning a rounded process called the apical cap is formed on the luminal cell surface; second, a horizontal dividing membrane is formed at the base of the apical cap; and finally tubules appear above the dividing membrane and fuse with one another leading to separation of the apical cap from the cell body. The formation of the dividing membrane is similar to the early observation of the so-called demarcation membrane from which the process may be detached (Minamitani, 1941b; Kurosumi et a l . , 1961), but in the case of Schaumburg-Lever and Lever (1975), the dividing membrane is not effective for separation of apocrine process. It is doubtful whether the tubules so described by these authors are sections through the cell surface invaginations at the constricted part of the process. If so, the illustrations in their paper did not show the final image of separation, but a slightly earlier stage before the true separation at the dividing membrane. They also showed a picture of destruction of glandular cells as evidence of holocrine secretion, but it is possible that this is an artifact during specimen preparation. f. Plasma Membrane Folding. The lateral intercellular boundaries of the axillary apocrine sweat gland cells are mostly smooth and straight, but their basal ends are folded in a complicated way. Furthermore, the basal surface abutting the basement membrane is much more complicated (Fig. 14). Such a folding of the basal plasma membrane of the human apocrine sweat gland was first described by Takahashi (1957). Then almost all researchers dealing with axillary apocrine sweat glands referred to the complicated folding of the basal plasma membrane (Charles, 1959; Kurosumi et a l . , 1959; Yamada, 1960; Hibbs, 1961; Yasuda er a f . , 1962; Munger, 1965b; Bell, 1974; Kurosumi and Kurosumi, 1982). These foldings may be formed at least partly by interdigitation with the adjacent cells as reported in other tissues such as the renal tubules (Rhodin, 1958) and salivary glands (Tamarin and Sreebny, 1965), but the true infolding of the plasma membrane into its own cytoplasm is really observed (Kurosumi and Kurosumi, 1982). In some papers, rows of vesicles are seen as continuous with the infolded double membranes (Kurosurni et al., 1959; Munger, 1965b). These vesicles might be formed artificially by simple osmium fixation through the fragmentation and vesiculation of the infolded plasma membrane. Recent papers using improved methods of fixation do not contain such features of membrane vesiculation. The ceruminous apocrine glands, however, contain no such complicated membrane foldings (Kawabata, 1964; Kurosumi and Kawabata, 1976), probably due to the fact that the cerurninous glands do not secrete sweat with much water content as secreted from the axillary glands. The plasma membrane may act as both the barrier and carrier of ions which may induce the movement of water being secreted as an important constituent of sweat. Biernpica and Montes (1965) reported high ATPase activity at the basal folded membranes as a result of comparative observations by electron micros-
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FIG. 14. The basal part of the secretory cell of the human axillary apocrine gland. Infolded plasma membranes are remarkable, but some of them are formed by interdigitation between the adjacent cells.
copy and light microscopic histochemistry on the human axillary apocrine glands. On the animal material, Matsuzawa and Kurosumi (1963) demonstrated ATPase and alkaline phosphatase activities on the basal infoldings of rat plantar eccrine sweat glands with electron microscopic histochemical techniques. We could not observe the basal infoldings in the sweat glands of neonatal rats, when sweating had not begun. Therefore the basal infolding may participate in the water transport directly correlated with sweating, but Bell (1974) reported that no changes in basal infolding were observed after epinephrine injection. It could be that either her stimulation was too weak or changes in enzyme activity would occur without any changes in morphology. 2 . The Myoepithelial Cell The other type of cells constituting the secretory portion of the apocrine sweat gland is the myoepithelial cells, which are interposed between the secretory cells and the basement membrane. The myoepithelial cells are long fiberlike cells about 100-150 pm in length and 3-5 pm in width (Kawabata and Kurosumi, 1976). They are arranged in parallel to each other, but they are not closely attached to one another leaving narrow gaps between the adjacent cells, looking like a louver. Electron microscopy of the myoepithelial cells of the human apocrine glands
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was first carried out by Taka.hashi (1957), and then many researchers on the ultrastructures of the apocrine sweat glands referred to the myoepithelial cells as well as the secretory glandular cells (Kurosumi et al., 1959; Yamada, 1960; Hibbs, 1962; Kawabata, 1964; Kawabata and Kurosumi, 1976). The general morphology and the ultrastructure of myoepithelial cells of the apocrine sweat glands are almost identical with those of eccrine sweat glands, but in the human skin, the myoepithelial cells are better developed in the apocrine glands than in the eccrine glands. The long axes of the myoepithelial cells are almost parallel to the long axis of the secretory tubule, but sometimes they are slightly oblique, and in such a case the general direction of myoepithelial arrangement is a loose spiral. Narrow gaps between the neighboring myoepithelial cells are filled with processes of secretory cells and attached directly to the basement membrane. A tangential section of the secretory coil through the myoepithelial gaps shows the narrow strips of secretory cells adjoined to each other with wavy interdigitating boundaries. The surface of the myoepithelial cells is usually smooth either abutting to the secretory cells or the cells of the same nature. The latter cell junctions are observed with the scanning electron microscope (Fig. 15), at the tips of long fibrous cells, where two adjoining cells attached to each other making V- or U-shaped end-to-end
FIG. 15. Scanning electron micrograph of the inner surface of the myoepithelial cells after the complete removal of secretory cells. The human ceruminous gland. (From Kawabata and Kurosumi, 1976.)
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junction or side-by-side junction on the lateral surfaces of the tapering tips of myoepithelial cells (Kawabata and Kurosumi, 1976). On the boundary between the myoepithelial cells and secretory cells, foldings of the plasma membranes are often observed. These folded membranes belong almost entirely to the plasma membranes of the secretory cells, and those of the myoepithelial cells only participating to the formation of a simple interlocking made by a small knobby spike projecting into the secretory cell cytoplasm. Sometimes narrow intercellular spaces are present between the myoepithelial cells and the secretory cells. The profile of the myoepithelial cells varies widely according to the cutting angles of these cells. When the secretory tubules are cut transversely, they appear either triangular, domelike, or clublike with rounded tips (Fig. 16). The nucleus is shifted toward the apex of the cell, and lies in the region of the greatest height of the cytoplasm. The lower half of the cytoplasm is filled with myofilaments running parallel to the long axis of the cell. Probably due to the abundance of myofilaments filling the cytoplasm, the overall density of the cytoplasm is higher than that of the secretory cell. Therefore, distinguishing between secretory cells and myoepithelial cells is easy even in low magnification electron micrographs. When the myoepithelial cell is cut longitudinally, the myofilaments run parallel to the long axis of the cell. They are sometimes straight but in some other cases they are remarkably undulating due probably to the contraction of filaments to a considerable extent. Myofilaments are usually divided into many bundles by dense longitudinal bands. Between the secretory cells and myoepithelial cells as well as between two adjacent myoepithelial cells desmosomes make their appearance. On the thickened plasma membranes at the desmosome, cytoplasmic filaments appear to be attached as in the case of desmosomes between epidermal keratinocytes. These cytoplasmic filaments attaching to the desmosomal plates are the intermediate (the so-called 10 nm) filaments and correspond to the tonofilaments in the keratinocytes. They are seen on both sides of the desmosome, namely, in the secretory cell and in the myoepithelial cell, but they are much more conspicuous in the former than in the myoepithelial cell. Along the basal surface of the myoepithelium abutting on the basal lamina are dense spots similar in morphology to the hemidesmosomes of the basal cell of the epidermis. They have been called dense zones (Ellis, 1965) or attachment devices (Tandler, 1965), but it may be proper to call these structures hemidesmosomes, because of their ultrastructural similarity to the hemidesmosomes of the epidermal basal cells. The occurrence of desmosomes and hemidesmosomes indicates the epithelial nature of the myoepithelial cells of the sweat glands, which are different in ontogeny from the ordinary smooth muscle cells. The basement membrane which is also called the basal lamina commonly covers the basal surfaces of both the myoepithelial cells and secretory cells.
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FIG. 16. A cross section of a myoepithelial cell of the human ceruminous gland. The basal half of the cytoplasm is filled with abundant myofilaments of two different thicknesses. Pairs of microtubules are also scattered (arrows). On the cell surfaces small vesicles probably concerning pinocytosis are observed. (From Kawabata and Kurosumi, 1976.)
There is a clear zone of about 40 nm in width between the basal plasma membrane of myoepithelial or secretory cells and the basal lamina. In this clear zone adjacent to the hemidesmosome can be seen a short delicate dense plate closely applied to the outer surface of the plasma membrane of the myoepithelial cells. The width of the clear zone is nearly uniform irrespective of the presence of hemidesmosomes, but the dense layer situated next to the clear zone, that is, the basal lamina proper, is variable in thickness, though it does not fall below about 200 nm. Sometimes, the basal lamina accompanying the clear zone invades the
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cytoplasm of the myoepithelial cells or the junction between the myoepithelial cell and the adjacent secretory cell. Such features indicate a very firm attachment of the myoepithelium to the basal lamina and periglandular connective tissue, which consists of strikingly developed collagen bundles. Along the myoepithelial cell surfaces, both on the apical and basal sides, particularly at the places where neither desmosomes nor hemidesmosomes are present, small vesicles gather or attach to the surface plasma membrane and they seem to open to the extracellular milieu. These vesicles may be formed by the invagination of the surface plasma membrane, taking a small amount of extracellular substance and carrying it deeply into the inside of the cell. This mechanism of cell drinking is often called micropinocytosis. As both the outer (basal) and inner (apical) surfaces of the myoepithelial cell are associated with these small vesicles, we can consider two possibilities: (1) the occurrence of micropinocytosis on these two opposite sides of the cell concomitantly, and ( 2 ) the vesicles are formed at one side of the cell and move toward the other side carrying the substance across the cell body, and finally expel the substance by exocytosis. Such a mechanism is called “cytopempsis” (Moore and Ruska, 1957) and transports a certain substance, probably the material for the synthesis of secretory substance by the glandular secretory cell (Kurosumi et al., 1959). However, these vesicles and pits which were assumed to be related to pinocytotic activity were also observed in the ordinary smooth muscle cells without connection to glandular cells. Therefore, the direct relation of these vesicles in the myoepithelium of the sweat gland to the secretory function may be ruled out. Thus the content of vesicles may be utilized in the myoepithelial cell itself, but the question of whether such a substance taken up by the mechanism of pinocytosis and contained in the vesicles may be a nutrient for the myoepithelial cell or a stimulative substance triggering the contraction of this cell has not been answered. The ordinary cell organelles are mostly situated in a narrow zone of the cytoplasm surrounding the nucleus. The nucleus is shaped like a spindle with its long axis oriented longitudinally. Observation with a scanning electron microscope demonstrated the strong protuberance of the cell body toward the luminal side of the gland. The nucleus is contained in such a protrusion (Kawabata and Kurosumi, 1976). The Golgi apparatus is always seen in the narrow perinuclear cytoplasm. They are not well developed. The rough endoplasmic reticulum is usually tubular and situated either around the nucleus or in the periphery of the cell. Slender mitochondria are also distributed along the cell surface. Dense irregular bodies probably corresponding to the lysosomes are also seen near the pole of the elongate nucleus. Free ribosomes are randomly scattered. Coated vesicles probably related to pinocytosis occur near the lateral cell surface. The basal half of the myoepithelial cell is filled almost exclusively with myofilaments, which are arranged parallel to the long axis of the cell. Scattered
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among the myofilaments many dense areas or zones are detected. They were thought to correspond to the Z-band of the striated muscle (Ellis, 1965). Yamada (1960) first reported the presence of two kinds of myofilaments in the human apocrine glands in the axillary region. We also found thin filaments about 50 A in thickness and thick filaments about 100 A in thickness in the myoepithelial cells of the ceruminous apocrine gland (Kawabata and Kurosumi, 1976). Intermediate filaments were reported in smooth muscle cells of other organs (Devine et al., 1972; Nonomura, 1976). It is not certain which of the filaments of the myoepithelium of the sweat gland corresponds to the intermediate filaments of smooth muscle cells. Microtubules also extend longitudinally in the myoepithelium and very often appear as twin filaments (Fig. 16) (Kawabata and Kurosumi, 1976). Ellis (1965), who studied the myoepithelium of the human eccrine sweat glands, reported only a single type of myofilament, but assumed that the microtubules might be equivalent to the thick filaments. As the thick filaments in the myoepitheliurn is similar in thickness to the intermediate filaments in other tissue, the microtubules may correspond to the thick filaments as suggested by Ellis (Kawabata and Kurosumi, 1976). Not only a similarity in ultrastructure to the ordinary smooth muscles, but also physiologically the ability of contraction was proved in the myoepithelial cells of the human sweat glands by Hurley and Shelley (1954). These authors observed the peristaltic waves along the secretory coil and the appearance of a sweat droplet upon the orifice, when myoepithelium was stimulated pharmacologically, electrically, or mechanically.
B. THE HUMANECCRINE SWEATGLAND The secretory portion of the human eccrine sweat gland is much more complicated than that of the apocrine sweat gland. There are two kinds of secretory cells: the superficially situated dark cells and basally situated clear cells. The myoepithelial cell is the third cell type and is most basally localized. The fundamental morphology of the myoepithelial cells is the same as that of the apocrine sweat glands, but the secretory cells are markedly different from those of the apocrine glands, and also are different from each other between the two cell types. Electron microscopy of this gland started at about the same period, in the latter half of the 1950s, as electron microscopy of the human apocrine sweat gland. Reports were then published, but not many, with only one or two papers appearing in each year from 1955 to now (Laden et al., 1955; Hibbs, 1958; Iijima, 1959; Charles, 1960; Kaname, 1960; Kurosumi et al., 1960; Munger, 1961; Ellis, 1962, 1967, 1968; Ito and Shibasaki, 1966a,b; Briggman et al., 1981; Kurosumi et al., 1982).
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In the early years of investigations of biological specimens by electron microscopy, almost all the specimens were fixed with a simple osmium fixation. The eccrine sweat glands were not an exception. The secretory granules of the superficial cells which characterize this cell type by their dark stainability with iron hematoxylin, from which the name “dark” cell (Montagna et al., 1953) originated, were not well preserved with simple osmium fixation and became similar to empty vacuoles (Ito and Shibasaki, 1966a,b). Therefore, the distinction between the dark cells and clear cells was vague in electron micrographs obtained with simple osmium fixation. For these specimens, as far as the human eccrine sweat gland is concerned, the initial nomenclature of Ito (1943) seems to be better than that of Montagna et al. (1953). Therefore, some of the relatively old literature on electron microscopic studies on the human eccrine sweat glands did not adopt Montagna’s nomenclature of dark and clear cells, but used Ito’s nomenclature, that is superficial and basal cells (Hibbs, 1958; Iijima, 1959; Kurosumi et al., 1960; Ito and Shibasaki, 1966a,b). Some years later, the double fixation method, consisting of a first fixation with glutaraldehyde and a second fixation with osmium tetroxide, became popular, since Sabatini et al. (1963) introduced glutaraldehyde as a fixative for electron microscopic cytochemistry . Today double fixation with glutaraldehyde and osmium is the routine method for specimen preparation for ultramicrotomy. Application of this double fixation to the eccrine sweat gland was performed by Terzakis (1964) on monkey eccrine glands and showed the characteristic dark staining of the secretory granules of the superficial (dark) cells, and complete coincidence between the light and electron microscopic findings concerning the stainability of secretory granules of this cell was achieved. Thus Montagna’s nomenclature was revived in electron microscopic cytology. It must be noted, however, that the clear (basal) cells which are usually seen as clear in routine electron micrographs because of the absence of dark secretory granules sometimes look dark, particularly if they contain a large amount of glycogen particles which are darkly stained after osmium fixation with phosphate buffer. 1 . The Dark (Superj5cial) Cell
In the human eccrine gland, the dark cell is shaped like an inverted triangle with the apex pointing toward the base of the epithelium. Thus the luminal surface is rather wide and on the contrary the basal slender process attaches either myoepithelial cells or the basement membrane. The apical cytoplasm contains numerous secretory granules about 300-500 nm in diameter, some of which are round but most of which are slightly deformed and irregularly shaped. As already mentioned, these granules are very dense after double fixation of glutaraldehyde and osmium (Fig. 17). If the specimen is fixed with simple osmium tetroxide solution, these secretory granules change to clear vacuoles. Sometimes a mixture
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FIG. 17. Luminal parts of dark cells of the human eccrine sweat gland, containing many dense granules, particularly in the apical cytoplasm. Some clear vacuoles (arrows) occur in the Golgi area ( G ) .Lipid droplets (LD) and lysosornes (LY) are observed. A small part of a clear cell cytoplasm (C) is seen at the bottom center. (From Kurosumi er a / . . 1982.)
of glutaraldehyde and osmium may be used, revealing secretory granules of medium density (Fig. 18). A triple fixation with osmium-glutaraldehyde-osmium also resulted in the vacuolization of secretory granules. The eccrine sweat glands of aged individuals often contain very few or no granules, being suggestive of a lower secretory activity in old persons (Kurosumi et al., 1960). The secretory granules of dark cells were thought to contain mucopolysaccharide, and hence Munger (1961) called cells of this type “mucoid cells” and Ellis (1968) narned them “mucous cells.” We observed that the
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FIG. 18. A part of the secretory coil of the human eccrine sweat gland fixed with a mixture of glutaraldehyde and osmium. Secretory granules of dark cells (D) are seen as vacuoles of medium density. Clear cells (C) contain many mitochondria but no secretory granules. Arrow indicates the continuity of the main lumen to the intercellular canaliculus between clear cells.
secretory granules may be intensely stained with methenamine silver, strongly suggesting that the secretory substance is mucopolysaccharide (Fig. 19) (Kurosumi et al., 1982). These granules are probably produced in the Golgi apparatus. The innermost cisterna (trans side) of the Golgi lamellae contains a dark substance, which is also stained with methenamine silver, implying the accumulation of a secretory mucoid substance in the trans cisterna of the Golgi stack corresponding to the socalled GERL of Novikoff (1964). From the results of electron microscopic cytochemistry, we considered that GERL is nothing but a specialized part of the Golgi apparatus, because secretory granules are produced in both the Golgi stack and GERL simultaneously (Inoue and Kurosumi, 1977; Kurosumi and Inoue, 1983). It may be concluded from this that the secretory granules of dark cells of the human eccrine sweat gland may be formed in the innermost cisterna of the Golgi stack which is also called GERL (Fig. 20a). Ellis (1967, 1968) described the presence of large vacuoles at the center of the Golgi area; the largest vacuoles contained flocculent material which was presumed to be a substance newly synthesized or recently conjugated within the Golgi apparatus. As the vacuoles matured, they were reduced in size and their contents increased in electron density. We also observed many vacuoles with variable contents either empty or
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Fic. 19. Luminal part of the dark cell of the human eccrine sweat gland. Methenamine-silver intensely stains the secretory granules and the content of Golgi cisternae and GERL (arrows). (From Kurosumi er al., 1982.)
FIG. 20. Golgi apparatus (G) and their neighborhood of the dark cells of the human eccrine sweat gland. The innermost sac of the Golgi stack (probably corresponding to GERL) contains a dark substance (arrow in a). Some immature granules (condensing vacuoles) with less dense content (arrows in b) occur in the Golgi area. (From Kurosumi ef al., 1982.)
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medium dense in the cytoplasm surrounded by the Golgi stack (Figs. 17 and 20b) (Kurosumi el al., 1982). They are probably immature secretory granules, but the relationship between the accumulation of dark substance in the innermost sac of the Golgi stack and formation of such large round vacuoles containing less dense substance is not clear. The progressive accumulation of material in the Golgi sac (or GERL) may result in the swelling and ballooning of the sac, but the concentration of secretory material may be lowered temporally as suggested by the low electron density of the material. The intensity of staining with methenamine silver is also slightly lower in such immature granules. This fact also suggests a low concentration of the polysaccharide-bound protein (glycoprotein) in these immature granules. But the matured granules are very dense in both ordinary sections and those stained with methenamine silver, so that the concentration of secretory substance proceeds as the granules are transported from the Golgi area to the cell surface. The secretory granules may come into contact with the inner aspect of the cell surface plasma membrane, the granule membrane may fuse to the plasma membrane, and a pore may be formed penetrating the fused membrane, through which the interior content of the granule may flow out (Fig. 21a and b) (Ellis, 1968; Kurosumi ef af., 1982). This mechanism is called “exocytosis” and was
FIG..21. Apical surface of the dark cell of the human eccrine sweat gland. Secretory granules are open tothe lumen (arrow in a) and release the content by the mechanism of exocytosis. The pit (arrow in b) on the surfqce may be the remnant of a secretory granule emptied of its content. (From Kurosumi et al., 1982.)
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classified as Type IV in the mechanism of secretion discharge (Kurosumi, 1961). It was also called “reverse pinocytosis,” but the use of the term, “merocrine secretion” for this release mechanism is erroneous, because merocrine secretion includes both the apocrine and eccrine secretion in the classification by Schiefferdecker (1917, 1922). An old term in light microscopy, eccrine secretion, may include various mechanisms invisible under the light microscope, that were classified by electron microscopy into Types 111, IV, and V (Kurosumi, 1961), later named microapocrine, eruptocrine (exocytosis), and diacrine secretion (Kurosumi, 1965). Ito and Shibasaki (1966a) proposed two different mechanisms for the release of the secretory substance from the dark (superficial) cell: exocytosis and apocrine secretion. They demonstrated a tonguelike projection extended into the gland lumen, whose base was transversed by a newly formed demarcation membrane. The projection may be separated from the main cell body at the demarcation membrane. This is the typical apocrine secretion repeatedly reported in the apocrine sweat glands, but it was also often observed in the eccrine sweat gland (Minamitani, 1941a; Ito and Iwashige, 1951). Furthermore, Ito and Shibasaki (1966a) observed bulbous swelling of the tips of microvilli, which may be pinched off as small droplets into the lumen. This mechanism is the microapocrine secretion (Type 111) of Kurosumi (1961). As Kurosumi et al. (1968) observed the concomitant occurrence of exocytosis and apocrine secretion in the rat lactating mammary gland, similar dual mechanisms of secretion release may occur in the eccrine and apocrine sweat glands as well. Ito and Shibasaki (1966a) reported that the membrane of secretory granules (vacuoles) might be ruptured and through such discontinuities the internal substance of granules might be diffused in the apical cytoplasm, and the accumulation of secretory substance might swell the apical part of the cell to form the apocrine projection. The rupture of the granule membrane, however, might occur as a result of inappropriate fixation with osmium tetroxide alone, for it is not observed after double fixation with glutaraldehyde and osmium. The rough endoplasmic reticulum is moderately developed, and the constituent cisternae are scattered through the cytoplasm. They are either irregularly dilated sacs or flattened sacs which are seen as double membranes. The roughsurfaced cisternae are often situated near the mitochondria. Free ribosomes are more numerous in the cytoplasm of the dark cells than in the clear cells. On the contrary, the smooth endoplasmic reticulum is scarcely observed in the dark cells, though it is markedly well developed in the clear cells. The development of tonofilaments is also one of the characteristics of the dark cells. Hibbs (1958) and Kurosumi er al. (1960) described the occurrence of tonofilaments in eccrine sweat gland cells. Ito and Shibasaki (1966a) demonstrated a tremendous amount of tonofilaments in superficial cells. They are distributed in the whole cytoplasmic area forming prominent bundles. In the
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apical cytoplasm, the filaments are oriented approximately parallel to the luminal surface and often attached to the desmosomes which are frequently found at the lateral cell boundaries. Glycogen particles are almost absent in the dark cells of the human gland, though in the dog eccrine gland glycogen is contained in the dark cells. Mitochondria of the dark cell are either round or elongate. The hypertrophy of mitochondria caused by the accumulation of some material in the mitochondria1 matrix seen in the apocrine gland cells does not occur in either dark or clear cells of the eccrine gland. Lysosomes and multivesicular bodies are often observed, especially in the Golgi area. Lipid droplets are frequently observed near the Golgi apparatus. There is a tendency for the lipid droplets to increase in size and number with aging (Kurosumi et al., 1982). As lysosomes are often attached to lipid droplets, the formation of lipid droplets may be related to the lysosomes. Sometimes small lipid droplets are contained in large lysosomes. Lipid droplets containing many vesicles are called either multivesicular or multilocular lipid droplets, while the simple droplets are monovesicular or monolocular lipid droplets (Ito and Shibasaki, 1966a). Iijima (1959) and Kurosumi et al. (1960) called these multilocular lipid droplets lipochondria, and thought that they might originate from the Golgi vesicles. The physiological significance of lipid droplets is not clear, but they seem to be pathological rather than physiological in nature. It is likely that the lipid droplets may arise as a result of aging (Kurosumi et al., 1960). The structure of lipochondria is like that of lipofuscin reported in other tissues and is also similar to the Type B of large dense granules found in the human apocrine gland. Ito and Shibasaki (1966a) demonstrated centrioles in the apical cytoplasm, which might be the basal body of a cilium, but they could not show the ciliary shaft in this gland. Microvilli are projected from the apical free surface into the gland lumen, but those of dark cells are relatively short and sparse, and sometimes replaced by apocrine secretory projections. The junctional complex which consists of the zonula occludens (tight junction), zonula adherens (intermediary junction), and macula adherens (desmosome) is situated at the apical end of the lateral boundaries. The basal half or two-thirds of the lateral cell boundary is provided with well-developed interdigitation. At the basal end of the basal processes either abutting on the basement membrane or lying upon the myoepithelial cells, infoldings of the plasma membrane are very poorly developed (Ito and Shibasaki, 1966a). 2. The Clear (Basal) Cell Because the second type of secretory cells of the eccrine sweat gland contains no stainable secretory granules, they are seen as clear both in light and electron microscopic images and are called clear cells (Figs. 2a and 22) (Montagna et al., 1953; Munger, 1961; Ellis, 1962). In the human eccrine gland, the clear cells lie
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FIG. 22. Clear cells of the human eccrine sweat gland. The intercellular boundary is strongly undulated due to interdigitation, but the cell boundaries near the main lumen and intercellular canaliculus (CA) are straight and provided with well-developed junctional complexes and desmosomes. Many large lipid droplets (L,D) and scattered glycogen particles are contained in the cytoplasm. Golgi apparatus ( G ) and smooth ER (SR)are seen near the cell surface. (From Kurosumi er al., 1982.)
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on the basement membrane or the myoepithelial cells, and are covered by the cell bodies of the dark (superficial) cells, so that they usually do not directly face the main gland lumen. Based on their localization, clear cells were named basal cells (Ito, 1943). Narrow intercellular canaliculi formed at the junction of two or three clear cells lead the secretory product of the clear cells to the main lumen of the gland. The cross-cut profile of the canaliculus is roughly round (Fig. 23a). Many microvilli extend into the canalicular lumen, and sometimes the lumen is almost obstructed by closely packed microvilli. They pour the secretion of clear cells into the cleft between the dark cells which are recesses of the main lumen. Rarely clear cells come into the superficial row and directly face the main gland lumen. In such a case, intercellular canaliculi open to the gland lumen without passing through the recess surrounded by dark cells. Microvilli of the clear cells are usually longer than dark cells, not only at the surface facing the canaliculi but also toward the main lumen (Fig. 18). Between the adjacent clear cells the junctional complexes are always observed around the cross-cut profile of the intercellular canaliculus. If the canaliculus were intracellular, no intercellular boundaries with junctional complexes would be associated with the canaliculus, but we have had no experience in finding such canaliculi. It may be concluded that no intracellular canaliculi exist in the human eccrine sweat glands. Likewise at the luminal corners of the lateral cell boundaries, well-developed junctional complexes are observed, as well as several desrnosomes arranged along the straight part of the cell boundaries. There are no desmosomes along the strongly folded interdigitating boundary (Fig. 22). Development of the rough endoplasmic reticulum is very poor in the clear cells, but the smooth endoplasmic reticulum is relatively well developed (Fig. 23a). Such a conspicuous development of smooth ER was first observed by Ito and Shibasaki (1966a), and Ellis (1968) also observed and ascribed it to the high degree of glycogen stores. The smooth ER is distributed uniformly throughout the entire cytoplasm except for the very superficial part of the cytoplasm subjacent to the free surface facing the intercellular canaliculi or the main lumen. The smooth ER consists of tubules which often branch and anastomose to form an irregular network. Vesicles are also intermingled with the tubular network. Most of vesicles may be the cross-cut profiles of the tubules but some are probably real vesicles. The Golgi apparatus of the clear cell is a dispersed type (diffuse type of Hirschler, 1927), and hence small complexes consisting of a few lamellae and vesicles are scattered at several places in the cytoplasm, particularly near the canaliculi. Sometimes it cannot be determined whether vesicles and tubules in the subapical zone around the canaliculus belong to the smooth ER or are derived from the Golgi apparatus. Ito and Shibasaki (1966a) suggested that the vesicles separated from the smooth ER might come into contact with the plasma membrane lining the intercellular canaliculi, and empty the internal substance into the canalicular lumen. Ellis (1967, 1968) described small clear vesicles present in
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FIG.23. Parts of the clear cells of the human eccrine sweat gland. (a) Intercellular canaliculus (CA) and the clear cell cytoplasm surrounding it, which contains Golgi apparatus (G) and smooth ER (SR).(b) Ample glycogen particles (GL) in the cytoplasm of a clear cell. (From Kurosumi et al., 1982.)
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the cytoplasm between the Golgi apparatus and the intercellular canaliculus, suggesting the transfer of secretory products from the Golgi zone to the canaliculus. These vesicles reported by Ito and Shibasaki (1966a) and Ellis (1967, 1968), however, are not secretory vesicles, but are undoubtedly a part of the smooth ER, especially well developed in the area between the Golgi apparatus and the intercellular canaliculus. Middle-sized vesicles or vacuoles might be formed by the dilatation of cisternae of the smooth ER due probably to an artifact during specimen preparation. In the human eccrine sweat glands, glycogen is accumulated exclusively in the clear (basal) cells (Ito and Shibasaki, 1966a,b; Kurosumi et af., 1982). The content of glycogen is variable from cell to cell. In most clear cells glycogen particles are scattered among the elements of smooth ER. The very surface of the cell is devoid of glycogen particles and other organelles, therefore, the peripheral zone of the cell is clear and amorphous (Fig. 22). Some clear cells contain a great amount of glycogen. Particles are gathered to form clusters and fill the cytoplasmic matrix among mitochondria and some other organelles (Fig. 23b). Tonofilaments are also seen in the clear cell cytoplasm, but they are less marked than in the dark cells. The clear cells also contain lipid droplets, lysosomes, and multivesicular bodies, the last of which is more conspicuous in the clear cells than in the dark cells. The basal plasma membrane infolds into the basal part of the cytoplasm and forms complicated foldings. In the specimens fixed with a simple osmium solution, rows of vesicles are associated with infoldings of the basal plasma membrane. Some authors conjectured that small parts of the infolded membrane are pinched-off and form free vesicles which might move from the basal cytoplasm toward the apical cytoplasm, and finally open to the free luminal surface conveying water and soluble substances across the gland cells (Iijima, 1959; Ito and Shibasaki, 1966a). After double fixation with glutaraldehyde and osmium, the above mentioned vesiculation of infolded membrane did not occur, so that such a phenomenon might be an artifact, and the transport of water and electrolites may be camed out by the diffusion of liquid, without morphological changes. Sometimes, there is the disappearance of microvilli and formation of blebs on the wall of the canaliculi. Ito and Shibasaki (1966a) thought that this feature was the result of the swelling and pinching-off of tips of microvilli, termed microapocrine secretion (Kurosumi, 1965). Ito and Shibasaki (1966a) also found basal bodies of cilia near the surface of the clear cell, facing the intercellular canaliculi, which may suggest the presence of a cilium protruding into the canalicular lumen. Briggman et al. (198 1) studied electron microscopic images of freeze fracture replicas. The tight junctions of the intercellular canaliculus and the main lumen in the secretory portion of the human eccrine sweat glands consist of approximately nine and six, closely spaced, parallel or anastomosing strands,
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respectively. These structures of the tight junctions are too complicated to suppose the paracellular route for transport of ions and molecules in secretion. Therefore, the hypertonic or isotonic precursor fluid of sweat may undoubtedly be produced in the secretory cells, especially clear cells, and transported through these cells toward the lumen of the intercellular canaliculi and main gland lumen.
3 . The Myoepithelial Cell There is no difference in the ultrastructure of the myoepithelial cells in the eccrine and apocrine sweat glands. Myoepithelial cells of the human eccrine sweat glands were studied with electron microscopes by Hibbs (1958), Iijima (1958), Kurosumi et al. (1960), Kaname (1960), Charles (1960), Ellis (1962, 1965), Ito and Shibasaki (1966a), and Kurosumi et al. (1982). Among these Ellis (1965) published the most detailed and comprehensive report on this cell type. Ellis (1965), however, indicated that the myoepithelial cells are restricted to the secretory segment and are not observed in the sweat duct, because he was unaware of the unique portion of the human eccrine sweat gland, the transitional portion, which is provided with myoepithelial cells (Kurosumi er a l . , 1982). According to Ellis (1963, the myoepithelial cells are usually elongate and sometimes branched, being alligned parallel to the long axis of the tubule. Binucleate myoepithelial cells are occasionally observed. Myofilaments fill the cytoplasm of the basal half of the cell, and the cell organelles are restricted to the narrow cytoplasm around the nucleus. Thin cores or columns of unmodified cytoplasm penetrate the filamentous mass. These cores of cytoplasm contain smooth endoplasmic reticulum, glycogen particles, and dense filamentous mitochondria. They are arranged parallel to the myofilaments, which are also parallel to the long axis of the cell. The base of each myoepithelial cell is irregularly serrated, and dense thickenings along the plasma membrane alternate with zones with pinocytotic pits and vesicles. These dense zones may serve as an attachment site for myofilaments. Pits or caveolae are not observed on the surface of the clear or dark secretory cells of the eccrine sweat gland, so that this characterizes the myoepithelial cells. Desmosomes are sparse but occasionally observed joining the myoepithelium to the secretory cells. Ellis (1965) observed only one kind of myofilament 50 8, in diameter, but they were intermingled with microtubules which were arranged almost parallel to the myofilaments. Not only the myofilaments but also the microtubules were thought to be contractile elements of this cell. Only Hibbs (1958) suggested the transition between the myoepithelial cells and the secretory cells of this gland, but other authors studying the human eccrine glands did not agree with this idea, because the myoepithelial cells seem to be quite different in ultrastructure from either clear cells or dark cells. Concerning the possible function of the myoepithelial cells, Ellis (1965) pre-
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sented a unique “valve” theory of the myoepithelial cells of the eccrine sweat gland. The contraction of the myoepithelial cells may open wide gaps in the myoepithelial pavement, which are filled with extentions of clear cells bearing highly folded plasma membrane. The myoepithelial cells may act as valves indirectly controlling the flow of sweat by regulating the surface area of the secretory cell that is exposed to the extracellular fluid. In a study of the myoepithelium of the human apocrine sweat glands distributed on the external auditory meatus (ceruminous glands), Kawabata and Kurosumi (1976) opposed Ellis’ valve theory and indicated that the contraction of myoepithelial cells might not bring about the decrease in thickness of this cell, from which he assumed the occurrence of widening of myoepithelial gaps where the basal foldings of secretory cells are present. The human ceruminous gland is provided with strikingly well-developed myoepithelium, though this gland bears almost no plasma membrane foldings at the base of secretory cells, unlikely in the case of axillary apocrine sweat glands and eccrine sweat glands distributed through the general body surfaces. In the latter glands, a transitional portion was observed (Kurosumi et al., 1982). The transitional portion is also provided with well-developed myoepithelial cells but the epithelial cells of this portion lack the foldings of the basal plasma membrane. As observed in various cases described above, the development of myoepithelial cells and that of basal infoldings is not parallel, and therefore the myoepithelium may not be correlated functionally with the foldings of the basal plasma membrane which are thought to be involved in water transport. It seems rather likely that the myoepithelium may press the gland tubule, and increase the internal pressure of the tubular lumen to transport the secretory product in the lumen toward the skin surface. C. EXCRETORY DUCTSOF
THE
HUMANSWEATGLANDS
Electron microscopists paid less attention to the excretory duct system of the sweat glands, as compared with the secretory portions. From a physiological point of view, however, ducts of the human sweat glands are important in elaboration of sweat, because the initial sweat produced in the secretory coil is not matured but modified during the passage through the duct. The duct of the eccrine sweat gland is a simple tubule, the longest part of which is straight and extends perpendicularly from the skin surface down to the subcutaneous tissue, where the duct twists and turns to form a glomerulum. The glomerulum of the human eccrine sweat gland consists of two major parts, the secretory coil and coiled duct, with a short minor part called the transitional portion intervening between the secretory coil and coiled duct (Ito and Enjo, 1949). In the apocrine gland, the duct is not usually involved in the formation of the glomerulum, and the transitional portion is also inconspicuous.
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The terminal segment of the sweat duct penetrates the epidermis with a remarkable spiral course like a cork screw. Thus the whole duct system of the human eccrine sweat gland may be classified into four parts: transitional portion, coiled duct, straight duct, and epidermal duct. The coiled duct and straight duct are collectively called the dermal duct. The apocrine glands usually open to the hair follicle, and therefore no epidermal duct is found in the ordinary apocrine sweat glands. As the ceruminous glands often open to the skin surface after passing through the epidermis, the epidermal duct exists in the ceruminous apocrine gland. 1. The Transitional Portion This special part of the human eccrine sweat ducts interposed between the secretory portion and the coiled duct was first found by Ito and Enjo (1949) with the light microscope (Fig. 2b). Hibbs (1958), in one of the earliest studies of human eccrine sweat glands with the electron microscope, observed every part of the eccrine gland tubules including both the secretory portion and excretory ducts. He found the transitional region by electron microscopy independently of the Japanese workers, because the paper of Ito and Enjo (1949) was unfortunately written in Japanese. Hibbs (1958) indicated that this portion consisted of simple columnar epithelial cells extending all the way from the basement membrane to the lumen. He also found the myoepithelial cells in the transitional portion, but these cells are absent in the duct proper. Shibasaki and Ito (1967) published the result of a systematic study on the duct of the human eccrine sweat gland, especially of the transitional portion. They found that the transitional portion consisted of the simple columnar or cuboidal epithelium associated with flattened basal cells, among which myoepithelial cells could be identified with the light microscope. By electron microscopy, however, they did not show the myoepithelial cells in the transitional portion, though they demonstrated a small part of cytoplasm of the “basal cell.” Kurosumi et al. ( 1982) later demonstrated well-developed myoepithelial cells in the transitional portion of the human eccrine sweat gland. The proximal segment of the transitional portion near the secretory coil has a narrow lumen and well-developed myoepithelial cells (Fig. 24). The narrowness of the lumen may be due to the contraction of the myoepithelial cells. On the other hand, the distal part of the transitional portion near the coiled duct has a very wide lumen and weakly developed myoepithelium (Fig. 25). Between the neighboring myoepithelial cells in the distal portion, relatively wide gaps are found, where the epithelial cells directly abut on the basement membrane. The epithelial cells of the transitional portion are either columnar or cuboidal, and arranged in a single layer. This mode of arrangement of epithelial cells
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FIG. 24. Cross section of a transitional portion of the human eccrine sweat gland. The lumen (arrow) is very narrow. Cuboidal epithelial cells are arranged in a single layer, and myoepithelial cells (ME) surround the duct almost completely. (From Kurosumi et al.. 1982.)
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FIG. 25. A distal part of the transitional portion of the human eccrine sweat gland. The myoepithelial cells (ME) are sporadically arranged. A winglike accumulation of filamentous substance is attached to the intermediary junction (arrows). Tubular smooth ER (SR) are seen in the apical cytoplasm. (From Kurosumi et al., 198;!.)
characterizes this portion of sweat glands, because the duct proper has twolayered epithelium and the secretory portion is provided with the pseudostratified epithelium consisting of two cell types. The epithelial cells are triangular or rectangular shaped, and the intercellular boundaries are almost straight, because infoldings and interdigitations are very poorly developed or sometimes absent. The lack of basal infoldings is the most prominent difference from the basal secretory (clear) cells of the secretory coil of
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this gland. Nuclei of the epithelial cells are characterized by their irregular contour, often possessing deep invaginations (Shibasaki and Ito, 1967; Kurosumi et al., 1982). The luminal surface of the epithelial cells is provided with poorly developed microvilli. Some cells at the distal segment showed no microvilli. The lateral intercellular boundaries are tortuous and folded, but the development of such lateral foldings or intercellular interdigitations is minimal in this portion as opposed to the secretory coil and dermal ducts. The basal infolding, which is one of the salient features of the secretory coil, cannot be found in the epithelial cells of this portion, and this characteristic is the useful criteria to differentiate the transitional portion from the secretory coil, though both possess myoepithelial cells. The general features of the epithelial cells of the transitional portion resemble the clear cells of the secretory portion. The epithelial cells of this portion also contain smooth endoplasmic reticulum at the apical region, and its constituent tubules are sometimes continuous to the lumen. The development of the smooth ER is much weaker in the cells of the transitional portion than in the clear cells of the secretory portion. The Golgi apparatus is usually situated just above the nucleus, which is slightly below the subapical smooth ER. Glycogen particles are very few or completely absent in the cytoplasm of the epithelial cells of this portion, and this characteristic differs very prominently from the large amount of glycogen in the clear cells of the secretory portion. Lipid droplets are also very few and are often lacking in the transitional portion. Mitochondria are scattered around the nucleus. Most of them are round or oval in shape, but a few are elongated. Kurosumi et al. (1982) found another characteristic in the epithelial cells of this portion, an accumulation of fine filaments attached to the intermediary junction (zonula adherens) (Fig. 25). The heaps of filaments look like wings of a dragonfly extending from both sides of the zonula adherens into the cytoplasm of the adjacent epithelial cells, but they are not continuous with another set of the same structure extended from the zonula on the opposite side of each cell. This accumulation of filamentous substance at the subapical portion of the epithelium is less developed in the proximal part, but it becomes more conspicuous as the transitional portion approaches the coiled duct, where the filament accumulation increases extraordinarily and forms a thick layer of filamentous feltwork at the apical zone of the luminal cells, designated as the terminal web or periluminal filamentous zone. The occurrence of the above mentioned accumulation of filaments attached to the zonula adherens may be one of the most convincing structures indicating that this portion is the “transitional” part between the secretory coil and the coiled duct. Shibasaki and Ito (1967) found conspicuous rodlike or spindlelike dense bodies in the epithelial cells of the transitional portion, but Kurosumi et al.
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(1982) did not find such elongated dense bodies, though spherical or oval dense bodies probably corresponding to lysosomes could be found in these cells. The ultrastructures of myoepithelial cells in the transitional portion are the same as those in the myoepithelial cells at the secretory portion of the same gland. The prominent development of myoepithelial cells entirely surrounding the initial part of the transitional portion may act as a sphincter at the neck of the duct system of this gland. The constriction and dilatation of this portion caused by the contraction and relaxation of the myoepithelium innervated by the autonomic nervous system may serve as a valve regulating the flow of sweat in each gland (Kurosumi et a l . , 1982). 2. The Dermal Ducts The proximal half of the dermal duct of the eccrine sweat gland is involved in the glomerular portion of the gland. This part is tortuous in course and therefore is called the coiled duct. Another half is rather straight ascending toward the epidermis, and therefore is called the straight duct. The fundamental structures of the coiled duct and straight duct are similar to each other. The wall of the dermal duct lacks the myoepithelium and is made up of two layers of the epithelial cells. They are cuboidal or somewhat flattened. The inner set of epithelial cells is called either the luminal cell (Zelickson, 1961; Hashimoto et a l . , 1966d; Kurosumi, 1977; Kurosumi et a l . , 1982), surface cell (Munger, 1961; Shibasaki and Ito, 1967), superficial cell (Hibbs, 1958; Ellis, 1967, 1968), or inner cell (Hashimoto et a l . , 1966a,b), while the outer set of the epithelial cells is called either the peripheral cell (Zelickson, 1961; Ellis, 1967; Kurosumi, 1977; Kurosumi et a l . , 1982), basal cell (Hibbs, 1958; Munger, 1961; Ellis, 1968; Shibasaki anid Ito, 1967), or outer cell (Hashimoto et a l . , 1966a,b). In this article, the terms “luminal and peripheral cells” are used, because the terms superficial and basal cells are the same as those designated by Ito (1943) for the dark and light cells of the secretory portion of the human eccrine sweat gland, and therefore can be confusing. Though the dermal ducts do not keratinize, the fundamental structures of the duct epithelium look like those of the epidermis. The cells of the dermal duct are also connected with one another by the desmosomes and these cells contain keratin-like cytoplasmic filaments (Kurosumi et a l . , 1982). The lumen of the dermal duct is usually wide. The luminal side of the duct epithelium consists of the “luminal cells” which do not attach to the basement membrane, on the other hand, the “peripehral cells” do not face the duct lumen. This arrangement of the epithelial cells is stratified and differs from the pseudo-stratified arrangement of secretory cells at the secretory portion of the human eccrine sweat glands. Such a two-layered stratified epithelium is observed in the dermal ducts of both the eccrine and .apocrine sweat glands (Kurosumi, 1977).
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In the dermal duct of the eccrine sweat gland, the luminal celIs are provided with well-developed microvilli, which are mostly short and thick. Sometimes the microvilli become irregular projections. As the microvilli of the secretory portion are usually long and slender, many more blunt microvilli of the duct cells are conspicuous (Shibasaki and Ito, 1967; Kurosumi, 1977; Kurosumi et al., 1982). Underneath the most apical part of the luminal cell, there is a specific layer of the cytoplasm filled with a tremendous amount of filaments (Fig. 26), which were called the cuticular border (Hibbs, 1958; Munger, 1961; Ellis and Montagna, 1961; Ellis, 1967; Shibasaki and Ito, 1967), periluminal filamentous zone (Hashimoto et al., 1966a,b), or terminal web (Kurosumi et al., 1982). Filaments in this zone are complicatedly interwoven and form a thick feltwork. Such a rigid ring of the filaments surrounding the lumen may prevent the collapse and the occlusion of the lumen (Ellis and Montagna, 1961; Hashimoto et al., 1966b; Kurosumi et al., 1982). Within the terminal web of the human eccrine sweat gland vesicles and granules were observed (Kurosumi, 1977; Kurosumi et al., 1982). Vesicles are 50-100 nm in diameter and are either empty or contain a dense core. Mediumsized granules are 150-300 nm in diameter, most of which are opaque, although some contain clear spots. Large vacuoles 400-800 nm in diameter are occasionally contained in this zone. These vacuoles often contain small vesicles (multivesicular bodies) or many dense granules, membranes, and some other substances. These vacuoles are presumably phagosomes and contain phagocytosed material taken up from the duct lumen, where cell debris probably derived from apocrine secretion are frequently seen (Kurosumi, 1977). Small vesicles seen in the subapical zone (terminal web) may be related to the pinocytotic absorption of sweat, and the medium-sized granules may be lysosomes. The cytoplasm encircling the nucleus appears clear because this part of the cytoplasm contains less tonofilaments, but a great number of mitochondria. The basal part of the luminal cell abutting the peripheral cell also contains many mitochondria (Fig. 27). The accumulation of mitochondria is not restricted to the luminal cells but in the peripheral cells there are relatively large numbers of mitochondria (Kurosumi, 1977; Kurosumi et al., 1982). Hibbs (1958) first observed such an accumulation of a large number of mitochondria more in the basal cells than in the luminal cells. The findings by Munger (1961) supported the findings of Hibbs (1958). Ellis (1967, 1968) described piles of mitochondria found only in the peripheral (basal) cells. However, Shibasaki and Ito (1967) observed that both cell types contain approximately equally numerous mitochondria. Kurosumi (1977) and Kurosumi et al. (1982) also found a gathering of mitochondria in both luminal and peripheral cells. Human eccrine sweat is known to be hypotonic and this characteristic may be brought about by the active absorption of sodium and chloride from the precursor sweat by the duct cells (Schwartz and Thaysen, 1956). The strong accumulation
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FIG 26 The dermal duct of the human eccnne sweat gland The duct wall consists of twolayered epithelial cells. The apical surface of the luminal cell is provided with blunt microvilli and the apical cytoplasm contains a filament-nch terminal web (TW) The intercellular boundaries of luminal cells are Very tortuous and are associated with many desmosomes <
of mitochondria in the epithelial cells is observed only in the human eccrine sweat glands, not in human apocrine glands (Hashimoto et al., 1966a; Kurosumi, 1977) and animal sweat glands (Munger and Brusilow, 1961). The hypotonicity is the characteristic of (he human eccrine sweat. Therefore, the mitochondrial spck may be concerned with the absorption and transport of ions, resulting in the hypotonic sweat. ,
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FIG.27. The basal half of the luminal cell cytoplasm containing a number of mitochondria (M). The coiled duct of the human eccrine sweat gland. (From Kurosumi, 1977.)
Ellis (1967, 1968) reported that the peripheral (basal) cells contain more mitochondria near the secretory coil, but toward the straight duct they contain fewer mitochondria, this reduction of mitochondrial number continuing along the course of the straight duct. He suggested that the basal cells are most active in sodium reabsorption near the secretory coil and that their capacity for reabsorption diminishes as the duct approaches the epidermis. Among the mitochondrial heaps in both luminal and peripheral cells, small vesicular or tubular elements of the endoplasmic reticulum partly rough and partly smooth are scattered. The Golgi apparatus consisting of a few lamellae and vesicles is observed in the perinuclear cytoplasm. A large number of desmosomes are developed along very tortuous cell boundaries between the adjacent luminal cells. Between the luminal cells and peripheral cells, desmosomes are relatively few in number. The cell boundaries between the adjacent peripheral cells are also tortuous, but almost no desmosomes are observed (Kurosumi et al., 1982). Hashimoto et al. (1965, 1966d) studied the development of eccrine sweat glands in the human embryos and found the difference in ontogeny between the epidermal and dermal parts of the eccrine sweat ducts. The epidermal duct lumen is formed by the coalescence and burst of intracellular cavities formed under the activity of pericanalicular lysosomes, while the lumen of the dermal duct is formed completely extracellularly by the dissolution of desmosomal attachment
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plaques between the cells composing the inner core of the duct germ in 15-weekold embryos. The secretory portion of the eccrine gland develops much later but its lumen is also formed, similarly to the dermal duct, by separation of desmosomal attachment plaques between two apposing luminal cells. Myoepithelial cells differentiate from the basal cells in 22-week-old embryos (Hashimoto et a l . , 1966d). Only a few papers on the fine structures of the human apocrine sweat ducts have been published. Hashimoto et al. (1966a) studied the axillary apocrine duct, and Kurosumi (1977) reported on the duct of the ceruminous gland (Fig. 28). Hashimoto et al. (1966a) indicated that the epithelial cells of the dermal apocrine duct consist of two or three layers, the inner, middle, and basal cells. The middle cells are sometimes absent. The number of microvilli increased in the direction of the pilosebaceous apparatus. Pinching-off of microvilli (microapocrine secretion) occurs more frequently in the upper portion than in the lower portion of the apocrine duct. Hashimoto et al. (1966a) argued that the apical plasma membrane on the cytoplasmic protrusion may break and a small amount of cellular contents may leak out into the lumen. Microapocrine secretion was also observed by Kurosumi (1977), but he found no disruption of the luminal plasma membrane. Kurosumi (1977) reported that the dermal duct of the ceruminous gland consists of two layers like those of the eccrine sweat glands, and called them luminal and peripheral cells. Adluminal condensation of tonofilaments corresponding to the terminal web of the eccrine duct was observed, but it was relatively inconspicuous in the apocrine duct as compared with the eccrine duct. No accumulation of mitochondria was observed in the apocrine duct (Hashimoto et a l . , 1966a; Kurosumi, 1977). The cell boundaries between the adjacent luminal cells and those between the adjacent peripheral cells are markedly tortuous, but only the former is associated with numerous desmosomes. The boundary between the luminal layer and the peripheral layer is almost straight. Accumulation of glycogen particles in the peripheral cells was reported by Hashimoto et al. (1966a). Peripheral cells in a part of the apocrine duct near the orifice to the hair follicle contain a large number of lipid droplets, becoming like sebaceous gland cells (Kurosumi, 1977). As both the apocrine sweat gland and sebaceous gland empty their secretions into the hair follicles, these two glands may be related to each other in an embryological way, and the accumulation of a large amount of lipid droplets in the apocrine duct may be regarded as a kind of metaplasia approaching the sebaceous gland. 3. The Epidermal Ducts Charles (1960) first performed electron microscopy of the epidermal ducts. He showed that the lumen of the eccrine sweat duct changes from the wide lumen of
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FIG.28. The dermal duct of the human ceruminous gland. The duct wall consists of two-layered epithelial cells. Small bulging of microvilli suggesting the microapocrine secretion is observed (arrow). (From Kurosumi, 1977.)
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the dermal duct to the almost occluded lumen of the epidermal duct. In contrast to the findings of Charles (1960), Zelickson (1961) demonstrated that the epidermal duct often remained patent throughout its course through the epidermis. The cells lining the duct lumen, called luminal cells, are particularly differentiated and surrounded by peripheral ductal cells which are slightly different from the luminal cell and from cells comprising the epidermis. The filamentous terminal web (cuticular border) is inconspicuous, but as the luminal cells keratinize at the more superficial position, filaments nearest the surfaces of the cell become more dense and form a ringlike structure (Zelickson, 1961). The development of the epidermal duct was studied by Hashimoto et al. (1965) in early human embryos. Anlagen of eccrine sweat glands is first seen in embryo 12-13 weeks old. Cells destined to line the lumen of the epidermal duct (inner cells) contain multivesicular dense bodies (pericanalicular lysosomes), which consist of an aggregation of small vesicles and a dense substance. These bodies may grow into intracytoplasmic cavities, which enlarge, coalesce, and break through the cell membrane; thus the extracellular lumen of the early epidermal duct is formed. The cross-cut feature of the luinen of the epidermal eccrine duct is shaped like a star due to several longitudinal folds (Fig. 29). The free surface of the luminal cells is provided with numerous microvilli, which are rather short and blunt. Some of them are club-shaped and their rounded tips are swollen and often pinched-off, and therefore the duct lumen contains a large number of vesicles and membranes, which are probably the cell debris (Kurosumi, 1977). In the apical part of the luminal cell a large number of small vesicles are found (Fig. 30a) (Kurosumi, 1977). Hashimoto et al. (1966b) suggested that these vesicles might enter the lumen as they passed through the cell surface, as a result of disruption of the cell membrane. It is hard, however, to conclude that these vesicles are secretory in nature, because the development of the rough ER and Golgi apparatus, suggestive of synthesis of secretory substance, is very poor. Therefore, we considered the possibility that these vesicles may be concerned with absorption instead of secretion (Kurosumi, 1977). The ordinary apocrine glands, such as those in the axillary skin, open to the hair follicle, and hence the epidermal duct is absent. But some apocrine glands in the external auditory meatus (ceruminous glands) sometimes open to the skin surface passing through the epidermis. Such specific apocrine glands are, thus, provided with the epidermal duct. The luminal cells of these epidermal apocrine duct have a few vesicles in the apical cytoplasmic zone (Fig. 30b). If these vesicles are absorptive in nature, it may be said that the epidermal eccrine duct reabsorbs some components of the sweat, but this function may be very weak or absent in the epidermal apocrine duct. As the ceruminous glands secrete a rather viscous or dry secretion, but not watery sweat, basal infoldings of the secretory cells are almost absent, and therefore reabsorption of ions and some other sub-
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FIG.29. The epidermal duct of the human eccrine sweat gland. The luminal cells surround a narrow stellate lumen. Both the luminal and peripheral cells contain irregularly shaped dense keratohyalin granules. Lamellar granules of Odland are contained in only peripheral cells (arrows). (From Kurosumi, 1977.)
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FIG. 30. The lumens of epidermal ducts of the human sweat glands. (a) The eccrine sweat duct, whose lumen is narrow and contains vesicular cell debris. The apical cytoplasm of the luminal cell contains a large number of small vesicles (arrows). (b) The apocrine sweat duct (ceruminous gland) whose lumen is very wide. The apical cytoplasm of the luminal cells contains only a few vesicles (arrows). (From Kurosumi, 1977.)
stances may not be performed in this gland, as opposed to ordinary eccrine sweat glands (Kurosumi, 1977). Desmosomes connect the luniinal cells with peripheral cells, and the latter with the surrounding epidermal cells, but these cells are finally separated from each other and shed into the duct lumen, as they keratinize. Both the luminal and peripheral cells contain irregularly shaped dense granules which correspond to the keratohyalin granules of the ordinary keratinocytes of the epidermis. In the epidermal ducts of the mouse plantar eccrine sweat glands, very regular spherical keratohyalin granules of various sizes were reported (Kurosumi and Kurosumi, 1970). The duct epithelial cells of the ceruminous glands occasionally contain con.;picuous lysosomes. Lamellar granules of Odland identical to Matoltsy’s membrane coating granules are observed in the peripheral cells, but are absent in the luminal cells. These granules are gathered near the cell surface toward the duct lumen. In keratinocytes of the stratum granulosum of the epidermis, Odland bodies (lamellar granules) are accumulated along the cell surface toward the skin surface. As the free surface of the duct epithelium corresponds to the body surface in the case of the epidermis, the preferential distribution of lamellar
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granules is equally toward the free surface of the epidermis as well as that of the duct epithelium. In the epidermal eccrine duct, the keratinization of the duct cells precedes that of the keratinocytes of the surrounding epidermis. On the contrary the keratinization of the epithelial cells of the epidermal duct of ceruminous gland are lagging behind the ordinary keratinocytes (Kurosumi, 1977). As the keratinization advances, the luminal cells are filled with loosely packed filaments and a few small vesicles and particles, but contain no nuclei. Keratohyalin granules are no longer observed. MicrovilIi continue to be present, though their numbers are reduced (Hashimoto er al., 1966b). Sometimes ghostlike microvilli, from which the content is lost and only the plasma membrane is left, were observed in the keratinized luminal cells of the mouse eccrine duct (Kurosumi and Kurosumi, 1970). In the middle section of the horny layer the lumind cells begin to shed into the lumen, and in the upper level the duct is lined by thin, completely keratinized peripheral cells which are also destined to be shed into the lumen (Hashimoto et al., 1966b). In the human eccrine duct (Hashimoto et al., 1966b) and in the mouse epidermal eccrine duct (Suzuki, 1973), it is known that the keratinization in the luminal cells is incomplete, and these cells shed prematurely into the lumen. The luminal cells of the mouse eccrine duct contain immature keratohyalin granules with a spherical shape and attached ribosomes; they contain no lamellar granules of Odland and the thickening of the surface plasma membranes does not occur. The keratin pattern characteristic for cornified cells of the epidermis was not observed in the lumind cells, though the peripheral cells underwent complete keratinization with a typical keratin pattern (Suzuki, 1973).
IV. Ultrastructural Cytology of the Secretory Activity in the Sweat Glands of Nonhuman Mammals Both morphological structures and biological or physiological functions of the sweat glands are remarkably different in nonhuman animals from those of the human sweat glands. Parallel with the difference in life style, both of concerning food uptake and reproduction, mammalian animals possess sweat glands with different structures and functions. Therefore, in order to understand very complicated structures of the human and animal sweat glands, comparative studies are very useful, though relatively fewer reports on the ultrastructures of sweat glands of nonhuman mammals have so far been published. A . ANIMAL APOCRINE SWEATGLANDS In the human being, most of the body surface is provided with eccrine sweat glands, and only limited parts of the skin contain apocrine glands. On the
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contrary, most nonhuman mammals have more apocrine glands than eccrine glands, because the latter is restricted to the glabrous skin on the plantar surface of feet and toes. But many mammals possess specifically differentiated skin organs consisting of well-developed apocrine glands in specialized parts of the body surface, such as the face, arms, and inguinal (perigenital) regions. These organs have been particularly well studied by morphologists as models for research concerning the mechanism of secretion. The controversy as to whether apocrine secretion, also called decapitation, is one of the physiological means of secretion release in the apocrine sweat glands or merely an artifact occurring during specimen preparation has been repeatedly discussed among cytologists studying animal and human apocrine sweat glands. Munger (1965a) studied the apocrine sweat glands of cats and monkeys and concluded that the so-called apocrine or decapitation secretion is an artifact, though he observed prominent protrusions of the cytoplasm into the gland lumen which he called apical caps. In a special apocrine gland of the rabbit which was named the “submandibular organ” (Schaffer, 1940) or “chin gland” (Mykytowycz, 1968), Kurosumi et al. (196 1) demonstrated conspicuous features of apocrine secretion, that is, the extension and constriction of the apical cytoplasm. The surface of the projection became smooth due to the disappearance of microvilli, and it contained neither granules nor organelles such as mitochondria. The smoothness of the surface of secretory projection is useful in differentiating it from the ordinary cell surface including the simple bulging of the luminal cell surface called “apical caps.” Occasionally rudiments of membrane are observed at the base of secretory projection, that is, the “demarcation zone” from which the projection will be separated off (Kurosumi et al., 1961). Such demarcation membranes were also demonstrated at a similar position, the base of the secretory processes of human apocrine (Schaumburg-Lever and Lever, 1975) and eccrine sweat glands (Ito and Shibasaki, 1966). As the apocrine secretory phenomena are rarely found in sections viewed through the transmission electron microscope as compared with observations by scanning electron microscopy (Kurosumi and Kawabata, 1976), many electron microscopists using only transmission microscopes argued that the apocrine secretion is an artifact. Heath (1974), who studied the same material as ours, the rabbit chin gland and aiso the brown inguinal gland, negated the apocrine mechanism of secretion release. However, Kneeland (1966) reported the results of electron microscopy of the specific apocrine sweat gland (antebrachial organ) of a primitive monkey, the ring-tailed lemur, and showed very beautiful evidence of apocrine secretion. In the apocrine sweat glands of other mammals such as the horse (Kurosumi et al., 1963; SBrensen and Prasad, 1973) and the bat (Sekine, 1966), secretion discharge by apocrine mechanism was reported.
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Among various nonhuman apocrine sweat glands, the dog’s apocrine sweat gland is most similar in ultrastructure to the human apocrine sweat gland (Kurosumi, unpublished observation). The apocrine gland of the dog’s dorsal skin consists of low cylindrical secretory cells and closely packed myoepithelial cells in a disposition similar to the human gland. The luminal surface of each secretory cell slightly bulges up like a dome and is provided with many short microvilli (Fig. 3 1).
FIG.31. The secretory portion of the dog’s apocrine sweat gland. The low cylindrical gland cell has a domelike apical surface provided with moderate numbers of microvilli. The basal surface of secretory epithelium is covered with myoepithelial cells (ME).
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Small vesicles and granules about 150-200 nm in diameter are accumulated in the cytoplasm beneath the surface plasma membrane (Fig. 32a). Small pits of the same size are often observed on the apical cell surface. Though pinocytosis cannot be ruled out, the pits on the surface most likely suggest the exocytotic release of the material contained in the small vesicles or granules appearing in the apical cytoplasm (Fig. 32a). These small granules or vesicles are of the same size as small secretory granules of the human apocrine sweat gland (Kurosumi and Kurosumi, 1982). Similar vesicles and granules are also found in the Golgi area of these cells of the dog’s gland. As compared with human glands, these presumably secretory granules of the dog’s gland are mostly clear vesicles, and dense granules of the same size are relatively fewer. This may be due to the difference in the preserving effects of fixation, though material from both man and dog was fixed by the same method, with glutaraldehyde and osmium. The dog’s apocrine gland cells also contain large dense granules, particularly in the supranuclear cytoplasm (Fig. 32b). They are very dense and round or slightly irregular with a corrugated contour. They are about 500-800 nm in diameter, which is far smaller than the large dense granules of the human apocrine gland. Multivesicular bodies and lysosomes with complicated contents are also observed. These secretory cells contain relatively large numbers of mitochondria, but the hypertrophy of mitochondria seen in the human gland was not observed in the dog’s gland. Cored vacuoles of the human apocrine glands were not seen in the dog’s gland, but small granule-containing cisternae of the rough ER were observed (Fig. 32c). The functional significance of these granule-containing cisternae is not clear, but this feature is very conspicuous and has not been reported in the sweat gland cells of other species. As in the human axillary apocrine gland, infoldings of basal plasma membranes are observed. Secretory cells of the monkey apocrine sweat glands contain a few relatively small secretory granules in the apical cytoplasm and in the Golgi area (Munger, 1965a). They are slightly larger than the secretory granules of the apocrine glands of man and dog, but far smaller than the secretory vacuoles, which are of enormous size, in the cat, horse, and bat apocrine glands (Munger, 1965a; Kurosumi et al., 1963; Serrensen and Prasad, 1973; Sekine, 1966). In the cat apocrine gland cells, Munger (1965a) found two types of granules: light secretory vacuoles of large size (more than 1 pm) which are gathered in the apical and supranuclear cytoplasm, and dense round granules 150-300 nm in diameter which are seen in both the secretory and myoepithelial cells. The latter dense granules are probably lysosomes. The antebrachial organ of the ring-tailed lemur (prosimian primate) is a sweat gland developed specifically for the purpose of scent marking and reproductive behavior. Secretory cells of this organ show a structure characteristic of apocrine sweat glands (Kneeland, 1966). They have many protrusions extended into the
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FIG. 32. Parts of the secretory cells of the dog’s apocrine sweat gland. (a) The apical cytoplasm containing many small vesicular and a few solid secretory granules (S) intermingled with microfilaments and scattered ribosomes. On the luminal surface, pits (arrows) are observed being suggestive of exocytosis. (b) The supranuclear cytoplasm containing large dense granules (D),mitochondria (M), and Golgi apparatus (G).(c) Arrows indicate dense round granules enclosed in the cisternae of rough ER.
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lumen, suggestive of the apocrine secretion called apocrine blebs. Some mitochondria are very large, like those of the human apocrine gland, though the degree of hypertrophy of mitochondria is not as high as the human gland. Secretory vacuoles are large and numerous, particularly in a stage of hyperactivity such as pregnancy in the female (Kneeland, 1966). The appearance of secretory vacuoles is quite different from the small secretory granules of man (Kurosumi and Kurosumi, 1982), but the cells contain a few dark granules, which were called lipopigment (Kneeland, 1966). The secretory cells also contain bundles of microtubules, whose functional significance was not elucidated. Large secretory vacuoles are similar to the apocrine glands of many other mammals, such as the cat, horse, and bat. The horse possesses the best developed and the most numerous sweat glands among various domestic animals, which are distributed over the whole body surface. These glands secrete a large amount of sweat after physical exercise such as running, and therefore it has been thought that the horse’s sweat glands are similar in function to the eccrine sweat glands of the human being, though the horse’s glands have the morphological characteristics of apocrine sweat glands, for example, the ducts empty into the hair follicles. The secretory cells contain a great number of lucent secretory vacuoles, which almost fill the entire cytoplasm (Kurosumi et al., 1963; Sgrensen and Prasad, 1973). After simple osmium fixation these vacuoles were seen as empty (Kurosumi et al., 1963), but double fixation with glutaraldehyde and osmium also revealed very clear vacuoles (Sgrensen and Prasad, 1973). The latter authors demonstrated a positive PAS reaction for these vacuoles and concluded that these vacuoles contain acid mucopolysaccharides. Kurosumi et al. (1963) called these secretory cells of the horse gland containing a large number of secretory vacuoles “vacuolated cells,” and they found another type of cell in the secretory coil of this gland and called them “dense cells.” The finding of two different types of cells in the horse apocrine sweat gland coincides with the findings by Ito et al. (1961) with the light microscope. The dense cells only rarely occurred, and looked dense, because they contained no vacuoles and the cytoplasm is relatively dense. The size of the dense cell is smaller than the vacuolated cell, as they have no secretory vacuoles and poorly developed cell organelles. Microvilli on the luminal surface are short. This appearance of dark cells suggested the possibility that they were formed from the active secreting vacuolated cells due to a kind of degeneration or hypofunction. Sgrensen and Prasad (1973) described a dense cell which has a smaller cell body containing a few secretory vacuoles with no sign of apocrine secretion. This cell somewhat resembled the dense cell of Kurosumi et al. (1963), but the authors wrote that they did not observe dense cells with poorly developed microvilli and without secretory vacuoles corresponding to the dense cells of Kurosumi et al. (1963).
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The vacuolated cell or active secreting cell of the horse’s gland contains welldeveloped cell organelles such as lamellar rough ER, well-developed mitochondria, and Golgi apparatus of moderate dimension. The infolding of the basal plasma membrane are also remarkable at the gaps between myoepithelial cells. From the luminal surface, many long microvilli are protruded and tips of some microvilli are expanded, suggesting pinching-off (microapocrine secretion). Larger protrusions without microvilli on their surface and containing homogeneous cytoplasm are often observed and are suggestive of typical apocrine secretion, as reported by both Kurosumi et af. (1963) and Seirensen and Prasad (1973). The latter authors described unique intracellular canaliculi lined with numerous microvilli which are apparently invaginations from the luminal plasma membrane, but Kurosumi et al. (1963) did not find such canaliculi. Sekine (1966) studied the apocrine sweat gland in the face skin of the bat (Rhinofophusferrum-equinumnippon) in detail. The secretory cells of this gland appear to be very active in secretory function, because numerous secretory granules fill the apical cytoplasm and the basal part of the cell contains exceedingly well-developed rough ER consisting of closely packed parallel lamellae, which look like pancreatic acinar cells (Fig. 33). On the luminal surface, microvilli are best developed among apocrine glands of various kinds of animals; they are long, closely packed, and some are branched. In the apical cytoplasm well-developed smooth ER consisting of tubules and vesicles is intermingled with tonofilaments and microtubules. Some centrioles were observed in the apical cytoplasm. Sekine (1966) did not observe the swelling of tips of microvilli suggesting microapocrine secretion, but found a large domelike protrusion without microvilli on the surface, which was suggestive of apocrine secretion (Fig. 34a). The Golgi apparatus of the bat apocrine gland cell is localized at the supranuclear region, and contains many vacuoles with clear content probably derived from the swelling of the Golgi cistemae forming the parallel lamellae (Fig. 34b). The Golgi vacuoles may be transformed into secretory granules, as the intermediary vacuoles between the Golgi vacuoles and the secretory granules are often observed (Sekine, 1966). Not only the apocrine mechanism of secretion discharge, but exocytosis of secretory granules as well as small vesicles from the smooth ER were observed in the bat apocrine gland cells by Sekine (1966). These ultrastructural features of this gland indicated that it is one of the typical protein-secreting glands and the secretory substance may be seromucous. But this gland is different from the exocrine pancreas or salivary gland, for example, in the presence of well-developed basal infolding and apocrine secretory phenomena. The rabbit submandibular organ (Schaffer, 1940) is one of the unique skin glands. Its name is similar to the submandibular gland, which is one of the
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FIG. 33. Low magnification view of the secretory cells of the bat facial apocrine sweat gland. Microvilli on the apical surface are very well developed. Secretory vacuoles (S) are packed in the apical and supranuclear cytoplasm. The basal part of the secretory cell is filled with a nucleus (N) and parallel arranged well-developed rough ER (ER). Myoepithelial cells (ME) are seen at the bottom. (From Sekine, 1966.)
salivary glands which conducts secretion into the oral cavity. The rabbit submandibular organ is a skin gland, and the secretory product is led to the skin surface. Some authors called this gland the chin gland (Mykytowycz, 1968; Heath, 1974), and the function of this gland is said to be to secrete a territorial marker (Mykytowycz, 1968). In our early study of this gland (Kurosumi et al., 1961), no limiting membrane bounding the secretory substance was observed. Smooth ER is well developed, consisting mostly of scattered vesicles. Rough ER is composed of irregular
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FIG. 34. The secretory cell of the bat apocrine gland. (a) A dome-shaped projection (AP) extending into the gland lumen, suggestive of apocrine secretion. (b) The Golgi area of the secretory cell containing less dense vacuoles which may contain the secretory substance (arrows). Mitochondria (M) and Golgi lamellae (G)are shown. (From Sekine, 1966).
cisternae distributed in the basal part of the cytoplasm. The Golgi apparatus is observed in the supranuclear region, but it is not as well developed and no sign of formation of secretory granules was observed. Instead of the Golgi apparatus and rough ER, mitochondria may play a role in the production of the secretory substance in this gland cell. At the resting stage, mitochondria are small and slender, but the cells in the active synthesizing stage, whose dimension becomes larger, contain many large mitochondria. Not only enlargement but also deformation occurs in these mitochondria of the active stage of secretory cells. Mitochondria are elongated extraordinarily and thinned at the central part. Frequently a mass of homogeneous or fine granulated substance is wrapped partly by thin sheets of mitochondria and sometimes continuity between the thin mitochondria and the secretion mass was observed. Kurosumi et al. (1961) concluded that the secretory substance of this gland may be initially synthesized within the mitochondria, and then discharged from them into the cytoplasmic matrix. Finally the secretory substance may diffuse in the apical cytoplasm and may be discharged by the mechanism of pinching-off of cytoplasmic projections, that is, typical apocrine secretion (Kurosumi et al., 1961). The secretory substance of this gland is presumably different in chemical nature from other apocrine sweat glands. It is assumed that the secretory substance in this gland may contain lipid in a relatively high rate, and hence the substance may be produced in mitochondria with an intimate relation to the smooth ER, with the rough ER and Golgi apparatus only indirectly involved. Heath (1974), who studied the same gland, reported that the mitochondria1 deformation was observed only in male rabbit but not in castrated male or in the
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female gland. Heath (1974) referred to this as a secretory lake for the accumulation of secretory substance occurring near the deformed mitochondria, but he reported that a membrane bounds the lake of secretory substance and the membrane is continuous to the smooth ER. He denied the presence of the apocrine secretory mechanism, and supposed that the secretion contained in the small vesicles might be released by exocytosis. In our later observation of this gland (Kurosumi, unpublished), the presence of membranes surrounding each mass of secretory substance was recognized, though the deformation of mitochondria near the secretory mass was marked (Fig. 35). The lack of a limiting membrane around the secretory mass in the previous study might be an artifact due to inappropriate fixation and embedding at that time, when the specimen technique had not been fully improved. There are still unsolved problems concerning the mechanism of secretion in animal apocrine sweat glands, which are not identical to the human gland, and await further morphological studies with advanced techniques.
B. ANIMALECCRINE SWEATGLANDS The first observations of sweat glands of nonhuman mammals with the electron microscope were our reports on the pig’s carpal organ (Kurosumi and
FIG.35. A part of the secretory cell of the rabbit submandibular organ (chin gland). Large secretory vacuoles containing the secretory substance ( S ) of moderate density are covered with a limiting membrane and partly surrounded by a deformed mitochondrion (arrow).
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Kitamura, 1958; Kitamura, 1958). In general, the eccrine sweat glands are restricted to the plantar skin of feet and toes in mammals other than humans and higher primates. In the pig, the carpal region corresponds to the foot pads of other mammals, and the skin gland in the carpal region of the pig, which is called the “carpal organ,” has been known to be conglomerated eccrine sweat glands as a result of light microscopy. Similar to the human eccrine sweat glands, the carpal organ has two different secretory cells, dark and clear cells. In our studies the specimens were fixed with simple osmium fixative, and therefore the secretory granules of the dark cell appeared as clear vacuoles, looking like the secretory (vacuolated) cells of the horse’s apocrine sweat glands; they also resemble the mucus-secreting goblet cells of the intestinal tract. The luminal surface of the secretory cells are provided with microvilli, which are longer and more numerous in the clear cells than in the dark cells. This relationship is the same as that in the human eccrine sweat glands. In the clear cells neither granules nor vacuoles resembling the secretory granules of the dark cells were observed, but small dense granules near the nucleus were reported as secretory granules. It is most likely that these granules may be lysosomes, though histochemical detection of acid phosphatase was not performed. Two types of secretory cells are not arranged in layers, but are arranged in a single layer. No intercellular canaliculi were developed. The cell boundaries are relatively complicated, that is, the interdigitations are seen on the lateral boundary and infoldings are developed on the basal surface. The invaginations or infoldings (P-cytomembrane of Sjostrand, 1956) of the surface plasma membrane were particularly stressed in the first report from our laboratory (Kurosumi and Kitamura, 1958), and then later similar structures, presumably for water transport, were observed in almost all the sweat glands of man and animals of either eccrine or apocrine type. It is reasonable to assume that the eccrine sweat gland of the monkey is very similar to that of humans. Terzakis (1964) used the sweat glands of African Green monkeys and tried many different fixatives, that is different combinations of glutaraldehyde, potassium permanganate, and osmium tetroxide. The monkey eccrine sweat glands have the same pattern of cell arrangement as that of the human eccrine sweat glands; the dark cells are usually situated on the apical side always facing the main gland lumen, while the clear cells are often situated at the basal part resting either upon the basement membrane or upon the myoepithelial cells, and usually border the intercellular canaliculi and only exceptionally face the main lumen (Terzakis, 1964). The secretory granules of the dark cell are preserved well by double fixation of glutaraldehyde and osmium, showing a very dense appearance identical to the secretory granules of dark cells of the human gland. These secretory granules evidently arise from the Golgi apparatus.
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The ultrastructure of the clear cells of the monkey is also similar to that of the human clear cells. These cells contain a few relatively small dense granules, to which Terzakis (1964) gave the name secretory granules. As already mentioned, the clear cells of the human eccrine sweat glands do not contain secretory granules. Such small dense granules resemble the dense granules in the clear cells of the pig’s carpal organ which were also called secretory granules. However, these small dense granules near the Golgi apparatus are doubtful as to lysosomes now. Abundant accumulation of glycogen particles in the clear cell is the same as in the human gland. Terzakis (1964) showed the intermediate cells between dark and clear cells. They have a moderate amount of glycogen, relatively small secretory granules of the clear cell type (doubtful as to lysosomes), and many secretory vacuoles (granules) in the apical cytoplasm whose diameter is intermediate between the granules of dark cells and clear cells. Such intermediate secretory cells were not observed in human as well as animal eccrine sweat glands other than the monkey. The eccrine sweat glands situated in the foot pads of the cat (Munger and Brusilow, 1961) and dog (Kurosumi, 1982) are similar to each other. The lumen is relatively wide and secretory cells are arranged in a single layer. Both the dark and clear cells face the gland lumen directly, but intercellular canaliculi develop between the adjacent clear cells (Fig. 36). The dark cell contains a large amount of secretory granules, which are seen as vacuoles after simple osmium fixation or with Dalton’s fixative (Munger and Brusilow, 1961), but are variable in density after successive glutaraldehyde and osmium fixation (Fig. 37a). The clear cells contain no secretory granules, but vesicles and tubules of smooth ER are observed in the apical cytoplasm (Fig. 37b). Munger and Brusilow (1961) indicated that the cytoplasm of the clear cell was granular and it was impossible to determine with accuracy the nature of the individual granules, although many of them resembled glycogen units. Curiously enough, glycogen particles were observed in the dog’s dark cells, but not in the clear cells (Kurosumi, 1982) (Fig. 37C). Plantar eccrine sweat glands of the rat and mouse were studied by some authors (Matsuzawa and Kurosumi, 1963; Wechsler and Fisher, 1968; Kurosumi and Kurosumi, 1970; Munger and Brusilow, 1971). These rodent eccrine sweat glands differ from conventional eccrine sweat glands of higher mammals such as man, monkey, cat, and dog, because the secretory portion of the former contains essentially one type of secretory cell. After glutaraldehyde and osmium double fixation, shrunken cells with exceedingly dense protoplasm were observed in the mouse eccrine gland (Fig. 38) (Kurosumi and Kurosumi, 1970). After simple osmium fixation, we could not find these very dense cells. It is highly probable that the occurrence of such dark cells may be the result of a fixation artifact or of some pathological imbalance in water metabolism. Matsuzawa and Kurosumi (1963) observed two types of cells, dark and clear
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FIG. 36. A low magnification view of ii cross section o l thc secretory coil of the eccrinc \weat gland in the dog's toot pnrt. Well-developed inyocprthclial cell3 ( M E ) \urrtrund thc tuhulc. TNOtypes o l sccrctory cells. dark cells (1)) and clear cells ( C ) ,arc observed. Interccllular caiialictili (arrows) appear hctwcen clear cells
cells, in 10-day-old rats, but no distinction between the dark and clear cells was made in the glands of 3-day-old infant as well as adult rats. 'The authors stated that the cells of the infant cccrine glands itre highly hydrated and look clear. but dark cells occur sporadically in the age of 10 days, whilc secretory cells all convert into dark cells by maturity.
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Ftc. 37. Parts of the eccrine sweat glands of the dog’s foot pad. (a) The apical cytoplasm of the dark cell containing secretory granules of varying density. One of the granules opens to the lumen suggesting the exocytosis (arrow). (b) The apical cytoplasm of the clear cell having rather long microvilli on the surface and smooth ER (SR) in the cytoplasm. (c) Dark cells of the dog’s eccrine gland, containing secretory granules ( S ) and glycogen particles (GL).
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Flc. 38. Secretory coil of the mouse plantar eccrine gland, a dark cell extending many thin processes penetrating into the surrounding clear cells. It is probable that this cell may be formed due to inappropriate fixation. (From Kurosumi and Kurosumi, 1970.)
Wechsler and Fisher (1968) observed only one type of secretory cell in the normal rat eccrine sweat gland, but they found dark and clear cells after salt or water overload and prolonged stimulation with mecholyl. These procedures brought out cells with lucent cytoplasm containing few RNA particles, sparse glycogen particles, and a slightly decreased number of mitochondria. The results of Matsuzawa and Kurosumi (1963) as well as those of Wechsler
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and Fisher (1968) suggested that the distinction between dark and clear cells may be brought about from the slight difference of water content at an immature stage of development or after the experimentally induced imbalance in water content in the secretory cells of the rat eccrine glands. Munger and Brusilow (197 1) clearly concluded that the secretory cells of the rat eccrine sweat gland are of only one type. But they found two populations of secretory cells on the basis of mitochondria1 morphology. Following stimulation with pilocarpine, these differences in mitochondria completely disappeared. Therefore, the secretory cells of the rodent eccrine gland may be essentially one type. The secretory cells of the adult rat eccrine gland are provided with remarkable foldings of plasma membrane which consist of interdigitation between the adjacent cell cytoplasm and infoldings of the basal plasma membrane. The neonatal rat eccrine glands have no differentiation of basal infoldings, and these foldings appear at about 10 days after birth. Before the time of the first appearance of membrane foldings, natural perspiration on the foot pads was not observed, and therefore it was thought that the folded membranes might be closely correlated to the function of sweating (Matsuzawa and Kurosumi, 1963). Electron microscopic histochemical investigation demonstrated that the activity of nonspecific alkaline phosphatase and ATPase was restricted to the folded plasma membranes at the cell base. These enzymes might be correlated to water transport. Furthermore, microapocrine secretion was demonstrated at the luminal surface of the secretory cells (Matsuzawa and Kurosumi, 1963). As already mentioned, in the eccrine sweat glands of other mammals including man, there are two types of secretory cells, the dark cells and clear cells. The dark cells contain secretory granules (vacuoles), while the clear cells contain well-developed smooth ER but no secretory granules. The secretory cells of the rat and mouse eccrine sweat glands contain both secretory vacuoles and smooth ER in the same cell (Fig. 39) (Matsuzawa and Kurosumi, 1963; Kurosumi and Kurosumi, 1970), but Munger and Brusilow (1971) thought that the vacuoles in the apical cytoplasm are not secretory vacuoles but are cross sections of pits or invaginations of the apical surface membrane. No intercellular canaliculi were observed in the rat and mouse eccrine glands. The fact that the secretory vacuoles and smooth ER appear in one and the same cell as well as the absence of intercellular canaliculi may indicate the eccrine glands of the rat and mouse are primitive in structure, and cell differentiation has not yet occurred (Kurosumi and Kurosumi, 1970). Ultrastructural studies on animal sweat ducts are few in number. Kurosumi and Kurosumi (1970) and Suzuki (1973) reported the fine structure as well as keratinization process of duct epithelial cells of the mouse plantar eccrine sweat gland. The substance of these papers was briefly referred to in an earlier part of this article in comparison to the morphology of the human eccrine sweat gland.
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FIG. 39. Apical part of a cell of the secretory portion of the mouse eccrine sweat gland. Ample microvilli are often swollen and detached as a result of microapocrine secretion (arrows). The apical cytoplasm contains both relatively small secretory vacuoles (V) and tubulous elements of smooth ER (SR). (From Kurosumi and Kurosumi, 1970.)
Our knowledge of the ultrastructure of animal sweat glands is still poor, because the sweat glands of many species of mammals have not been explored with the electron microscope. It seems necessary to widen our knowledge using many other samples to attain final conclusions concerning the correlation between the structure and function of the sweat glands.
V. Concluding Remarks Though each gland is small in dimension, the sweat glands are distributed over the entire body surface in many mammals, and also form specialized glandular organs in certain regions of the body surface in most animals. Therefore, the total dimension of the sweat glands in one individual animal body is enormous. Thus the function of this exceedingly large glandular system cannot be neglected in animal or human physiology. Another characteristic of the sweat glands is wide variations in structure and function. The difference between the apocrine and eccrine glands is well known, but the fine structural differences between these two types of sweat glands have been clearly shown rather recently. The discovery of two types of secretory cells
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in the human eccrine sweat glands by It0 (1943) opened the door for study of the modem cytology of this gland, and made clear differences in the fine structure between the eccrine and apocrine gland. Introduction of electron microscopy to the field of cytology of sweat glands sometimes brought forth confusion in understanding ultrastructure, but a tremendous amount of information on human and animal sweat glands in this quarter century led to a detailed knowledge of the morphology of this skin gland system. It is noteworthy that both apocrine and eccrine sweat glands of almost all mammalian species studied secrete mucopolysaccharides. It is reminiscent of the skin glands secreting a large amount of mucus onto the body surface of fish and amphibians. The primary function of the sweat gland may be the protection of the body surface with its mucous secretion. In higher mammals, secretion of a large amount of water mixed with mucus makes the well-known function of regulation of body temperature prominent. The differentiation of clear cells is concerned with the appearance of this function in the human eccrine sweat glands. But in the sweat glands of the horse, mucoid-secreting cells may also be involved with the function of water transport, because the gland in this species belongs to the apocrine sweat glands. The majority of apocrine sweat glands, however, are involved in functions important in the reproductive and social lives of animals by their odorous secretion. The dependency of the development of some apocrine glands upon sexual hormones indicates their functional significance in reproductive behavior. The odor of some apocrine glands may act as a pheromone. Large gaps are still left in our knowledge of the ultrastructure and function of sweat glands of man and animals, because only a few scientists have been interesting in this skin gland to now. As mentioned above, this gland contains many more interesting structures, which were not completely explained from a physiological points of view. We hope that the field of the cytology of this gland will continue to be explored in the near future.
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Index
A Adenosine triphosphatase, in mitotic spindle Ca*+-ATPase, 91-93 calmodulin regulation of, 95-96 cytochemistry, 86-87 dynein, 87-90 models of mitosis and, 96-100 myosin, 90-91 other ATP hydrolyzing enzymes associated with microtubules, 94-95 Adenylate cyclase, capping and, 213 Apocrine sweat gland light microscopic cytology, 256-259 ultrastructures and functional significance in human cored vacuoles, 268-270 large dense granules, 263-268 large less dense granules, 270 plasma membrane folding, 276-277 secretion mechanism, 273-276 small secretory granules, 270-273 myoepithelial cell, 277-282 ultrastructural cytology of secretory activity in nonhuman mammals. 309-3 I8
C Calcium ions-calmodulin, capping and, 209-210 Calmodulin, mitotic spindle ATPase and, 95-96 Cancer cells, nucleoli of, 144-147 Cundidu
steady state model, information required, 12-13 transition model, information required, 13-14 Cundida ulbicuns. carbohydrate metabolism in: a system to model, 10-12
33 1
Capping general phenomena ligand-depehdent processes, 196- 197 ligand-independent processes, 197- 198 intermediate filaments and, 207-208 mechanisms of, 213-217 microfilaments and, 198-203 microtubules and, 203-207 related regulatory molecules and enzymes, 208-209 Ca*+-calmodulin, 209-210 cyclic AMP and adenylate kinase, 213 myosin light chain kinase, 210-213 Carbohydrate metabolism, in Cundidu ulbicans: a system to model, 10-12 Cell(s), differentiation, maturation and aging, nucleolus and, 140-143 Cell-cell communication, via diffusion-mediated trapping, 62-64 Cell cycle, nucleolar ultrastructural morphology during interphase, 126- 129 mitosis, 129-136 Cell membrane, proteins, slow mobility and immobility, 25-26 Chromatin, nucleolus-associated composition, 122-123 ultrastructure, 122 Circadian rhythm, nucleoli and, 140 Citric acid cycle, in Dictyostelium discoideum: a steady state model, 1-4 Cyclic AMP, capping and, 213 Cyclic AMP-mediated hormonal response, protein diffusion and, 59-61 Cytochemistry, of mitotic spindle, 86-87 Cytoskeleton, protein diffusion and, 26-29
332
INDEX
D Dicryostelium discoideum, citric acid cycle in:
a steady state model, 1-4 Diffusion barriers, intrinsic, 29-30 Dynein, mitotic spindle and, 87-90
E Eccrine sweat gland light microscopic cytology, 259-263 ultrastructures and functional significance in human, 282-283 clear (basal) cell, 289-294 dark (superficial) cell, 283-289 myoepithelial cell, 294-295 ultrastructural cytology of secretory activity in nonhuman mammals, 318-325 Excretory ducts, ultrastructures and functional significance in human, 295-296 dermal ducts, 300-304 epidermal ducts, 304-309 transitional portion, 296-300 Electron transport, protein diffusion and, 49-59 Embryogenesis, in animals, nucleolus and, 137 Energy requirements, for mitosis, 84-86 Epididymus, contribution to sperm surface heterogeneity, 179-182 Extracellular matrices, entrapment in, 40-41
F Fibrillar centers, nucleolar composition, 110-120 ultrastructure, 109-1 10 Fibrillar component, nucleolar composition, 120- 12 I ultrastructure, 120 Filaments, intermediate, capping and, 207-208
G Glycocalyx, extracellular influences, protein diffusion and, 31-32 Glycogen metabolism, in rat liver and Morris hepatoma: transition models, 4- 10 Granular component, nucleolar composition, 12 I- 122 ultrastructure, 121
Growth, normal, muscle satellite cell content and, 238-243
H Heterokaryons, nucleoli and, 143- 144 Hypertrophy, compensatory, muscle satellite cell content and, 244-245 1
Immobilization, contact-induced, 64 Information flow, in membrane plane, 64-66 1
Leukocytes, degranulation, protein diffusion and. 61-62
M Macromolecules, contractile, 35 Meiosis, in germinal cells, nucleolar ultrastructural morphology during, 136- 137 Membrane bulk flow of, 36-37 components, localization by electric fields, 37-38 diffusion-mediated trapping and, 41-45 reduction of dimensionality and, 45-49 Microfilaments, capping and, 198-203 Microtubules ATP hydrolyzing enzymes associated with, 94-95 capping and, 203-207 Mitosis energy requirements for, 84-86 models of, spindle ATPase and, 96-101 Mitotic spindle ATPase in Ca2+-ATPase, 91-93 calmodulin regulation, 95-96 cytochemistry, 86-87 dynein, 87-90 models of mitosis and, 96-100 myosin, 90-91 other ATP hydrolyzing enzymes associated with microtubules, 94-95 Moms hepatoma, glycogen metabolism in transition models, 4- 10
INDEX Muscle satellite cell activation stimulus, 245-246 cell location cardiac muscle, 231 skeletal muscle, 226-231 fine structure, 233-238 gross morphology, 231-233 situations affecting cell content normal growth, 238-243 nutrition, 243 regeneration and compensatory hypertrophy, 244-245 Myosin, mitotic spindle and, 90-91 Myosin light chain kinase, capping and, 2 10-2 I3
333
Protein diffusion implications in membrane biology biological signaling at the membrane, 59-66 pattern creation and maintenance, 34-45 reactions in two dimensions, 45-59 self-assembly and sorting, 66-7 1 known and unknown biological scope of measurements, 21 -22 diffusional constraints, 25-34 experimental techniques, 20-21 short- versus long-range diffusion, 24-25 temperature dependency, 22-24
R N Nucleolar interstices composition, 122 ultrastructure, 122 Nucleolar matrix, 123- 124 Nucleolus components, 108-109 functional significance, 124- I26 morphology and composition, 109- 124 exportation of RNP, 148-150 variations in ultrastructural morphology during cell cycle, 126- 136 functional significance, 147-148 during meiosis in germinal cells, 136- I37 under physiological or experimental conditions, 137- 147 Nutrition, muscle satellite cell content and, 243
P Phagocytosis, protein diffusion and, 71 Photosynthesis, protein diffusion and, 56-59 Plants activation of quiescent cells in, nucleolus and, 137-139 seasonal modifications in nucleoli, 139 Protein(s) ligand-free and ligand-bound, diffusion of, 30-3 I solubility and aggregation, 35-36
Rat liver, glycogen metabolism in transition models, 4-10 Regeneration, muscle satellite cell content and, 244-245 Respiration, protein diffusion and, 49-50 biochemicalikinetic evidence, 54-55 early evidence on chain dynamics, 50-52 lateral diffusion measurements, 52-54 rotational diffusion measurements, 55-56 Ribonucleoprotein (RNP), exportation from nucleolus, 148- I50
S Sperm biogenesis of membranes, I7 1- 172 acrosomal , 176- 179 plasma, 172-176 functional aspects of membrane heterogeneity fusion interactions and, 183- 188 surface modification, 182- 183 structural differentiation in membranes head, 161-168 tail, 168-171 summary of structure and function, 160-161 surface heterogeneity, epididymal contribution, 179-182 Sweat gland historical survey of light microscopic studies of morphology apocrine glands, 256-259
334
INDEX
eccrine glands, 259-263 general histology, 254-256 ultrastructural cytology of secretory activity in glands of nonhuman mammals apocrine, 309-318 eccrine, 318-325 ultrastructures and functional significance in human apocrine, 263-282 eccrine, 282-295 excretory ducts, 295-309
T Thylakoid stacking, protein diffusion and, 69-7 1 Tight junctions, segregation and, 38-40
V Virus budding, protein diffusion and, 66-69 Viscous limit, determination of, 32-34
Contents of Recent Volumes and Supplements Volume 72
Volume 70
Cycling F? Noncycling Cell Transitions in Microtubule-Membrane Interactions in Cilia and Flagella-wILLIAM L. DENTLER Tissue Aging, Immunological Surveillance. The Chloroplast Endoplasmic Reticulum: StrucTransformation, and Tumor Growthture, Function, and Evolutionary SignifiSEYMOUR GELFANT The Differentiated State of Normal and Maligcance-SAKAH P. G l B B S DNA Repair-A. R. LEHMANNA N D P. nant Cells or How to Define a "Normal" Cell in CUltUre-MINA J. B1ssk:i.L KARKAN On the Nature of Oncogenic Transformation of Insulin Binding and Glucose Transport-RusSELL H i u . L A U R I K. E SOHGE,A N D ROGER CellS4ERALD L. CHAN J. G A Y Morphological and Biochemical Aspects of Adhesiveness and Dissociation of Cancer CellsCell Interactions and the Control of DcvelopA N D YASUJI ISHIMAKLI Hiueo HAYASHI ment in Myxobacteria Populations-DAVID The Cells of the Gastric M U C ~ S ~ - H ~ R B EF.K T WHIT^ HELANDER Ultrastructure, Chemistry. and Function of the Bacterial Wall-T. J . BEvkxiDGt Ultrastructure and Biology of Female Gametophyte in Flowering Plants-R. N . KAPILA N D I N D E X A. K . BHATNACAK INDEX
Volume 73 Volume 71 Integration of Oncogenic Viruses in Mammalian CellS
Protoplasts of Eukaryotic AIgBe--MAKTHA D. BERLINER Polytene Chromosomes of Plants-WALTER NAGL. Endosperm-Its Morphology, Ultrastructure, and Histochemistry-S. P. BHATNAGAH AND VEENA SAWHNEY The Role of Phosphorylated Dolichols in Membrane Glycoprotein Biosynthesis: Relation to Cholesterol Biosynthesis-JOAN TUGEND H A ~ TMILLS A N D ANTHONY M. ADAMANY Mechanisms of lntralysosomal Degradation with Special Reference to Autophagocytosis and Heterophagocytosis of Cell OrganellesHANS GLAUMANN, JAN L. E. ERICSSON, A N D LOUISMARZELLA Membrane Ultrastructure in Urinary TubulesLELIOORCI, FABIENNE HUMBERT, DENNIS BROWN,A N D ALAINPERRELET Tight Junctions in Arthropod Tissues-NANCY J. LANE Genetics and Aging in PrOtOZoa-JOAN SMITHSONNEBORN
INDEX
INDEX
335
336
CONTENTS OF RECENT VOLUMES AND SUPPLEMENTS
Volume 74
Organization and Expression of Viral Genes in Adenovirus-Transformed Cells-S. J. FLINT The Plasma Membrane as a Regulatory Site in Highly Repeated Sequences in Mammalian GenOmeS-MAXlNE F. SINGER Growth and Differentiation of Neuroblastoma CdlS-SIEGFRIED w. DE LAAT Moderately Repetitive DNA in EvolutionAND PAULT. VAN DER SAAG ROBERTA. BOUCHARD Mechanisms That Regulate the Structural and Structural Attributes of Membranous Organelles Functional Architecture of Cell Surfacesin Bacteria-CHARLEs C. REMSEN JANETM. OLIVER A N D RICHARD D. BERLIN Separated Anterior Pituitary Cells and Their ReGenome Activity and Gene Expression in Avian sponse to Hypophysiotropic HormonesErythroid Cells-KARLEN G . GASARYAN CARLDENEF,Luc SWENNEN, AND MARIA Morphological and Cytological Aspects of Algal ANDRIES Calcification-MICHAEL A. BOROWITZKAWhat Is the Role of Naturally Produced Electric Naturally Occumng Neuron Death and Its RegCurrent in Vertebrate Regeneration and Healing?-RICHARD 8 . BORGENS ulation by Developing Neural PathwaysTIMOTHY Metabolism of Ethylene by PlantS-hHN J . CUNNINGHAM A. HALL DODDSAND MICHAEL The Brown Fat Cell-.fAN NEDERGAARD AND OLOVLINDBERG INDEX INDEX
Volume 75
Volume 77
Mitochondria1 Nuclei-TsuNEYosHi KUROIWA Slime Mold Lectins-JAMES R. BARTLES, WILLIAMA. FRAZIER,AND STEVEND. ROSEN Lectin-Resistant Cell Surface Variants of Eukaryotic Cells-EvE BARAKBRILES Cell Division: Key to Cellular Morphogenesis in the Fission Yeast, SchizosaccharomycesBYRONF. JOHNSON,CODE B. CALLWA, ZUKER,AND I A N BONGY. Yoo, MICHAEL I . MCDONALD Microinjection of Fluorescently Labeled Proteins into Living Cells, with Emphasis on Cytoskeletal PrOteinS-THOMAS E. KREIS AND WALTERBIRCHMEIER Evolutionary Aspects of Cell DifferentiationR. A. FLICKINGER Structure and Function of Postovulatory Follicles (Corpora Lutea) in the Ovaries of Nonmammalian Vertebrates-SRiNivAs K. SAI-
Calcium-Binding Proteins and the Molecular Basis Of Calcium Action-LINDA J . V A N ELD I K , JOSEPH G. ZENEDEGUI, DANIELR. MARSHAK,AND D. MARTINWATTERSON Genetic Predisposition to Cancer in Man: I n Vifro Studies-LEVY KOPELOVICH Membrane Flow via the Golgi Apparatus of Higher Plant Celh-DAViD G . ROBINSON AND U w KRISTEN Cell Membranes in Sponges-WERNER E. G . MULLER Plant Movements in the Space EnvironmentDAVID G. HEATHCOTE Chloroplasts and Chloroplast DNA of Acefubularia mediterraneu: Facts and HypothesesANGELALUTTKEAND SILVANO BONOTTO Structure and Cytochemistry of the Chemical SynapSeS-sTEPHEN MANALOV AND WLADIMIR OVTSCHAROFF INDEX
DAPUR INDEX
Volume 78 Volume 76 Cytological Hybridization to Mammalian ChromOSOmeS-ANN s. HENDERSON
Bioenergetics and Kinetics of Microtubule and Actin Filament Assembly-DissassemblyTERRELLL. HILLAND MARCW. KIRSCHNER
CONTENTS OF RECENT VOLUMES AND SUPPLEMENTS Regulation of the Cell Cycle by Somatomedins-HOWARD ROTHSTEIN Epidermal Growth Factor: Mechanisms of Action-MANJUSRI DAS Recent Progress in the Structure, Origin, Composition, and Function of Cortical Granules in Animal Egg-SARDUL S . GURAYA
337
Immunofluorescence Studies on Plant C e l l d . E. JEFFREE, M. M. YEOMAN,A N D D. C. KILPATRICK Biological Interactions Taking Place at a LiquidSolid InterfaCe-ALEXANDRE ROTHEN INDEX
INDEX
Volume 81 Volume 79 Oxidation of Carbon Monoxide by BacteriaYOUNGM. KIMA N D GEORGED. HEGEMAN The Formation, Structure, and Composition of the Mammalian Kinetochore and Ki- Sensory Transduction in Bacterial Chemotaxi S a E R A L D L. HAZELBAUER A N D SHlGEAKl netochore Fiber
Volume 80 DNA Replication Fork Movement Rates in Mammalian Cells-LEON N. KAPP A N D ROEERTB. PAINTER Interaction of Virsues with Cell Surface Receptors-MARC TARDIEU.ROCHELLEL. EPSTEIN, A N D HOWARD L. WEINER The Molecular Basis of Crown Gall lnductionW. P. ROBERTS The Molecular Cytology of Wheat-Rye Hybrids-R. APPELS Bioenergetic and Ultrastructural Changes Associated with Chloroplast Development-A. R. WELLBURN The Biosynthesis of Microbodies (Peroxisomes, G1yoxysomes)-H. KINDL
Volume 82 The Exon:lntron Structure of Some Mitochondrial Genes and Its Relation to Mitochondria1 EVOhtion-HENRY R. MAHLER Marine Food-Borne Dinoflagellate ToxinsDANIELG. BADEN Ultrastructure of the Dinoflagellate AmphieSma-LENITA c . MORRILLAND ALFREDR. LOEBLICH 111 The Structure and Function of Annulate Lamellae: Porous Cytoplasmic and Intranudear Memb~ane%RlCHARD G. KESSEL Morphological Diversity among Members of the
338
CONTENTS OF RECENT VOLUMES AND SUPPLEMENTS
Gastrointestinal MiCrOflOra-DWAYNE SAVAGE
C.
INDEX
Cell Surface Receptors: Physical Chemistry and Cellular Regulation-DOUGLAS LAUFFENBURGER A N D CHARLES DELIS1 Kinetics of Inhibition of Transport Systems-R. M. KRUPKAAND R. D E V ~ S
Volume 83
INDEX
Transposable Elements in Yeast-VALERIE MoVolume 85 ROZ WILLIAMSON Techniques to Study Metabolic Changes at the Cellular and Organ Level-ROBERT R. DE- Receptors for Insulin and CCK in the Acinar Pancreas: Relationship to Hormone ActionFURlA A N D MARYK. DYGERT IRA D. GOLDFINE A N D JOHNA. WILLIAMS Mitochondria1 Form and Function Relationhips in Vivo: Their Potential in Toxicology and The Involvement of the Intracellular Redox State and pH in the Metabolic Control of StimPathology-ROBERT A. SMITH A N D MURIEL ulus-Response COUphg-ZYGMUND ROTH, J. ORD NAOMl CHAYEN, A N D SHABTAY DlKSTElN Heterogeneity and Territorial Organization of Regulation of DNA Synthesis in Cultured Rat the Nuclear Matrix and Related StructuresHepatoma Ceh-ROELAND V A N WlJK M. BOUTEILLE,D. BOUVIER,A N D A. P. Somatic Cell Genetics and Gene Mapping-FASEVE TEN KAO Changes in Membrane Properties Associated with CellUlar Aging-A. MACIEIRA-COELHO Tubulin Isotypes and the Multigene Tubulin Families-N. J. COWANA N D L. DUDLEY Retinal Pigment Epithelium: Proliferation and Differentiation during Development and Re- The Ultrastructure of Plastids in RWtS-JEAN M. WHATLEY generation-OLcA G. STROEVAA N D VicThe Confined Function Model of the Golgi TOR I. MITASHOV Complex: Center for Ordered Processing of INDEX Biosynthetic Products of the Rough Endoplasmic Reticulum-ALAN M. TARTAKOFF Problems in Water Relations of Plants and Volume 84 Ceb-PAUL J. KRAMER Controls to Plastid Divsion-J. V. POSS~NGHAMPhagocyte-Pathogenic Microbe InteractionsANTOINElTE RYTER A N D CHANTAL DE AND M. E. LAWRENCE CASTELLIER Morphology of Transcription at Cellular and INDEX I’UVIONMolecular kVdS-FRANCINE DuTiLLEuL An Assessment of the Evidence for the Role of Volume 86 Ribonucleoprotein Particles in the Maturation of Eukaryote mRNA-J. T. KNOWLER Toward a Dynamic Helical Model for the InfluDegradative Plasmids-J. M. PEMBERTON ence of Microtubules on Wall Patterns in Regulation of Microtubule and Actin Filament Plants-CLivE W. LLOYD Assembly-Disassembly by Associated Small Cellular Organization for Steroidogenesisand Large MOIeCUleS-TERRELL L. HILL PETERF. HALL AND MARCW. KIRSCHNER Cellular Clocks and Oscillators-R. R. Long-term Effects of Perinatal Exposure to Sex KLEVECZ,S. A. KAUFFMAN,A N D R. M. Steroids and Diethylstilbestrol on the ReSHYMKO productive System of Male MammalsMaturation and Fertilization in Starfish YASUMASA ARAI,TAKAO MORI. YOSHlHlDE OOCYte+LAURENT MEIJER AND PIERRE SUZUKI,AND HOWARDA. BERN GUERRIER
CONTENTS OF RECENT VOLUMES AND SUPPLEMENTS Cell Biology of Trypanosoma Cruzi-WANDERLEY DE SOUZA The Neuronal Organization of the Outer Plexiform Layer of the Primate Retina-ANDREW P. MARIANI INDEX
Supplement 10: Differentiated Cells in Aging Research
339
Thyroid Cells in CUhlr+FRANCESCO s. AMBESI-IMPIOMBATOA N D HAYDENG . COON Permanent Teratocarcinoma-Derived Cell Lines Stabilized by Transformation with SV40 and SVmSA Mutant virUSe+wARREN MALTZMAN, DANIELI. H. LINZER, FLORENCE BROWN,ANGELIKA K. TERESKY,MAURICE A N D ARNOLD J . LEVINE ROSENSTRAUS, Nonreplicating Cultures of Frog Gastric Tubular Celk&ERTRUDE H. BLUMENTHAL AND DINKAR K. KASBEKAR
INDEX Do Diploid Fibroblasts in Culture Age?-EuG E N E BELL, LOUIS MAREK,STEPHANIE SHER, CHARLOITE MERRILL, DONALD Supplement 1lA: Perspectives in Plant Cell LEVINSTONE, A N D IAN YOUNG and Tissue Culture Urinary Track Epithelial Cells Cultured from Human Urine-J. S. FELIX A N D J . W. Cell Proliferation and Growth in Callus CulLITTLEFIELD tures-M. M. YEOMAN A N D E. FORCHE The Role of Terminal Differentiation in the Finite Culture Lifetime of the Human Epider- Cell Proliferation and Growth in Suspension mal KeratinOCyte-JAMES G. RHEINWALD Cultures-P. J . KING Long-Term Lymphoid Cell Cultures-CkoRcE Cytodifferentiation-RICHARD PHILLIPS F. SMITH, PARVIN JUSTICE, HENRI Organogenesis in Virro: Structural, Physiological, and Biochemical Aspects-TREVOR A. FRISCHER, LEE K I N CHU,A N D JAMESKROC THORPE Type II Alveolar Pneumonocytes in VirrWILLIAMH. J. DOUGLAS, JAMES A. Chromosomal Variation in Plant Tissues in Culture-M. W. BAYLISS MCATEER,JAMES R. SMITH,A N D WALTER Clonal Propagation-INmn K. VASIL AND R . BRAUNSCHWEIGER VlMLA VASlL Cultured Vascular Endothelial Cells as a Model System for the Study of Cellular Senes- Control of Morphogenesis by Inherent and Exogenously Applied Factors in Thin Cell cence-ELLIOT M. LEVINEAND STEPHEN Layers-K. TRANTHANHVAN M. MUELLER Vascular Smooth Muscle Cells for Studies of Androgenetic HaPlOidS-INDRA K. VASlL Cellular Aging in V i m ; an Examination of Isolation, Characterization, and Utilization of Mutant Cell Lines in Higher Plants-PAL Changes in Structural Cell L i p i d d L G A 0. MALIGA BLUMENFELD,ELAINE SCHWARTZ,VERONICA M. HEARN, AND MARIE J. SUBJECT INDEX KRANEWOL Chondrocytes in Aging ReSearCh-EDWARD J. Supplement 11B: Perspectives in Plant Cell MILLERAND STEFFANGAY and Tissue Culture Growth and Differentiation of Isolated Calvarium Cells in a Serum-Free MediumJAMESK. BURKSAND WILLIAMA. PECK Isolation and Culture of Protoplasts-INoRA K. VASILAND VIMLAVASIL Studies of Aging in Cultured Nervous System Tissue-DONALD H. SILBERBERC AND Protoplast Fusion and Somatic Hybridizationh 0 SCHIEDER AND INDRA K. VASlL SEUNGU. KIM Genetic Modification of Plant Cells Through Aging of Adrenocortical Cells in Culture-PE1. KADOAND Uptake of Foreign DNA<. TER J . HORNSBY, MICHAELH. SIMONIAN, AND GORDONN. GILL A. KLEINHOFS
340
CONTENTS OF RECENT VOLUMES AND SUPPLEMENTS
Nitrogen Fixation and Plant Tissue CultureKENNETH L. GILESA N D INDRA K. VASIL A. Preservation of Germplasm-LuNosEv WITHERS lntraovarian and in Vitro Pollination-M. ZENKTELER Endosperm Culture-B. M. JOHRI,P. S . SRIA N D A. P. RASTE VASTAVA, The Formation of Secondary Metabolites in Plant Tissue and Cell Cultures-H. BOHM Embryo Culture-V. RAGHAVAN The Future-CkoRc MELCHERS
Supplement 13: Biology of the Rhizohiaceae The Taxonomy of the Rhizobiaceae4ERALD H. ELKAN Biology of Agrobacterium tumefaciens: Plant Interactions-L. W. MOORE A N D D. A. COOKSEY Agrobacterium tumefaciens in Agriculture and Research-FAwzi EL-FIKIA N D KENNETHL. GlLES
Suppression of, and Recovery from, the Neoplastic State-ROBERT TURGEON Plasmid Studies in Crown Gall TurnorigenesisSUBJECT INDEX A N D RICK L. STEPHENL. DELLAPORTA PESANO Supplement 12: Membrane Research: Classic The Position of Agrobacterium rhizogenesJESSEM. JAYNES AND GARYA. STROBEL Origins and Current Concepts Recognition in Rhizobium-Legume Symbioses,-TERRENCE L. GRAHAM Membrane Events Associated with the Generation of a BhStOCYSt-MARTIN H. JOHNSON The Rh;zobiurn Bacteroid State-W. D. SuTTON, C. E. PANKHURST, A N D A. S . CRAIG Structural and Functional Evidence of Cooperativity between Membranes and Cell Wall in Exchange of Metabolites and Energy between Legume and Rhizobium-JOHN IMSANDE BaCteria-MANFRED E. BAYER KONPlant Cell Surface Structure and Recognition The Genetics of Rhizobium--ADAM DOROSI A N D ANDREW W. B. JOHNSTON Phenomena with Reference to SymbiosesIndigenous Plasmids of Rhizobium-J. DEENS. REISERT PATRICIA ARIEE, P. BOISTARD,FRANCINE CASSEMembranes and Cell Movement: Interactions of DELBART, A. G. ATHERLY, I. 0. BERRY, Membranes with the Proteins of the AND P. RUSSELL Cytoskeleton-JAMES A. WEATHERBEE Electrophysiology of Cells and Organelles: Nodules Morphogenesis and DifferentiationWILLIAM NEWCOMB Studies with Optical Potentiometric IndicaMutants of Rhizobiurn That Are Altered in A N D PHILIP c. tOTS-JEFFREY c. FREEDMAN Legume Interaction and Nitrogen FixationLARIS L. D. KUYKENDALL Synthesis and Assembly of Membrane and Organelle PrOteinS-HARVEY F. LODISH, The Significance and Application of Rhizobium WILLIAM A. BRAELL,ALANL. SCHWARTZ, in Agriculture-HAROLD L. PETERSON AND THOMASE. LOYNACHAN GER J. A. M. STROUS, A N D ASHER INDEX ZILBERSTEIN The Importance of Adequate Fixation in Preservation of Membrane UltrastmctureRONALDB. LUFTIGAND PAUL N. Mc- Supplement 14: Intracellular Symbiosis MILLAN Liposomes-As Artificial Organelles, To- Some Eco-evolutionary Aspects of lntracellular Symbiosis-F. J . R. TAYLOR pochemical Matrices, and Therapeutic Carrier SyStemS-PETER NICHOLLS Integration of Bacterial Endosymbionts in Amoebae-KwANc W.JEON Drug and Chemical Effects on Membrane Transport-WILLIAM 0.BERNDT Perspective on Algal Endosyrnbionts in Larger FOraminifera-JOHN J. LEE INDEX
CONTENTS OF RECENT VOLUMES AND SUPPLEMENTS
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The Biology of the Xenosome, an Intracellular Supplement 15: Aspects of Cell Regulation Symbiont-A. T. SOLDO Endosymbionts of Euplores-KLAUS HECK- Cellular Factors Which Modulate Hormone ReMANN sponses: Glucocorticoid 4ction in PerspectiVe-ROBERT w . HARRISON, 111 Endonuclear Symbionts in Ciliates-HANs-DIETER GORTZ Regulation of Genetic Activity by Thyroid HorMetabolic Interchange in Algae-Invertebrate mones-A. ABDUKARIMOV SymbiOSiS
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