International Review of Cytology, A Survey of Cell Biology, Vol. 191

VOLUME 191

SERIES EDITORS Geoffrey H. Bourne James F. Danielli Kwang W. Jeon Martin Friedlander Jonathan Jarvik

1949-1 988 1949-1 984 19671984-1 992 1993-1 995

EDITORIAL ADVISORY BOARD Eve Ida Barak Rosa Beddington Howard A. Bern Robert A. Bloodgood Dean Bok Stanley Cohen Rene Couteaux Marie A. DiBerardino Laurence Etkin Hiroo Fukuda Elizabeth D. Hay P. Mark Hogarth Anthony P. Mahowald

M. Melkonian Keith E. Mostov Andreas Oksche Vladimir R. Pantic Jozef St. Schell Manfred Schliwa Robert A. Smith Wilfred D. Stein Ralph M. Steinman M. Tazawa Donald P. Weeks Robin Wright Alexander L. Yudin

Edited by

Kwang W. Jeon Department of Biochemistry University of Tennessee Knoxville. Tennessee

VOLUME 191

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Front cover photograph: Electron micrograph of axon terminals bordering the perivascular space which surrounds capillaries of the primary portal plexus in the neurohypophyseal median eminence. (For more details, see Chapter 5, Figure 1.)

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CONTENTS

Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ix

Role of Activin and Other Peptide Growth Factors in Body Patterning in the Early Amphibian Embryo Makoto Asashima, Kei Kinoshita, Takashi Ariizumi, and George M. Malacinski I. II. Ill. IV. V. VI. VII. VIII.

Introduction . . . ................................... Mesoderm-Inducing Factors and Their Modifiers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effects of Activin on the Regional Expression of Specific Axial Patterning by Activin(s) . . . . . . . . . . . . . . . . . . . . . . . Life History of Activin Signaling Mechanisms of the Embryo . . . . . . . . . . . . . . Proposed Molecular Models for Activin's Role in Signal Tr Activin Causes a Broad Array of Dlfferentiations in Vitro ........................ Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............ References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......

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38 40 42

Calcium Regulation of the Actin-Myosin Interaction of Physarum polycephalum Akio Nakamura and Kazuhiro Kohama I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Calcium Inhibition of Motile Events Related to Actornyosin ...................... 111. Calcium Inhibition of the Actin-Myosin Interaction of Physarum as Detected In L/ltro.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Ca-Binding Properties of Physarum Myosin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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CONTENTS

Phosphorylated State and Calcium Inhibition of Physarum Myosin . . . . . . . . . . . . . . . . Actin-Binding Proteins of Physarum That Are Involved in .............................. Calcium Inhibition . . . . . . . . . . . . . VII. Ca-Binding Proteins in Physarum .................... VIII. Ameba1 Myosin and Arnebal-Plas .................... IX. Concluding Remarks . .................... References . . . . . . . . . . . ................................. V. VI.

72 77 03 00 90 92

Characteristics of Skeletal Muscle in Mdx Mutant Mice Sabine De La Porte, Sophie Morin, and Jeanine Koenig I. II. 111. IV. V. V.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . Animal Models . . . . . . . . . . . . . . . . . . . . . . . . Dystrophin, Utrophin, and Associated Proteins Mdx Muscle Cells . , , , Therapeutic Projects . . .......... Concluding Remarks . . .......... References . . . . . . . . .

Regulation of Phosphate Transport and Homeostasis in Plant Cells Tetsuro Mimura

111.

Distribution of Inorganic Phosphate

V. Homeostasis and Detection of Pi Status in Plant Cells VI. Concluding Remarks

.......... .......... ............... ............... ...............

149 151 152 157 184

...............

194

Synaptic-like Microvesicles in Mammalian Pinealocytes Peter Redecker I. II. 111. IV. V.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Mammalian Pineal Organ: A Mediator of Darkness . . . . . . . . . . . . . . . . . . . . . . . . . UltrastructuralObservations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Molecular Aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Emergence of Functional Concepts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

201 203 205 21 1 224

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CONTENTS

VI. VII.

Biogenesis of Synaptic-like Microvesicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding Remarks and Future Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

236 237 241

Invertebrate Integrins: Structure, Function, and Evolution Robert D. Burke I. 11. 111. IV.

Introduction ............................ Invertebrate lntegrins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Evolution of lntegrins . . . . . . . . . . . . . . . . . . . . Concluding Remarks ..................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

257 259 266 281 281

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Numbers in parentheses indicate the pages on which the authors' contributions begin.

Takashi Ariizumi (1)1 CREST Project, University of Tokyo, Komaba, Meguro, Tokyo 153, Japan Makoto Asashima (l), Department of Life Science and CREST Project, University of Tokyo, Komaba, Meguro, Tokyo 153, Japan Robert D. Burke (257),Department of Biology, University of Victoria, Victoria, British Columbia, Canada V8W 3N5 Sabine De La Porte (99), Laboratoire de Neurobiologie Cellulaire et Mo/eculaire, CNRS UPR 9040, 91 198 Gif sur Yvette Cedex, France Kei Kinoshita (l), CREST Project, University of Tokyo, Komaba, Meguro, Tokyo 153, Japan Jeanine Koenig (99), lnstitut de Myologie, Groupe Hospitalier Pitie-Salpetriere, 75651 Paris Cedex 13, France Kazuhiro Kohama (53),Department of Pharmacology, Gunma University School of Medicine, Maebashi, Gunma 371-8511, Japan George M. Malacinski (1), Department of Bio/ogy, Indiana University, Bloomington, Indiana 47405 Tetsuro Mimura (149),Biological Laboratory, Hitotsubashi University, Naka 2-1, Kunitachi, Tokyo 186-8601, Japan Sophie Morin (99), Laboratoire de Neurobiologie Cellulaire, Universite de Bordeaux 11, 33405 Talena Cedex, France Akio Nakamura (53) Department of Pharmaco/ogy,Gunma University School of Medicine, Maebashi, Gunma 371-8511, Japan Peter Redecker (201), Department of Anatomy 1, Hannover Medical School, 0-30625 Hannover, Germany ix

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Role of Activin and Other Peptide Growth Factors in Body Patterning in the Early Amphibian Embryo Makoto Asashima,*lt Kei Kinoshita,t Takashi Ariizumi,t and George M. Malacinski$ *Department of Life Science and ?CREST Project, University of Tokyo, Komaba, Meguro, Tokyo 153, Japan, and $Department of Biology, Indiana University, Bloomington, Indiana 47405

The amphibian body plan is established as the result of a series of inductive interactions. During early cleavage stages cells in the vegetal hemisphere induce overlying animal hemisphere cells to form mesoderm. The interaction represents the first major body-patterning event and is mediated by peptide growth factors. Various peptide growth factors have been implicated in mesoderm development, including most notably members of the transforming growth factor-p superfamily. Identification of the so-called "natural" inducer from among the several candidate peptide growth factors is being achieved by employing several experimental strategies, including the use of a tissue explant assay for testing potential inducers, cloning of marker genes as indices of early induction events, and microinjection of altered peptide growth factor receptors to disrupt normal embryonic inductions. Activin emerges as the most likely choice for assignment of the role of endogenous mesoderm inducer, because it currently best fulfills the rigorous set of criteria expected of such an important embryonic signaling molecule. Activin, however, may not act alone in mesoderm induction. Other peptide growth factors such as fibroblast growth factor might be involved, especially in the regional patterning of the mesoderm. In addition, several genes (e.g., Wnt and noggin), which are expressed after the mesoderm is initially induced, probably assist in further definition of the mesoderm pattern. Following mesoderm induction, the primary embryonic organizer tissue (first described in 1924 by Spemann) develops and contributes further to body patterning by its action as a neural inducer. Peptide growth factors such as activin may also be involved in the inductive event, either directly (by facilitating gene expression) or indirectly (by serving to constrain pathways). Inrrmurronal Review of CyruluKy. V d 191

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KEY WORDS: Mesoderm induction, Animal-cap explants, Activin, Fibroblast growth factors, Peptide growth factors, Embryogenesis. 0 1999 Academic Press.

1. Introduction The major aim of contemporary developmental biology research is to understand the mechanisms which guide formation of the body plan of a complex, multicellular animal from the single-celled egg. Inductive interactions, which involve signaling between groups of cells, are widely considered to play a fundamental role in the establishment of the basic body plan. A variety of such inductive interactions operating at different times and in different places in the embryo most likely combine to generate the changes in the overall form, shape, and regional specialization features which characterize morphogenesis. A research focus on the nature of inductive interactions began (for amphibian embryos) with the pioneering studies of Hans Spemann. Together with his student Hilde Mangold he described the role the so-called primary embryonic organizer tissue plays in orchestrating the formation of body plan in early embryos. For several decades the biochemical identity of the molecules which are responsible for the remarkable properties of the primary embryonic organizer remained unknown. Recently, dramatic progress has not only been made in identifying specific proteins (e.g., peptide growth factors) as probable inducers but also in uncovering other subcellular components (e.g., receptors) which play roles in establishing the body plan of the early embryo. In order to provide background information for understanding the role that peptide growth factors play in embryonic induction, and to appreciate the inherent complexity of the regulatory circuitry which comprises inductive interactions, this review will summarize some of the key early events which lead up to and follow the action of Spemann’s primary embryonic organizer tissue. A. Embryonic Axis Specification

Amphibian eggdembryos represent the experimental system of choice for most studies on polarization phenomena for obvious reasons: The eggs are large (approx 1-3 mm diameter), readily collected, easily manipulated, and conveniently pigmented [animal (upper) hemisphere = dark; vegetal (lower) hemisphere = light]. Originally, urodele embryos (e.g., newts and

ROLE OF ACTlVlN IN BODY PATTERNING

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axolotls) were researched, but recently anuran embryos (especially Xenopus) have become more popular as a ready source of large numbers of eggs. Most of the information included herein was collected from Xenopus studies, although comparisons of anuran inductive interactions to urodele embryos reveal strong similarities (Malacinski et al., 1996). Axis specification actually begins during oogenesis, the period during which, while in the ovary, the egg develops from a tiny, cytoplasm-poor cell to a large cell which consists mainly of yolk-laden cytoplasm. During oogenesis the oocyte becomes polarized along its animal (darkly pigmented)-vegetal (lightly pigmented) axis. Typically, the egg contains large, densely packed yolk platelets in the vegetal hemisphere and smaller, more loosely arranged yolk platelets in the animal hemisphere (Ubbels, 1977; Neff et al., 1984). The egg cytoplasm is organized in a radially symmetrical fashion about the animal-vegetal axis. That is, all meridia are identical, and each has the potential to form future dorsal or ventral structures (Fig. 1). It is in this state that a mature oocyte awaits fertilization. Fertilization breaks the egg’s radial symmetry. The entering sperm causes (i) the egg cortex to contract toward the side of the egg where the sperm entered, and (ii) the underlying cytoplasm to shift with it. This cortical rotation establishes the bilateral symmetry of the egg. The side opposite the sperm entrance site becomes the dorsal side and the sperm entrance side the ventral side (Gerhart et al., 1989). Right and left halves are thus simultaneously established vis-a-vis the animal-vegetal polarization mentioned previously. Experimental verification of this scenario has been achieved by disrupting the cortical rotation with ultraviolet irradiation and rescuing “depolarized” eggs by subsequently reorienting them 90” along the earth’s gravitational axis for a brief period (Chung and Malacinski, 1980; Elinson and Rowning, 1988; Gerhart et al., 1989). The molecular basis of dorsal-ventral polarization was speculated by Gerhart et al., (1991) to involve the activation of “dorsal determinants of polarity.” This activation is thought to occur as the vegetal hemisphere cortex encounters the animal hemisphere cytoplasm on the future dorsal side and the animal cortex meets the vegetal cytoplasm on the prospective ventral side. This speculation was further extended to encompass, as a subsequent effect of the activation reaction, the formation of a dorsoventral signaling center in the vegetal hemisphere (Nieuwkoop, 1969), previously known as the “Nieuwkoop organizing center.” This organizing center is generally believed to induce mesoderm formation in some of the equatorial cells of the early cleaving egg. Finally, the mesoderm is considered the source of an inductive signal to overlying cells, which causes them to acquire the features of Spermann’s primary embryonic organizer. Thus, establishment of body patterning is not a single-step process, even during its earliest phases. Rather, body patterning is the product of a series

(a) Unfertilized egg animal

(b) After fertilization

( c ) Morula

ventralwdorsal

(d) Blastula ventral ectoderm

------

]mesoderm DV marginal zone


P

cortical rotation

(e) Late blastula

(f)

endoderm

mesoderm induction

Gastrula - neurula

(g) h - v a

sumptive neural regi

dorsalization &

neural induction

ventralization FIG. 1 Early events in amphibian (e.g., Xenopus) development. The unfertilized egg is radially symmetrical about the animal (darkly pigmented hemisphere)-vegetal (lightly pigmented hemisphere) axis (a). Rotation of the egg cortex driven by the fertilizing sperm breaks that radial symmetry and establishes a dorsal-ventral axis prior to the first cleavage division (b). This event represents the beginning of body patterning. Mesoderm induction occurs during early cleavage stages (c) and presumptive dorsal and ventral mesoderm regions are determined by the early blastula stage (d; shown in cross section in e). Neural induction and dorsalization occur progressively during gastrulation (f [cross section]). Following early inductions. cell differentiations establish the regional features of the larva (g).

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of events, many of which involve cell-cell signaling and are hence referred to as “inductive interactions.” Most probably, the key outcome of these early inductive interactions is the development of mesoderm cells (Tiedemann et al., 1996; Asashima et al., 1997).

B. Mesoderm Induction Presumptive endoderm cells play the key role in inducing the overlying cells to develop into mesoderm. This important fact was established by a series of classic tissue recombination experiments (Nieuwkoop, 1969). In isolation, explanted pieces of animal and vegetal-pole cells form only ectoderm and endoderm, respectively. However, when animal and vegetal cells are combined and cocultured, various mesodermal tissues differentiate within the animal cap’s progeny cells. This observation indicates that vegetal hemisphere (endoderm) cells produce a signal which induces equatorial zone (animal hemisphere) cells to form mesoderm. Specifically, vegetal endoderm induces (in tissue recombinants) a dorsal-ventral patterning in responding equatorial cells (Nieuwkoop, 1969; Dale and Slack, 1987). Dorsal-vegetal cells induce dorsal mesoderm, including muscle, notochord, and Spemann’s organizer. In fact, when a dorsal-vegetal blastomere is transplanted to the prospective ventral side of the early embryo, it acts as a Nieuwkoop organizing center and induces ectopic axial structures. Conversely, ventral-vegetal cells induce ventral mesodermal features, such as mesothelium and blood cells. Thus, the vegetal hemisphere of the egg contains a signaling system, presumably inherited from oogenesis, which, once activated, is responsible for programming mesoderm pattern in overlying cells. C. Activation of the Zygote Nucleus

The foregoing events which lead to mesoderm formation take place, remarkably, in the absence of any zygote nucleus transcription. These events occur during the early cleavage stages, when the embryo consists of 32,64, or 128 cells. It is at approximately the 12th cleavage (-10,000 cells) that the embryo’s genome is transcribed for the first time following fertilization. At the 12th cleavage, the so-called midblastula transition (MBT), the synchrony of exponential cell division ends, the adhesiveness of cells increases, and various genes are transcribed (Newport and Kirschner, 1982a,b). These facts imply that the zygotic genome is activated and that cell differentiation begins thereafter. Some of this gene expression is presumably responsible for body patterning in the embryo.

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D. Neural Induction Following mesoderm induction, a second major inductive event occurs: anterior-posterior body patterning and specification of the central nervous system (CNS). During the cell movements of gastrulation, axial mesodermal structures are formed from the dorsal marginal zone. This region is the leading edge of the involuting tissue and undergoes convergent extension along the future anteroposterior axis. As a result of these cell movements, three so-called germ layers can be recognized in the embryo: ectoderm (primarily on the embryo’s surface), mesoderm (“middle” zone of tissue), and endoderm (yolk-laden “inner” mass of cells). These cell rearrangements set the stage for CNS development because the CNS originates from an interaction between dorsal ectoderm and involuting dorsal mesoderm. Neural development was first studied extensively by Spemann and Mangold (1924) in newt embryos. Their early discoveries led to the notion that dorsal mesoderm acts vertically on overlying ectodermal cells to induce (i.e., specify) neuroectoderm. This inductive interaction is both powerful and dramatic, as they demonstrated by the following tissue transplantation procedure. When the dorsal mesodermal tissue from the vicinity of where gastrulation begins (blastopore lip region) is transplanted into the ventral side of a host embryo, a complete second body axis develops (Asashima et al., 1998). The transplanted tissue becomes head mesoderm and notochord, whereas the host’s ventral tissue becomes organized into primary axial structures such as somites and neural cells. Hence, the term “primary embryonic organizer” was coined t o describe the dorsal mesoderm cells in the blastopore lip region. Organization of a secondary axis is of course more complicated than just described. For example, the blastopore lip from an early gastrula-stage donor induces mostly head structures. When taken from a late gastrulastage donor, the blastopore lip induces mainly trunk and tail structures. Thus, the early lip has been termed “head organizer” and the later lip the “trunk/tail” organizer. This distinction was further refined when Mangold (1933) demonstrated that dorsal mesoderm from different anteroposterior positions displayed varying inducing properties. When such different tissue pieces were implanted into the blastocoel of recipient embryos, the portions of a secondary embryo (e.g., neural tissue) which were induced corresponded to the anteroposterior location of the donor (implanted) tissue. Taken together, these results indicated that a “vertical signaling system” is most likely responsible for the natural inducing properties of the primary embryonic organizer. The signal was presumed (by intuition) to be diffusible. Its biochemical identification has of course been intensely pursued,

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and the the search for natural inducing molecules has been popular for contemporary developmental biologists. In addition to vertical signaling, recent evidence indicates that signals also travel horizontally within the plane of the ectoderm (Doniach et al., 1992). The existence of this “planar signaling system” was recognized only recently, mainly because the extent of morphogenesis which occurred during various exogastrula experiments with newt embryos could be interpreted without invoking such a second signaling system. Recent data with molecular markers indicate that a planar signal emanates from the organizer region (of Xenopus) embryos and moves to the animal hemisphere ectoderm (Dixon and Kintner, 1989). Although weaker than the vertical signal, this second signal is believed to induce neural tissue and to specify the anteroposterior pattern in the neural plate. Working in the opposite direction (backwards from the primary embryonic organizer toward a portion of the presumptive mesoderm) another signaling system is postulated to exist. It “dorsalizes” part of the mesoderm and thereby changes the specification of lateral mesodermal tissues from ventral (e.g., blood and mesenchyme) to more dorsal fates (e.g., muscle, heart, and pronephros). It is believed to act during gastrulation (Slack, 1994).

II. Mesoderm-Inducing Factors and Their Modifiers Having interpreted the data from classical embryological studies on inductive phenomena to indicate the existence of diffusible signal molecules, research has proceeded in earnest in order to isolate, characterize, and identify the proteins, RNAs, or other molecules which comprise these signaling systems. The “mesoderm-inducing’’ factors (MIFs) have been especially sought after (Smith, 1993; Asashima, 1994) because they are believed to play pivotal roles in establishing the subsequent pattern-specification events. As is typically the case for such purification endeavors, the source of starting material and the nature of the assay systems have been crucial components in the research strategies.

A. Identification of Factors: Bioassay Procedures The most widely used bioassay in present-day research employs pieces of presumptive ectodermal tissue excised from the animal cap of a blastula-

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stage embryo (Fig. 2a). These pieces are bathed in a solution of potential inducing substance, cultured, and examined for differentiated features. Typically, for the assay of mesoderm-inducing activity Xenopus stage 8 or 9 blastulae are employed. This assay is usually referred to as an “explant” assay or more specifically as the “animal cap” assay. The explanted tissue is ordinarily cultured for 2 days. In the absence of any added “inducing substance” it forms a cell cluster, more or less spherical in shape, which rotates in the culture fluid through the action of cilia which grow out of the surface cells. This tissue mass shows no evidence of cytodifferentiation and is referred to as “atypical epidermis.” It serves as a “control” for actual assays of prospective inducing substances. By soaking animal caps in a range of concentrations of inducing substances, the relative potency of a given substance can be compared with the activity of other substances.

(a) Animal cap assay

inactive

atypical epidermis mesoderm formati on

(b)RNA injection assay

< inactive active

(c) RNA injection assay + animal cap assay inactive

atypical epidermis

.--)

mesoderm formation FIG. 2 Bioassay procedures for morphogenetic substances such as MIFs. (a) The animal cap of a blastula-stage embryo (c/s) is cultured in a MIF and examined for histological differentiation features. (b) a MIF mRNA is injected directly into the early embryo. (c) Tissue from an injected embryo is cultured.

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FIG. 3 Animal cap assay for MIFs, using stage 8 or 9 Xenopus embryos as donors of the animal cap tissue. It is bathed in saline solutions containing various concentrations of MIFs (in this example, activin). Control tissue develops as a cluster of epidermis, whereas treated cultures display a range of differentiation patterns. Note: Activin and retinoic acid also act synergistically on chick blastoderm cells (Knezevic et u/., 1995). ILl1, interleukin 11; SCF, stem cell factor: RA. retinoic acid.

Figure 3 illustrates the animal cap assay and provides descriptions of typical results obtained with activin. Explants treated with an active inducing substance usually elongate as they form a vesicle. Their surface becomes smooth (rather than wrinkled, as is the case for control explants). Accurate scoring is best achieved when a combination of histological techniques and molecular probe analyses are paired. Figure 4 illustrates such scoring data. The competence of animal cap cells to respond to mesoderm inducing substances recedes by the gastrulation stage. The capacity of animal cap cells to respond to neural induction signals increases, however, concomitant with the decrease in mesoderm-forming ability. If a prospective neuralinducer substance is indeed active, neural differentiation features can be recognized in the cultured explant. Other bioassays have also been employed, including coagulating a prospective inducing substance with an inert protein such as bovine serum albumin and implanting it into the blastocoel of a recipient embryo, much the same way tissue explants have been inserted into host embroys. For mRNAs, injection directly into the blastocoel has also been employed (Figs. 2b and c). Reviews by Ariizumi and Asashima (1995a) and Asashima et al. (1998) describe various bioassay procedures in detail.

FIG. 4 Combined histological and molecular analysis of the effects of activin on animal cap explant. (a) Section of control (atypical epdermis) shows no differentiation of mesoderm features. (b) Section of activin-treated explant (mus. muscle; epi, epidermis; neu, neuron). (c) Immunofluorescence staining of a section of the explant shown in b with an antibody specific to muscle myosin. (d) Expression of muscle-specific actin gene assayed by RNase protection assay. Lane 1, control explants; lane 2, explants cultured for 10 h after activin treatment; lane 3, explants cultured for 20 h after activin treatment (reproduced from Asashima, 1994).

ROLE OF ACTlVlN

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In the early years of the search for inducing substances (using these bioassays) a number of what were then termed “vegetalizing factors” were recognized in extracts from such diverse sources as chick embryo extract, guinea pig bone marrow, and carp swim bladder. Today, some of these vegetalizing factors have been reexamined and, as will be detailed later, correspond to activin, a well-described and authentic MIF (Asashima et al., 1991a,b; Tiedemann et al., 1996).

B. Rigorous Criteria for Identifying the “Natural” Inducer As will be discussed later, the various bioassays have served to identify a variety of substances as inducers. Most but by no means all of them are “peptide growth factors.” They each have at least some capacity to induce animal cap explants to differentiate into one or another type of mesodermal tissue. Thus, choosing from among them the best candidate(s) for designation as the natural (endogenouslin vivo) inducer molecule(s) becomes both an experimental and an intellectual challenge. Candidate MIFs for assignment as a natural inducer should meet most of the following minimal experimental criteria: exist in the embryo (as either protein or mRNA) in adequate concentrations prior to the onset of mesodermal induction (morula stage, as described by Jones and Woodland, 1987); cause animal cap cultures to differentiate distinct mesodermal features; convert nonmesoderm cells (e.g., animal cap) into mesodermal tissue as the result of experimental loading of the egg with either prospective inducer protein or mRNA; and interruption of the MIF’s signaling pathway (e.g., by interfering with its receptor) should inhibit mesoderm differentiation. Additional criteria might also be employed, such as employing MIF maternal effect mutants of amphibia to demonstrate that mesoderm differentiation is erased in the absence of specific MIFs. Such absolute criteria, however, are not practical yet since amphibian developmental genetics is still in its infancy. Nevertheless, using as many criteria as possible should aid in distinguishing between bona fide natural inducing substances and those factors which serve instead to modify various inductive responses. Also, these criteria will help establish the extent to which combinations of signaling systems act in an inductive event instead of just one individual signal molecule. Finally, these criteria provide for intellectual challenge. If more than one signal molecule meets the criteria, and if combinatorial action is suspected, the issue of the identity of the natural inducer might need to be reframed in terms of multiple inducers, overlapping signaling systems, or redundancy in signal pathways.

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Initially, however, it is perhaps most useful to frame the issue in the simplest terms, such as “Which of the candidate MIFs is most likely endogenous inducer?” With this approach in mind, a number of candidate molecules which have been proposed to function to different extents as endogenous (natural) inducing molecules will be reviewed. C. Characteristics of Proteins Which Induce Mesoderm Differentiation

Various proteins have been purified and/or identified which act in the bioassays mentioned previously to induce mesoderm. Several of these will be individually reviewed, and then an assessment will be attempted in order to determine which one(s) most likely is the natural (endogenous) inducing factor. In the early experiments a number of crude extracts and partially purified “factors” were demonstrated to have mesoderm-inducing activity. These included one MIF extracted from Xenopus gastrulae by Faulhaber (1972). It was characterized as having a molecular mass of between 65 and 115 kDa. Being unstable and relatively low in activity, it was not purified further. For these reasons and others, most of the earlier extraction procedures employed heterologous sources of starting material. For example, chick embryo extract (Geithe et al., 1981), mammalian liver and bone marrow (Toivonen and SaxCn, 1955; Hayashi, 1956; Yamada, 1962), and carp swim bladder (Kawakami, 1976; Asashima et al., 1986) were used. Although never purified to homogeneity, these factors were acknowledged to be highly active as mesoderm inducers, and their use to quantitate mesoderm induction served to set the stage for recent and much more highly refined studies. The impact of these investigations has been largely superseded by the discovery that various peptide growth factors, some directly available as pure preparations from commercial supply houses, have MIF activity. These include, most notably, fibroblast growth factors (FGFs) and activins [members of the transforming growth factor+ (TGF-P) superfamily]. They are reviewed below:

1. Fibroblast Growth Factors FGF is the first of the peptide growth factors to have been recognized as having MIF activity in the Xenopus animal cap assay. Slack et al. (1987) reported that heparin-binding basic fibroblast growth factor (bFGF) obtained from bovine brain possessed MIF activity and thereby began a search for other peptides with MIF activity. Animal caps incubated in a solution

ROLE OF ACTlVlN IN BODY PATTERNING

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of 5-20 ng/ml bFGF differentiated preferentially into ventral mesoderm tissues such as mesothelium and blood cells. Only minor amounts of dorsal mesodermal muscle mass and, in fact, no notochord were observed in histological sections. Thus, FGF induction of mesoderm is incomplete, and the existence of other MIFs was therefore postulated. Nevertheless, FGF research continued. Transcripts and proteins corresponding to several forms of FGF and FGF receptors (also proteins) were discovered to be maternally expressed. Kimelman et al. (1988) demonstrated that bFGF mRNA exists in unfertilized eggs, disappears during cleavage, and reappears at the neurula stage. Western blotting using antiFGF antibody detected a 15-kDa peptide in a heparin-bound fraction of unfertilized eggs. Slack and Isaacs (1989) found a 14- to 19-kDa bFGF peptide in the ovary and early embryo in a high enough amounts to account for ventral mesoderm induction. In addition, Shiurba et al. (1991) demonstrated immunohistochemically that both bFGF and acidic FGF exist in the embryo throughout oogenesis and up until the blastula stage. Staining was faint in the animal hemisphere but very obvious in the equatorial region. Thus, there is little doubt that FGF is present in the uncleaved egg (and therefore fulfills one of the key criteria of an endogenous inducer; see Kimelman et al., 1988). The gene for the receptor of Xenopus bFGF was first cloned by Musci et al. (1990). Amaya et al. (1991) reported that expression of a dominant inhibitory bFGF receptor protein caused malformation of the posterior and lateral region of the embryo, without severely affecting anterior development. The case for bFGF as a natural inducer was thereby further strengthened. A number of studies indicate that bFGF also plays a role in the induction of neural tissue in the ectoderm of gastrula stage embryos (Kengaku and Okamoto, 1993; Lamb and Harland, 1995). These studies imply that bFGF might comprise at least part of the primary embryonic organizer activity. Data collected by Slack et al. (1992) suggested, however, that int-2 and embryonic FGF (eFGF), which possess signal sequences, are better endogenous inducer candidates than bFGF. eFGF is maternally expressed in Xenopus and is a potent MIF. It is most highly expressed in the mesoderm during gastrulation, so it is more likely to be involved in induction processes which occur later than the events associated with early mesoderm specification. In fact, it has been implicated in maintaining the properties of the mesoderm in the gastrula stage (Slack et nl., 1992; Kengaku and Okamoto, 1993; Isaacs et al., 1994). 2. Activins

Activins were recognized as having MIF activity shortly after FGF was implicated in embryonic induction. In 1987, Smith reported that conditioned

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culture medium from a Xenopus tadpole cell line (XTC cell) could transform ectoderm to mesoderm. This MIF was characterized as being a heatstable protein of approximately 16 kDa. [It would later be identified as the peptide growth factor activin (Smith et al., 1990)l. Animal cap explants treated with this MIF differentiated into dorsal mesoderm, including muscle and notochord (vs FGF, which induced ventral mesoderm and did not include these two tissues). At about the same time that the XTC MIF was generating enthusiasm, Kimelman and Kirschner (1987) reported that another peptide growth factor, TGF-P1, which alone has no mesoderm-inducing activity, acts synergistically with bFGF to induce dorsal mesodermal tissues. Complicating hopes for a simple, straightforward identification of the natural inducer was the report of Rosa (1989), which demonstrated that yet another peptide growth factor, TGF-/32, possessed mesodermal-inducing activity. These studies also demonstrated that the XTC MIF and TGF-02 have similar biological properties. Taken together, these data suggest a model in which the endogenous primary embryonic organizer activity is composed of a combination of peptide growth factors, including FGF (which induces ventral mesoderm) and TGF-P (which induces dorsal features of the mesoderm). While these models were being discussed, Asashima’s laboratory succeeded in extracting a potent MIF from culture media conditioned by mammalian cell lines (Nakano et al., 1990). It was activin, and it unmistakably induced dorsal mesoderm (Fig. 3 and 4). The structure of activin is illustrated in Fig. 5. It is a dimer and exists in three subunit configurations (A, B, and AB). Each subunit is 13 kDa in size. It is a member of the TGFP superfamily (Ling et al., 1986; Eto et af., 1987), which also, of course, includes TGF-PI and -P2 mentioned previously. The activins were originally extracted during the purification of inhibins (A and B) from mammalian ovarian follicular fluid (Ling et af., 1986). Activins actually share common subunits with inhibins, which are composed of an A and PAor PB subunit, as illustrated in Fig. 5. Activin A consists of two inhibin PAsubunits. Despite the structural homologies of activins and inhibins, their biological effects are antagonistic. Activins are gonadal proteins which stimulate the release of pituitary follicle-stimulating hormone (FSH). Activins are also equivalent to erythroid differentiation factor (EDF) (Asashima et af., 1990a,b). Inhibins are produced by the gonads and serve to suppress the release of FSH. In the animal cap assay these two proteins are not antagonistic, however. Using purified human recombinant activin A and inhibin A, Asashima et al. (1989, 1990a) demonstrated that although activin A induces mesoderm at concentrations <1 ng/ml, inhibin A does not. Inhibin A, when mixed with activin. did not interfere with its action.

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FIG. 5 Structure of TGF-P superfamily proteins. Activins are dimers of A, B. or A/B subunits. Inhibins are composed of one of the same subunits found in activins and a second subunit. TGFP proteins contain seven cysteine residues, which are well conserved throughout the family.

The Xenopus genome contains several activin-related genes, as revealed when a genomic library was screened with a human activin cDNA probe (Ueno et al., 1991). Thus, it is perhaps not surprising that a Xenopus cell line (XTC) was discovered to secrete an activin-like protein (Smith et af., 1990). Also, the discovery of multiple genes encoding activin broadens the scope of the debate over which, if any, of the peptide growth factors fully qualify as the natural inducer. Using cDNA probes, Thomsen et al. (1990) examined Xenopus embryos for the presence of mRNAs which code for the activin PA and activin PR subunits. mRNA for the PB chain first appears in blastulae, whereas PA chain mRNA does not appear until the late-gastrula stage. In both instances, therefore, their appearance is too late to be considered a part of the normal mesoderm signal system. Recently, however, a Xenopus cDNA which codes for a new member of the TGF-P superfamily, activin PD, has been isolated and used to probe various embryonic stages (Oda et al., 1995). PD mRNA has been identified in unfertilized eggs and early embryonic stages. Interestingly, it is uniformly distributed throughout the early embryo rather than being concentrated in one or another region of the embryo. Microinjection of a synthetic Po mRNA into ventral blastomeres induced the forma-

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MAKOTO ASASHIMA E T AL.

tion of a partial secondary body axis (see Fig. 2b). Also, injection of the synthetic mRNA into eggs, from which the animal cap was later dissected out and cultured alone, revealed that activin PD is a powerful inducer (Fig. 2c). Activin protein(s) is also present in Xenopus eggs and early embryos. Its existence in blastulae was first suggested by van den Eijnden-Van Raaij et al. (1990). Asashima et al. (1991~)succeeded in extracting activin directly from early Xenopus embryos. Using thousands of unfertilized eggs or blastulae as the starting material for high-performance liquid chromatography fractionation, Asashima and coworkers found some fractions that exhibited the retention times of activin A and gave positive results in the animal cap assay. Identification of these fractions as bona fide activin was also demonstrated because they were inhibited by follistatin (an activin-binding protein) and shown to possess EDF activity. Approximately 1pg of activin, quantitated with an E D F assay, was found in a single egg (which corresponds to approximately 0.5 ng/ml of egg cytoplasm). Recently, the Asashima laboratory succeeded in extracting three types of activin (A, AB, and B; see Fig. 5 ) from early Xenopus embryos (stages 1-5) (Fukui et al., 1994). Thus, the important criterion for assignment of “endogenous inducer” status-the presence of adequate amounts in the early embryo-has clearly been met for activin. Additional support for this status has been provided by studies in which the activin receptor protein was manipulated. HemmatiBrivanlou and Melton (1992) reported that overexpression of the receptor in the early embryo results in the formation of ectopic dorsal axial structures. Furthermore, introduction of a defective (truncated) receptor inhibited mesoderm induction/formation of axial structures in these studies. As straightforward as these data might appear, they have been challenged by Schulte-Merker et al. (1994) on the grounds that truncated activin receptors might interact with and inhibit the function of related, nonactivin members of the TGF-/3 family such as Vgl. 3. Vgl as a Mesoderm Inducer

Vgl is a maternally inherited protein which is localized to the vegetal hemisphere of Xenopus eggs (Melton, 1987). Its homology to members of the TGF-P family suggests that it might function as a natural MIF. Several features of its biology and biochemistry are problematic for this role. Expression of Vgl protein in the animal hemisphere following injection of synthetic mRNA has no effect on development. Vgl protein also fails to form dimers and is not processed to release the biologically active form of the peptide. Furthermore, it has been demonstrated that the N-terminal signal peptide of Vgl is not cleaved following translocation into the endoplasmic reticulum, which may explain the failure to dimerize. Thus, the

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17

role Vgl plays in early embryonic pattern formation remains obscure. More promising data for assigning Vgl an important role as an endogenous MIF has been obtained by Kessler and Melton (1995). They injected Xenopus oocytes with achimeric activin-Vgl mRNA, which encodes the proregion of activin Pe fused to the mature region of Vgl, and observed processing and secretion of mature Vgl. Treatment of animal pole explants with this mature Vgl protein yielded differentiation of dorsal mesodermal tissues. Therefore, Vgl, under somewhat “contrived” experimental conditions, displays features which make it a candidate for natural inducer status.

D. Factors Which Modify the Action of MlFs on Mesoderm Patterning Microinjection experiments, in which a mRNA or protein known to have effects on patterning in other organisms (e.g., Drosophila) is injected into Xenopus embryos, have been very revealing. These experiments have indicated that a variety of signaling molecules, other than peptide growth factors, may play a role in mesoderm patterning, especially by modifying the response of cells of MIFs. These signaling molecules have been termed “modifiers” by Moon and Christian (1992) since they act on mesoderm patterning and do not have mesoderm-inducing activity. Some of these modifiers do not dorsalize the mesoderm pattern, but instead ventralize it in a manner that results in a diminished capacity to form notochord (Harland, 1994). Several, but by no means all, of these modifiers will be briefly reviewed in the following sections.

1. Wnts and Their Downstream Gene Products Wnts represent a family of related glycoproteins found in both vertebrates and invertebrates. They were first identified when a protooncogene (int-I) was identified as being activated in response to proviral insertion of a mouse mammary tumor virus. Then an int-1 homolog was recognized in Drosophila wingless mutants, and the term Wnts was coined as an amalgam of in?and wingless. McMahon and Moon (1989) reported that ectopic expression of mouse Wnt-1 in Xenopus embryos generates a set of secondary axial structures. The Xenopus homolog of Wnt-Z (Xwnt-8)has also been cloned, and it has been demonstrated that microinjection of its mRNA into ultraviolet-ventralized embryos can rescue them (Sokol et al., 1991; Smith and Harland, 1991). Thus, a normal role for a Wnt gene in formation of either the Nieuwkoop center or the Spemann’s primary embryonic organizer is thereby suggested. However, Xwnt-8 is normally expressed on the ventral side of gastrula embryos (Christian et af., 1991) and therefore is perhaps

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best conceptualized in terms of a ventral mesoderm patterning morphogen. This notion is reinforced by the observation that ectopic expression of Xwnt-8 in organizer cells after the midblastula transition by injection of plasmid DNA ventralizes the fate of these cells (Christian and Moon, 1993). Since similar effects have also been reported for lithium (Yamaguchi and Shinagawa, 1989), it can reasonably be speculated that Wnt proteins act by repressing the IP-3 cycle in the same manner that lithium represses it. Two other Xenopus Wnts, Xwnt-1 and Xwnt-3A, have been shown to possess potent dorsal axis inducing activity. However, neither are known to be expressed maternally (Wolda et al., 1993). Other Xenopus Wnts, including Xwnt-SA, Xwnr-11, and Xwnt-8b, have nevertheless been discovered to be maternally expressed (Moon et al., 1993; Ku and Melton, 1993; Cui et al., 1995). However, incomplete interpretations are encountered here: Xwnt-SA and Xwnr-11 display relatively weak activity. Neither can for example, induce a complete body axis. Injected embryos actually generate defects in dorsoanterior structures without inducing a secondary axis. Xwnt11 mRNA is found in the oocyte vegetal cytoplasm and is accumulated at high levels in the dorsal marginal zone in the late blastula, in accordance with expectations for the Nieuwkoop center. However, ultraviolet-irradiated embryos which are microinjected with Xwnt-11 lack a notochord and anterior head structures. Conversely, microinjection of Xwnt-8b can completely rescue dorsal development of ultraviolet-irradiated (ventralized) embryos (Cui et al., 1995). Xwnt-8b transcripts, however, are concentrated in prospective ectodermal cells of the animal, rather than in the vegetal hemisphere, during early cleavage stages. These transcripts, therefore, represent excellent candidates for roles in the development of competence to respond to other signaling systems such as the those of activin or FGF. Indeed, evidence exists that Wnt signals are certainly involved in multiple developmental processes in Drosophila and probably also in other organisms. The gene products of disheveled, zeste-white3/shaggy, and Armadillo act downstream of wingless (Wnt-1) signaling events. Their homolog in Xenopus have been cloned and studied. Microinjection of mRNA encoding the Xenopus Xdsh (a homolog of disheveled) protein into prospective ventral mesodermal cells triggered formation of a complete dorsal axis (Sokol et al., 1995). Dominant-negative mutant forms of the mRNA of the XGSK3 gene (a Xenopus homolog of zeste-white 3/shaggy) induced dorsal differentiation (He et al., 1995; Pierce and Kimelman, 1995), whereas wild-type XGSK-3 mRNA induced ventralization. The Drosophila protein encoded by wingless serves to signal through a pathway that antagonizes the effects of the zeste-white 3/shaggy’s serine/threonine kinase. It is also plausible that cellular responses to Xwnt signals include changes in cell adhesion. Injection of synthetic P-catenin in mRNA into the ventral side of the early Xenopus embryo causes formation of a complete body axis (Guger and

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Gumbiner, 1995; Funayama et al., 1995). P-Catenin was originally identified as a cytoplasmic cadherin-associated protein required for adherin adhesive function and has homology to the Drosophila segment polarity gene Armadillo (McCrea and Gumbiner, 1991). Wnt action may be inhibited by axin, the product of the Drosophila Fused locus. Among the pleiotrophic effects of mutations in the mouse Fused locus is the duplication of axial structures. The way in which such a dramatic effect on patterning is thought to occur is through the action of axin (protein product of the Fused locus) on the endogenous Wnt signaling pathway (Zeng et al., 1997). This interference might be effected by axin’s ability to be upregulated by a glycogen synthetase kinase-3P-mediated phosphorylation (Kishida et al., 1998) and, as demonstrated in Xenopus embryos, axin’s subsequent binding of P-catenin (Yamamoto et al., 1998). Thus, complex regulatory circuits which involve-for the formation of dorsal mesoderm-the action of several gene products likely exist. Wnt gene products are good candidates for playing roles, in coordination with peptide growth factors, in this circuitry. For example, cooperation between the activin and Wnt signaling pathways leading to expression of the Siamois homeobox gene (involved in Spemann organizer action) has recently been reported. As will be discussed later (e.g., see Fig. 8), Smad2 is phosphorylated by an activin receptor and subsequently acts as a transcription regulator. Wnt expression can enhance the role that Smad2 plays. When the activin receptor activity is experimentally inhibited, the cooperation between activin and Wnt is erased, and the expression of Siamois is substantially reduced (Crease et al., 1998). In normal embryogenesis Wnts signaling ability is inhibited by negative regulators (e.g., axin) which interrupt the Wnt signaling pathway. The interrelationships between the various pathways are therefore extensive and need to be considered more seriously when attempting to formulate models to describe the molecular mechanisms which comprise individual inductive interactions. For example, although activin alone, when experimentally applied to an ectodermal explant, is capable of programming a complete Spemann organizer (Ninomiya et al., 1998), multiple signaling pathways can be assumed to be triggered. It is unlikely that one single MIF regulates all primary organizer activity.

2. Maternal noggin Gene Products Noggin is a gene which codes for a secreted glycoprotein; therefore it, like the Wnt proteins, strictly speaking is not a peptide growth factor. Smith and Harland (1992) identified this gene in a screen for factors capable of rescuing the dorsal axis in ultraviolet-ventralized Xenopus embryos. Its transcripts are expressed uniformly in the oocyte and early embryo. When

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ectopically injected into early embryos, noggin can induce dorsal development and also modify the response of animal hemisphere cells to MIFs. The maternal noggin transcripts do not, however, appear to be translated during early cleavage stages (Schroeder and Yost, 1994), which would be expected if noggin were to be assigned a role as a natural inducer. Thus, it has been suggested that noggin protein changes the fate of lateral mesodermal tissues from a ventral to a more dorsal fate (Smith et af., 1993). In effect, it appears to modify the response of cells to natural inducing stimuli. For example, it has been reported to act by directly inhibiting the action of bone morphogenetic proteins (Wilson et al., 1997). 3. Bone Morphogenetic Proteins

Bone morphogenetic proteins (BMPs) are peptide growth factors of the TGF-P superfamily (Fig. 5) and are believed to play an indirect role in dorsal mesoderm patterning via their action as ventralizing agents. BMP4 is closely related to the Drosophila decapentapfegic (dpp) gene. Both transcripts and protein of the Xenopus BMP-4 homolog modulate the effects of mesoderm induction by enhancing ventral tissue development (Dale et al., 1992; Jones et af., 1992). Its ability to ventralize both whole embryos and activin-treated animal caps is well known. When a truncated BMP-4 receptor (“dominant-negative” protein) was injected into embryos, the fate of some of the prospective ventral mesoderm was altered to dorsal mesoderm (Maeno et af., 1994b; Suzuki et al., 1994). Zygotic expression of BMP-4 begins after the onset of gastrulation and initially is localized in the ventral marginal zone (Fainsod et af., 1994). Ectopic expression of BMP-4 inhibits formation of dorsal and lateral mesoderm and reduces the size of the neural plate (Schmidt et af., 1995). BMP4 is therefore likely to act by opposing the action of dorsalizing signals such as those that originate in the dorsal organizer region. It has also been proposed that BMP’s role in ventral mesoderm patterning is mediated through the action of the Xvent genes (Onichtchouk et al., 1998). The existence of negative regulators of BMP has revealed that, as is the case for several of the peptide growth factors mentioned previously, regulatory circuits may act to modulate the activity of BMP. For example, Xenopus FKBP, a cytoplasmic protein which binds to various enzymes, when injected into eight-cell embryos, can cause secondary axis formation. It presumably acts by inhibiting endogenous BMP signaling (Nishinakamura et al., 1997). In addition, BMP signaling is likely mediated by specific Smad (DNAbinding) proteins (Nakayama et al., 1998), some of which (e.g., Smad8), are believed to act in a negative feedback loop in which BMP first induces Smad8 expression only to be inhibited as the level of Smad8 protein in-

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creases (Nakayama et al., 1998). Misexpression studies have implicated other Smad proteins (e.g., SmadS) as BMP’s downstream inducers of ventral mesoderm development (Suzuki et al., 1997). It should be kept in mind, however, that most of the gene expression markers such as members of the Smad family were discovered in Drosophila and Caenorhabditis elegans and then used to clone homologs from amphibia. Regulatory circuits which resemble those of invertebrates are therefore necessarily being proposed for peptide growth factor action in vertebrates. Transcriptional regulators and pathways which may be unique to amphibia are thus-due to technical limitations imposed by the lack of amphibian genetics-left undiscovered. A sort of “invertebrate bias” governs the design of regulatory circuits for amphibians. 4. Other Modifying Factors

The strategy employed to discover the previously mentioned modifying factors, not to mention the strategy employed to elucidate the important role(s) that peptide growth factors play in amphibian body patterning, is perhaps best described as “random musings.” That is, a systematic, genetically based approach to identifying the key components of the amphibian embryo’s body patterning, cell signaling systems is not possible due to the lack of adequate genetic systems in amphibia. Instead, genes which have been identified in other pattern specification systems in other organisms (especially Drosophila) have been used to clone homologs from amphibia. The search for modifying factors is therefore heavily biased by the dependence on an invertebrate organism for gene probes. Clearly, substantial conservation of gene structure between Drosophila and amphibia exists. It is, however, unclear whether the detailed features of the regulatory circuits which comprise pattern specification in these organisms are also highly conserved. It is entirely possible, therefore, that many additional modifying factors exist which are unique to amphibian patterning and do not play important roles in Drosophila patterning: They await discovery. As mentioned previously, due to the lack of good amphibian genetic systems, current technology (other than tedious “differential screening” methods) is unfortunately not adequate for recognizing such modifying factors. The general scheme which is emerging will therefore continue to maintain its invertebrate features bias.

E. A Simple Working Hypothesis: Activin Acts in Concert with Modifying Factors to Provide Regionalized Pattern Specification Review of the great extent t o which activin meets the “rigorous criteria” mentioned in Section 11, B, combined with consideration of the fact that

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various modifying factors such as Wnt and noggin gene products influence patterning, suggests a reasonable approach to formulating a hypothesis or model. As stated previously, this working hypothesis accounts for specific features which are known and also provides opportunities for future research. As an experimental approach to testing the previous hypothesis, research on the effects of activin on the expression of a variety of marker genes is currently being carried out in several laboratories.

111. Effects of Activin on the Regional Expression of Specific Genes De novo gene expression begins in the early embryo during the blastula stage (MBT). Some of this de n o w gene expression appears to be spatially localized in the early embryo. For example, some genes are expressed uniformly in the equatorial region, others in the dorsal region (including the organizer), and yet others in nondorsal regions (excluding the equatorial region). Many of these genes are believed to code for transcription factors, whereas others are thought to affect mesodermal or neural patterning in other, less direct ways. Most likely, a cascade of gene expression events is set in motion by the early transcribed genes which encode transcription factor proteins: Secondary signaling molecules are synthesized/secreted, position-specific homeobox genes are then induced, and tissue-specific expression of the genes which give various regions of the embryo their unique differentiated patterns occurs. Activin has dramatic effects in the animal cap assay. It triggers gene expression cascades which include markers of the type documented in Table I. The time course of the expression of these genes in animal cap assays mimics the sequence in whole, intact embryos. Comparing the schematic diagram of Fig. 3, which summarizes the remarkable range of histological features induced by activin, with the list in Table I provides a compelling argument for assigning activin a key role as a natural inducer in the normal amphibian embryo.

A. Activin Induces Expression of General Mesoderm Marker Genes Marker genes (see Table I) are expressed broadly in the embryo’s vegetal or marginal zone and, of course, in activin-treated animal caps. Examples include Mix. I (Rosa, 1989) and Bruchyury ( X b r a ) (Smith et al., 1991). Mix. I is a homeobox gene which is highly diverged from the Drosophila Antennapedia-class genes. Although Mix. I is induced by activin, FGFs

TABLE I Genes Expressed in the Xenopus Animal Caps after Activin Treatment

Gene Mix1 Mix.2 milk goosecoid Xbra Xlim-1 XFKHl Xwnt-8 Xnot noggin k? chordin follistatin cerverirs Xhox3

Defining feature of gene

Onset of expression 30 min 30 min" 30 min" 30 min 1.5 h 2h

3 ha 3 h" 3h" 3 ha 3 ha 3 h 6 h"

Xprx-I XMyoD XlHboxl

6h 6 ha 11 h"

XIHbox6

13 h"

a-uctin N-CAM XlHbox8

19 h 19 h 36 ha

Homeobox gene, relative of Drosophila paired Homeobox gene, relative of Drosophila paired Homeobox gene, relative of Xenopus Mix.] and Mix.2 Homeobox gene, relative of Drosophilu gooseberry and bicoid Homologue of mouse Brachytcry ( T ) Homeobox gene with LIM domain Homeobox gene. relative of Drosophila forkhead Relative of Drosophilulmouse oncogene int-l Homeobox gene. relative of Drosophilu empty spiracles BMP-binding protein BMP-binding protein Activin and BMP-binding protein Novel gene, head inducer Homeobox gene, relative of Drosophila even-skipped paired-related homeobox gene Homolog of mouse MyoD Homeobox gene, relative of Drosophilu untennapedia, homolog of mouse Holr C6 Homeobox gene, relative of Drosophilu abdominalB, homolog of mouse Hox B9 Cardiac and early axial muscle actin gene Neural cell adhesion molecule gene Homeobox gene, expressed in pancreas

~~

" Approximate time after initiation of activin treatment.

Reference Rosa (1989) Vize (1996) Ecochard et al. ( 1998) Cho et al. (1991a) Smith et al. (1991) Taira et al. (1994) Dirksen and Jamrich (1992) Christian et a/. (1991) von Dassow et al. (1993) Smith and Harland (1992) Sasai et ul. (1994) Hemmati-Brivanlou et al. (1994) Bouwneester et al. (1996) Ruiz i Altaba and Melton ( 1989) Takahashi et al. (1998) Hopwood et ul. (1989) Cho and De Robertis (1990) Cho and De Robertis (1990) Kinoshita and Asashima (1994) Kinoshita and Asashima (1994) Gamer and Wright (1995)

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have no effect on its expression. In normal embryogenesis Mix. I is expressed transiently. It begins at the MBT and ceases before the neurula stage. It is expressed mostly in the future endoderm of the midblastula stage embryo, so it can be considered (at this early stage) to represent an endodermal marker. This fact suggests that activin might actually possess “vegetalizing” signaling ability as well as mesoderm signaling properties. The Brachyury (T> gene is required for the cell-autonomous formation of posterior mesoderm in both mouse and zebrafish embryos. The high degree of sequence conservation represented in the Xenopus homolog (Xbra) suggests that it has the same function in amphibian embryogenesis. Smith et al. (1991) demonstrated that Xbra is expressed in the ring of involuting mesoderm during gastrulation and is also expressed in the notochord and posterior tissues at later stages. Its expression in animal caps can be induced by either FGF or activin. Xbra mRNA injection experiments demonstrated that it is able to induce formation of ventral mesodermal tissue (Cunliffe and Smith, 1992).

6. Activin Induces Expression of Dorsal Marker Genes The expression of several genes can be experimentally induced by activin, Vgl, and Wnt but not by FGF. Goosecoid (gsc) is one example. It is a homeobox gene which encodes a DNA-binding specificity similar to that of the Drosophila bicoid protein (Blumberg et al., 1991). Treatment of animal caps with high concentrations of activin generates rapid (within 30 min) expression of gsc. Its rapid appearance is not inhibited by the protein synthesis inhibitor cycloheximide, so gsc is thought of as an early response gene. Distribution of gsc mRNA in normal embryos, as revealed by in situ hybridization, closely corresponds to the primary organizer region (Cho et al., 1991b). gsc mRNA disappears shortly after gastrulation. Microinjection of gsc mRNA into ventral blastomeres leads to the formation of an additional body axis including head structures. Approximately 10% of the secondary axes are complete (Cho et al., 1991a). The progeny of goosecoid mRNA-injected cells participate in secondary axis formation. Other homologs of Drosophila homeobox genes, such as the Fork head/ HNF3-related Pintallavis/XFC-I and XFKHUXFD-I genes, are expressed rapidly upon activin treatment (Dirksen and Jamrich, 1992; Ruiz i Altaba and Jessel, 1992). In normal embryos they are localized in the dorsal blastopore lip. Pintallavis encodes a member of the HNF-3/fork head transcription factor family. It is expressed in the organizer, notochord, and in the midline neural plate cells which give rise to the floor plate. The expression of fork head genes in midline cells may contribute to the establishment of the floor plate fate.

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A transcript of the LIM class, another homeobox gene (Xfim-Z),has been discovered in the unfertilized Xenopus egg. The transcript becomes concentrated in the organizer region of gastrulae (Taira et al., 1992). Then it appears in prechordal mesoderm and notochord during gastrulation, and it slowly disappears from these tissues during the neurula stages, except for the posterior tip of the notochord, where it lingers. These observations suggest that Lim-I plays a role in early mesoderm formation and later in the specification of the differentiation pattern of the CNS. Expression of Xlim-Z mRNA is induced by either activin or retinoic acid. Treatment of animal caps with both factors gives a synergistic effect (Moriya et af., 1993; Uochi and Asashima, 1996). C. Secondary Gene Expression Responses of Dorsal Marker Genes

As verification of the gene expression cascade theme mentioned previously, several recent observations of dorsal-related gene expression sequences have been reported. For example, expression of the homeobox gene chordin begins in the organizer region subsequent to goosecoid expression (Sasai et af., 1994). The cordin gene was isolated by differential screening. Its expression can be activated by gsc and Xnot2 genes. Since its induction by activin requires de n o w protein synthesis, chordin expression is best considered to represent a secondary response. Microinjection of chordin mRNA induces a secondary axis and can completely rescue axial development in ventralized embryos. The Xenopus chordin gene has been identified as the functional homolog of Drosophifa sog, which antagonizes the effects of dpp, a homolog of the vertebrate peptide growth factor BMP-4 (Holley et al., 1995; Francois and Bier, 1995). Chordin is thought to be a potent dorsalizing factor which is expressed at the right time and in the right place to participate in organizer functions. Noggin transcripts, initially restricted to the presumptive dorsal mesoderm, reach their highest levels at the gastrula stage in the organizer region (Smith and Harland, 1992). In the neurula stage, noggin is transcribed in the notochord and prechordal mesoderm. When gastrula animal cap is placed in a high concentration of noggin protein, it acts as a neural inducer without displaying any mesoderm induction (Lamb et al., 1993; Knecht et af., 1995). Finally, we discuss one more secondarily expressed gene of interest: the Xenopus homolog of sonic hedgehog (Xhh). It is detected in both normal embryos and activin-treated animal caps (Yokota et al., 1995). It is not induced by bFGF. Sonic hedgehog is expressed in mammalian tissues with known signaling capacities, such as the notochord, floor plate of the CNS, and the zone of polarizing activity in the limb. Expression of Xhh, which

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is induced in animal caps by activin, is not inhibited by cyclohexamide. This suggests that Xhh represents an immediate early response gene. D. Later (Postgastrulation] Gene Expression Promoted by Activin

Following gastrulation several position-specific genes are expressed, probably in response to the action of an initial signal molecule such as a MIF. Activin likely initiates a cascade, which eventually includes the expression of several markers for anterodorsal and posteroventral mesoderm. XlHboxl is expressed in anterior mesoderm and neural tissues, for example, in response to activin treatment (De Robertis ef al., 1989). Likewise, Xprs-I, another homeobox gene, is induced by activin in Xenopus animal caps (Takahashi et al., 1998). Later in development additional gene transcripts can be identified. Two of the most convenient markers are the mesoderm-specific muscle actin (detected in the dorsolateral mesoderm) and blood island globin (ventral mesoderm). Activin can induce both in animal caps. Neural markers such as N-CAM are also expressed, presumably as an indirect effect of activin treatment. That is, activin probably first induces mesoderm, which in turn induces the formation of the embryonic organizer region, which then induces neural tissue in ectoderm cells. Feedback loops probably act to regulate each of the steps just mentioned. For example, Smad7, which is induced by MIFs, inhibits both activin and BMP signaling and therefore interferes with mesoderm formation and axis development. At appropriate concentrations Smad7 can act as a neural inducer (Casellas and Brivanlou, 1998). It thus appears that although researchers place their major emphasis on the discovery and study of the positive (inducing) aspects of MIF action in order to understand the molecular features of patterning in early amphibian embryogenesis, extensive parallel, concomitant negative (inhibitory) regulatory mechanisms likely play roles that are equally as important. The inhibitory Smads provide a paradigm for future research designed to elucidate the nature of what may eventually prove to be extensive networks of mechanisms which downregulate the positive aspects of MIF activities.

IV. Axial Patterning by Activin(s) Based on experiments with heterogeneous inducing factors, Toivonen and SaxCn (1955) proposed a “dual-gradient’’ model to explain how neuralizing and mesodermalizing agents act to generate axial patterning in the early

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amphibian embryo. Recently, a “three-signal’’ model has been proposed by Slack (1991). No doubt more complex versions will be developed as the regulatory circuitry becomes better understood. The models proposed to date share a common feature-concentration gradients of a limited number of signal molecules play key roles in specifying regional patterns. Experimental evidence for endorsing activin as a key player in gradient/multiple signal models has so far been compelling. Most of these models will, however, likely need at least minor modification because it has recently been discovered that superimposed on “forward-acting’’ signaling models is a set of inhibitory intermediates such as Smad proteins. These anti-Smads act to inhibit both activin and BMP signaling by binding to MIF receptors (Casellas and Brivanlou, 1998). A. Animal Cap Responses t o Various Activin Concentrations

Mesoderm patterning is widely acknowledged to be influenced by the concentration of MIF, especially in animal cap explant cultures (Green and Smith, 1990; Ariizumi et al., 1991a,b; et al., 1992). The mesoderm is more dorsal in character as concentrations of MIF are increased (Fig. 6).

FIG. 6 Spectrum of tissue types (scored by conventional histology) which are induced in animal cap explants cultured in increasing concentrations of activin (a, control, no activin; b, 0.3 ng/ml: c, 5 ng/ml: d. SO ngiml). bl, blood cells; cg. cement gland; co, coelomic epithelium; epi, epidermis: mes, mesenchyme; rnus, muscle; not. notochord.

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The studies of Ariizumi et af. (unpublished data) revealed that low concentrations of FGF (0.5-10 ng/ml) differentiate ventral mesoderm, including mesenchyme, blood, and coelomic epithelium. Higher concentrations (30-120 ng/ml) induce small pieces of muscle but no notochord. In contrast, activin induces all types of mesoderm, including muscle and notochord (Ariizumi et al., 1996). Low activin concentrations (0.1-1 ng/ml) induce ventral mesoderm tissue, whereas higher concentrations (1-10 ng/ml) induce dorsal mesoderm. Notochord, the tissue with the most dramatic dorsal character, is induced at 50 ng/ml. At this high concentration muscle formation was reduced. It therefore appears that activin can act alone, without the aid of, for example, FGF, to induce a broad range of mesodermal tissues. The three activin types (see Fig. 5) induce similar mesodermal tissues. Activin is also very likely responsible for the development of neuralinducing activity since activin does not induce neural tissue directly. Animal caps treated with activin develop organizer activity and can therefore induce neural tissue secondarily in gastrula animal caps (Ariizumi and Asashima, 1995b).

6. Toward an Understanding of Activin’s Mechanism of Action The animal cap’s responses to different concentrations of activin might depend on the proportion of the activin receptors on the surface of animal cap cells which become saturated. Longer treatments with activin can mimic the effects of shorter treatments with higher activin concentrations (Ariizumi el al., 1991b). Further insight into the mechanism of action will likely come from the use of marker genes to track activin’s effects and thereby sort out early from late responses of a tissue to activin treatment. Green et ul. (1990, 1992) obtained several insights using this approach. Animal cap cells become more sensitive to activin treatment in terms of marker gene expression when blastomeres are dissociated into single cells. Using this system they demonstrated that narrow dose ranges of activin, bounded by sharp thresholds, induce at least five different states of differentiation ranging from posterolateral mesoderm to dorsoanterior organizer mesoderm. Thus, the notion that activin acts in the normal embryo in graded concentrations is reinforced. Most likely, activin acts to specify regional mesoderm features. Following such regional specification of the mesoderm, genes which play roles in mesoderm patterning that are somewhat limited compared to the more general role just proposed for activin are expressed. Bruchyury is probably one of them. It is required for differentiation of the notochord (the archetypical dorsal mesodermal tissue) in mouse and fish embryos,

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but, even in high concentrations, injection of Xenopus brachyury mRNA does not induce notochord formation in animal caps. Xbra alone, however, is sufficient to specify ventral mesoderm in animal caps, as evidenced by the expression of Xhox3 and low levels of muscle actin (Cunliffe and Smith, 1992). It has recently been demonstrated that injection of Xbra mRNA into embryos at low doses induces animal caps to develop mesothelial smooth muscle vesicles and mesenchyme. At higher doses somitic muscle is formed (O’Reilly et al., 1995). Coexpression of Xbra with either noggin or Xwnt-8,however, does induce animal caps to develop dorsal mesodermal features. Coexpression of Xbra mRNA with noggin mRNA in animal caps yields dorsal tissues such as muscle, notochord, and neural structures. Coexpression with Xwnt-8 converted animal caps to muscle masses. Cunliffe and Smith (1994) concluded that Xbra defines a cell state in the embryo which can respond to diffusible dorsal signals such as noggin and Xwnt-8 and thereby differentiate dorsal mesodermal features. In addition, O’Reilly et al. (1995) demonstrated that coexpression of Pintallavis (but not goosecoid) with Xbra causes formation of dorsal mesoderm, including notochord. The effect of Pintallavis, like that of Xbra, is dose dependent. Thus, it appears that MIF action serves as a trigger for deployment of the complex regulatory circuits of the types just described that further refine (e.g., spatially localize) the pattern of mesoderm differentiation. The manner in which gradient components such as activin might impose pattern on early embryos is of course not well understood. It is generally assumed that diffusible morphogens activate, at various threshold concentrations, different genes. In the case of activin cell-cell contact appears to be required for a multithreshold response to occur (Green et al., 1994; Wilson and Melton, 1994). In fact, it appears that cells recognize their positions in a gradient of activin by indirect mechanisms since brief (e.g., 3 h) exposures to activin fail to show the graded response detected after a longer (e.g., 15 h) exposure. These mechanisms might involve the establishment of cell-cell contacts. There is difficulty in interpreting data derived from dissociated or cultured embryonic cells since the natural cellular adhesions and local environmental cues which are presumably so important for morphogenesis are absent. Thus, it is perhaps not surprising that a contradictory view of the activin actiodcell-cell contact issue has emerged. Gurdon and Mahony (1995) demonstrated, based on in situ analysis of blastula tissue containing activin-loaded beads, that cells respond directly to various morphogen concentrations. Their conclusion is that the response of animal cells is direct and depends on the highest concentration of MIFs to which they are exposed during their period of “competence to respond.”

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Regardless of which model is correct, it is likely that eventually it will be surmised that defined concentrations of activin (e.g., see Fig. 6) induce expression of various sets of genes, a proportion of which specify mesodermal features directly and a proportion of which function to prepare the secondary signals which further refine patterning. In virtually all scenarios of the mechanism of action of activin, competence to respond and signal timing are no doubt vitally important. C. Competence [Prepatterns) and Signal (Induction] Timing

Several studies (Sokol and Melton, 1991; Bolce et al., 1992; Christian and Moon, 1993) have shown that the prospective dorsal and ventral regions of the blastula animal cap respond differently to the same concentration of activin. These observations reveal that a dorsoventral competence prepattern to activin responsiveness exists in the animal blastomeres. In order to initiate an investigation of dorsoventral prepatterning, Kinoshita et al. (1993, 1995) assayed the responsiveness to activin of Xenopus dorsal and ventral blastomeres isolated at the 8-cell stage. These embryo segments constitute a “half-animal assay” and are useful for the study of mesoderm patterning because they contain the prospective (uninduced) mesoderm region which the conventional animal cap assay lacks (Masho, 1988; Vodicka and Gerhart, 1995). The isolated half-animal explants cultured without MIF form atypical epidermis, like conventional animal caps similarly treated. This of course implies that the prospective mesoderm region has not yet received induction signals at the 8-cell stage. Even when they were exposed to activin at later times they did not respond until they reached the 32-cell stage (Kinoshita el al., 1995), which corresponds to the starting period of mesoderm induction in vivo (Nakamura and Takasaki, 1970; Jones and Woodland, 1987). When the isolated dorsal and ventral blastomeres were separately treated with activin, dorsal blastomeres differentiated both dorsal and ventral mesoderm, whereas ventral blastomeres formed only ventral mesoderm. These results indicate that a competence prepattern for responding to MIF exists as early as the eight-cell stage, Since this prepattern is absent in ultravioletirradiated embryos (Sokol and Melton, 1991), it is possible that the prepattern requires a cortical rotation (which is known to be diminished in irradiated embryos). As candidate molecules that create the prepattern, we suggest that the dorsal determinants and dorsal modifiers (described in Section II,D), which can affect mesoderm patterning but do not induce mesoderm, be considered. The prepattern, in whatever form it exists in the embryo, can be

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experimentally modified by lithium treatment in such a way that ventral blastomeres form dorsal tissues (Kinoshita et al., 1995j. This observation might prove useful for future studies directed at understanding competence prepatterning. The fact that the half-animal assay deals with uninduced presumptive mesoderm enables the examination of the early events of mesoderm patterning. Kinoshita and Asashima (1995) reported that the timing of induction affects mesoderm patterning. Half-animal embryo segments (both dorsal and ventral halves), treated with activin before the MBT, preferentially form dorsal mesoderm including notochord. However, after the MBT they differentiate mostly ventral mesoderm. Despite the low responsiveness to activin, the cleavage-stage half-animal segments preferentially expressed the dorsal marker gsc rather than the ventral marker Xwnt-8. Our supposition is that the altered response of animal cells represents not simply the loss of competence but rather the emergence of a dorsal determination process which occurs in animal blastomeres before the MBT stage. There are several reports that support this notion: Embryo dorsalization caused by lithium is lost after the MBT (Yamaguchi and Shinagawa, 1989), ventral marker Xwnt-8 mRNA acts as a dorsal determinant before the MBT (Christian et al., 1991; Smith and Harland, 1991), and induction of erythroid (ventral marker) differentiation by ventral mesoderm does not occur earlier than the MBT (Maeno et al., 1994a). An alternative view-that FGF is a competence factor for the activin signaling system-has been proposed. In studies performed by Cornell et al. (1995) various molecular markers, rather than indices of histological differentiation (as employed by Kinoshita er al., 1993,1995), were followed in vegetal hemisphere explants. Addition of FGF induced ectopic expression of the mesodermal markers Xbra and MyoD. This is due in part, at least according to these authors, to the enhancement that FGF provides to the endogenous activin levels present in vegetal cells.

V. Life History of Activin Signaling Mechanisms of the Embryo A comprehensive view of the manner in which activin functions in axis determination should include considerations of how it is delivered to the egg, how it contributes to a signaling system, and how various signaling systems are linked or networked. Beginning with its synthesis in follicle cells and accumulation in the oocyte, activin will be traced through the early embryo (Fig. 7).

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FIG. 7 Hypothetical scheme which describes the “life history” of activin in amphibia. It is synthesized in the ovary, testis, kidney, liver, brain, and other organs in adults (origin 2) and transported through blood vessels to oocytes, where it is complexed with the yolk protein vitellogenin (which is synthesized in the liver). Follistatin is also synthesized in reproductive organs, brain, and follicle cells. Activin is synthesized in follicle cells (origin 1) and transported into the growing oocyte. Activin is also detected in oocyte at stages 1 or 2 (origin 3). There it remains, presumably stored, until early cleavage stages, when it serves as a signal which induces animal hemisphere cells to form mesoderm. Later, neural induction, which may also involve activin (perhaps indirectly; see text) occurs. By then the body pattern is fully established.

A. Accumulation of Activin[s] as Maternal (Oocyte] Information Activin proteins as well as follistatin (its inhibitory binding protein) exist in the unfertilized egg, presumably having arrived there during oogenesis. Both activin and follistatin are synthesized in follicle cells and then accumulate in the early (stage VI) oocyte. Activin mRNAs have been detected in ovarian follicle cells (Dohrmann et al., 1993; Rebagliati and Dawid, 1993), and electron microscopic immunolabeling of gold colloidal particles (Uchiyama et al., 1994) provides evidence for the synthesis of these proteins in follicle cells. Both molecules were found to be localized in yolk platelets but not in cytoplasmic organelles. Furthermore, it appears that activin and follistatin bind preferentially to vitellogenin. Thus, it is likely that yolk platelets act as a reservoir for activin (and other MIFs?) in the early embryo. How it is released, in an active form, remains a question for further research. Most likely, a mechanism comes into play which uncouples activin from follistatin, thereby permitting activin to initiate a signaling process. Such a mechanism must be signaled since yolk platelets exist everywhere in the

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early embryo, yet the activin induction system functions in a localized (e.g., dorsal) region. Once released in an active form, activin is then conceptualized as interacting with its protein receptor.

B. Activin Receptors and Signal Transduction The prospective geographical location of MIF receptors was first examined in urodele (newt) gastrula animal caps (Asashima and Grunz, 1983). The newt gastrula ectoderm consists of two cell layers, which can be conveniently separated.The outer layer has a lower competence to form dorsal mesoderm structures compared to the inner layer. When monitored with activin AI”’, receptors were found to be localized on the surface of inner-layer cells (M. Asashima, unpublished data). Receptors are therefore nonuniformly distributed in the embryo. This finding is consistent with notions about the regionalization of competence described previously. Substantial information about the receptors for members of the TGF-@ family exists. They represent a heteromeric complex composed of two types of transmembrane serinehhreonine kinases (types I and 11) (Kingsley, 1994). Type I1 receptor is thought to be responsible for initially binding the ligand. Then a type I receptor is recruited and subsequently phosphorylated by the type I1 receptor (Wrana et al,, 1994a). The phosphorylated type I receptor in turn phosphorylates intracellular substrates and in the process initiates a signal cascade. A number of type I and I1 receptors have been shown to exist in vertebrates, including receptors for activin, TGF@, and BMP. Two types of activin receptors are known in Xenopus (XactRs): type I (ALK-2 and ALK-4; Armes and Smith, 1997) and type I1 (Kondo et al., 1991; Mathews et al., 1992; New et al., 1997). In addition, various subtypes of activin receptors are known and likely generate different downstream gene expression patterns, as suggested by New et af. (1997). The Xenopus BMP-4 receptor is type I (Graff et al., 1994), as is another TGF@-relatedreceptor (Mahony and Gurdon, 1995). Overexpression studies with activin receptors in Xenopus embryos indicate that they are indeed involved in transmission of the activin signal. XactRZZB, cloned by Mathews et al. (1992), is a homolog of mouse ActRZZB. When XactRZZB mRNA was injected into the early embryo’s ventral blastomeres, a secondary body axis was induced. X A R l , which is highly homologous to AactRZZB (Hemmati-Brivanlou and Melton, 1992), is expressed continuously in the ovary, unfertilized egg, and neurula embryo. Maternal XactRZZB mRNA is found uniformly in the early embryo but is restricted in distribution to the neural plate at the neurula stage. These observations imply that activins play roles later in neurogenesis as well as earlier in mesodermal induction.

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C. A Conflicting Opinion on Activin’s Role

An opinion which contrasts sharply with the view that activin/activin receptors play key regulatory roles in mesoderm/neural patterning has, however, recently been offered. It has been suggested that activin signaling may be unnecessary for mesoderm formation. Both Schulte-Merker et al. (1994) and Kessler and Melton (1995) have demonstrated that truncated versions of Xenopus activin receptors also interfere with the activity of mature Vgl protein. This observation presents the possibility that such modified receptors may block the function of several members of the TGF-fi family and not just activin. Thus, it is possible that the effects of truncated activin receptors on Xenopus development can be explained not by activin inhibition but rather by inhibition of Vgl signaling. However, swinging the pendulum back toward activin as the dominant PGF signal, Dyson and Gurdon (1997) prepared an activin receptor construct which selectively blocks activin but not Vgl action. They claim it inhibits mesoderm differentiation. Clearly, the use of defective receptors as inhibitors for amphibian embryonic induction research requires further study, especially in view of the fact mentioned previously that numerous closely related and cross-reacting receptors likely exist. In the mouse, however, gene targeting recently has been used to inactivate a type I activin receptor. Embryos failed to gastrulate properly, providing genetic evidence for a role for activin in early embryonic patterning (Gu et al., 1998). Whether extrapolation to amphibia is warranted is a matter of speculation. It should be noted that follistatin (an activin-binding protein) mRNA injection fails to block mesoderm induction in the whole, intact embryo (follistatin does not inhibit Vgl signaling). A lack of a follistatin effect, although it represents “negative evidence,” argues against a function for activin in mesoderm induction. Finally, it should be mentioned that if the “truncated receptor” data are to be heavily weighed, so as to discount the role of activin in normal mesoderm development, more compelling data for assigning to Vgl the role previously ascribed to activin should be generated. After all, as mentioned earlier, arguments promoting Vgl as a natural mesoderm inducer have several shortcomings.

D. Networks and Cascades of Inductive Signals Various studies have suggested that natural induction and patterning processes of mesoderm development involve a complex system involving multiple signal molecules. For example, mesoderm induction by activin (Cornell and Kimelman, 1994; LaBonne and Whitman, 1994) and processed Vgl

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(Schulte-Merker et al., 1994) is facilitated by FGF-mediated signals. In several studies it has been shown that a number of the components of the FGF signal transduction pathway are important for activin signaling in the early embryo (LaBonne and Whitman, 1994;MacNicol et al., 1993; Whitman and Melton, 1992). For example, activation of the tyrosine kinase FGF receptor appears to activate the small GTPase p2lras, which in turn activates the cytosolic kinases raf-Z and mitogen-activated protein (MAP) kinase. As with the dominant inhibitory F G F receptor, dominant inhibitory mutants of raf and ras inhibit mesoderm induction by both FGF and activin (LaBonne and Whitman, 1994; Whitman and Melton, 1992). Despite the apparent need €or a functional FGF signaling pathway for activin induction, activin does not cause a significant activation of MAP kinase or p2lras (Graves et al., 1994); LaBonne and Whitman (1994) indicated that activin does not directly stimulate this pathway. These data suggest that early signaling responses can be used to distinguish pathways in vivo and thus help define the roles played by individual factors or families of factors. Recent studies using MAP kinase-specific phosphatases (LaBonne et al., 1995; Gotoh et al., 1995; Umbhauer et al., 1995) demonstrated that MAP kinase activation is required for mesoderm induction by FGF and activin. In fact, MAP kinase is sufficient for the induction of mesodermal markers (Umbhauer et af., 1995). Presumably, MAP kinase acts downstream of the initial signaling caused by FGF or activin. As more signaling molecules are identified, mesoderm induction is increasingly being viewed as an intrinsically complex phenomenon. The following are recent additional entries in the mesoderm-induction circuit “derby.” The Xenopus homolog of the mouse nodal gene ( X n r s ) has been reported to act in mesodermal patterning (Smith et al., 1995; Jones et al., 1995). Nodal is a diverged member of the TGF-P superfamily gene and is expressed in the mouse node during gastrulation. Xnrs appears transiently during embryogenesis and is specifically expressed in the Spemann organizer at the early gastrula stage. When X n r mRNAs were injected into early embryos, they rescued the embryonic axis in ultraviolet-ventralized embryos, dorsalized ventral marginal-zone explants, and induced muscle differentiation.Although no maternal Xnr mRNAs were found, the activities of the X n r proteins suggest that a signaling pathway involving nodalrelated peptides is an essential element in mesoderm differentiation and axial patterning. It has been discovered that a TGF-P-related type I receptor, XTrR-1, (Mahony and Gurdon, 1995) is expressed in all regions of embryos throughout early development. Overexpression of this receptor does not affect ectoderm or endoderm but dorsalized the mesoderm such that muscle appears in ventral mesoderm and notochord appears in lateral mesoderm

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normally fated to become muscle. In addition, overexpression of XTrR-1 in irradiated embryos is able to cause formation of a partial dorsal axis. These results suggest that XTrR-1 encodes a receptor which responds in normal development to a TGF-like ligand so as to promote dorsalization. Its function would therefore be to direct mesodermalized tissue into muscle or notochord. There is at least one more interesting gene which is known to be expressed in the embryo’s dorsovegetal region-the homeobox-containing gene Siarnois. Its RNA is first detected shortly after the midblastula transition, earlier than mRNAs for goosegoid or Xbra, and is present most abundantly in the dorsal endoderm of early gastrulae (Lemaire et af., 1995). Embryos injected with it in a ventral-vegetal blastomere develop a complete secondary axis. The progeny of Siarnois-injected cells do not participate in secondary axis formation. It would be easy to imagine Siarnois as playing a role in the formation of the Nieuwkoop center.

E. Inhibition of Neural Induction by Activin? Although activins induce neural tissue in animal caps, they do not act directly as a neural inducer. Microinjection of low doses of activin A protein directly into the blastocoel (see Fig. 2b) generates a secondary embryo. High doses yield a headless embryo with an outgrowth of tail (Ariizumi et al., 1991b). These observations hint that activin might interfere with the regulatory circuitry which comprises normal neural induction. Indeed, expression of a dominant inhibitory form of the activin type I1 receptor, which blocks mesoderm-inducing activity of activin, induces the formation of anterior neural tissue in animal cap explants (Hemmati-Brivanlou and Melton, 1994). Also, overexpression of this truncated receptor in ultravioletirradiated embryos yielded embryos with neural structures but that lacked major axial structures. In addition, expression of follistatin, a specific inhibitor of activin, in animal caps results in expression of neural markers (Hemmati-Brivanlou et al., 1994). Since Xenopus follistatin is expressed first in the gastrula organizer and later in the notochord and anterior nervous system, these localizations coincide with the tissues which would be expected to produce neural inducing factors. Thus, activin’s role in normal embryogenesis might extend beyond early dorsal mesoderm induction to include later inhibitory effects on neural induction. A key to understanding these different roles might lie in the mechanisms which regulate the endogenous levels of activin protein at various developmental stages. Accordingly, Klein and Melton (1995) investigated the translational control of activin in Xenopus embryos. They conclude that maternal

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factors regulate translation of activin mRNA by interacting with the 3‘untranslated region of the mRNA.

VI. Proposed Molecular Models for Activin’s Role in Signal Transduction Pathways As mentioned previously, the lack of a systematic approach (e.g., genetics) to elucidate the regulatory circuits in which activin is involved generates models which are likely to be incomplete. Nevertheless, such models are usually very valuable because they provide testable features and can be used as a point of departure for further studies. A few are illustrated in Fig. 8. They should not activin Smad2

activin

$.(+)

FAST-1

actkiin signal; Mix.2 expression type I1

kinase

signal transduction expression

activin

activin signal FIG. 8 Various ways of viewing the role of activin in signal transduction pathways. (Top) Two membrane receptors (types I and 11) are required for activin action. They each have a cytoplasmic domain which acts as a serinelthreonine kinase. The type I receptor becomes phosphorylated and is subsequently responsible for propagating the signal downstream [see Chen et al. (1997) for more complex versions of this model]. (Middle) The expression of the goosecoid (gsc) gene is initiated by the action of both activin and Wnf. The product of the gsc gene then triggers further steps in the gene expression cascade, which ultimately leads to development of Spemann’s primary organizer on the embryo’s dorsal side. (Bottom) BMP is employed as a negative regulator of activin action.

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be considered as competing models. Rather, they represent models which have been developed by using different databases, and consequently they depict different views of activin’s role in signal pathways. One model, based on the data of Chen et al. (1996) and Wrana et al. (1994a,b), proposes that activin interacts with its type I1 receptor, which in turn phosphorylates the activin type I receptor. This subsequently leads to phosphorylation of a cytoplasmic protein (Smad2) (Lo et al., 1998). It is translocated to the nucleus, where it interacts with a transcription factor (FAST-1) and binds to a promoter region [the so-called activin response element (ARE), a 290-base pair sequence], leading to transcription of the Mix.2 gene. M h . 2 is a homeobox gene which presumably plays a role in initiating a gene expression cascade. Thus, in this model activin is a key component in upregulating gene expression by virtue of its ability to activate a set of “transcriptional partners.” These transcriptional partners-when present in sufficiently high concentrations-may also act in a negative feedback loop to control activin action (Nakao et aL, 1997). Also, additional coactivators (CBP and p300) have recently been discovered to be involved in a cooperative mechanism which promotes Smad2 activation (Janknecht et al., 1998). Similar models which link activin and Smad2 are reported to be active in patterning the early mammalian (mouse) embryo in ways which mimic those described previously for amphibian embryos (Nomura and Li, 1998; Zhao et al., 1998). Another model emphasizes the manner in which activin and other peptide growth factors induce gsc gene expression early in a signaling pathway (Watabe et al., 1995). It proposes that the gsc promoter has a distal element (DE) and a proximal element (PE) which respond to activin (DE) and Wnt (PE) action. Simultaneous but independent interactions at both these promoter sequences is believed to be required for the guusecuid gene expression. A third model depicted in Fig. 8 emphasizes the role activin plays in promoting the expression of the early response gene X F K H l (Kaufman et al., 1996). The expression of this gene is postulated to comprise part of the Spemann primary organizer. An A R E located in the promoter region of the X F K H l gene is depicted as responding to activin. In addition, a second promoter region [BIE (BMP-activated)] in this gene is thought to respond to BMP signaling and override the activity of the activin-ARE system. Thus, both positive and negative regulation are included in this model.

VII. Activin Causes a Broad Array of Differentiations in Vitm One of the most remarkable features of activin is its ability to generate a diverse array of tissue differentiations in animal cap explant cultures when

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applied at various doses (see Fig. 3). Its ability to induce the differentiation of such a broad spectrum of tissue differentiations provides one of the most compelling arguments, when viewed in terms of the rigorous criteria (that it largely fulfills) discussed earlier, for considering it to be an endogenous inducer. A few examples of these differentiations are reviewed in the following sections. A. Induction of Endoderm by Activin

The group of MIFs from heterogeneous tissues (e.g., chick embryo extract) have been termed vegetalizing factors based on their ability to induce endoderm and mesoderm differentiation in newt animal cap explants (Grunz, 1979; Grunz and Tacke, 1989; Kocher-Becker and Tiedemann, 1971). At low concentrations they typically induce mesoderm, whereas at high concentrations they preferentially induce endodermal differentiations. When a pellet of vegetalizing factor is implanted into the blastocoel, a secondary axis is induced. Thus, these vegetalizing factors mimic the action of the inducing signal from the Nieuwkoop center. Do activins possess the functions expected of the Nieuwkoop center? Activin induces the expression of an endoderm marker gene (Mix. I ) and antigens recognized by endodermal-specific antibodies (Jones et al., 1993). Also, high concentrations of activin (100 ng/ml) can induce histological differentiation of endoderm in newt animal caps (Ariizumi and Asashima, 199%). For example, endodermal epithelia and, occasionally, a beating heart develop. Heart induction has been shown to require signals derived from both endoderm and organizer tissue in Xenopus ventralmesoderm explants (Nascone and Mercola, 1995), so a role for activin is very plausible. Furthermore, activin-treated newt animal caps can induce mesoderm in uninduced animal caps (Ariizumi and Asashima, 199%). We can therefore propose the following model for these in vitro results. Activins act strongly on a portion of the animal cap to induce endoderm. This endoderm in turn induces mesoderm in the rest of the (uninduced) animal cap. This model is clearly compatible with activin serving as a key component in the embryo’s natural Nieuwkoop center.

6. Activin-Induced Organizer Guides Neural Patterning The vertical signal of neural induction implies that the involuting dorsal mesoderm signals the overlying ectoderm to become neural tissue. The anteroposterior pattern in neural structures is established during this period,

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presumably as a result of patterned induction from mesoderm. The results of an organizer graft (Spemann and Mangold, 1924; see Fig. 2b) can be mimicked by the action of activin. Ariizumi and Asashima (1995b) treated newt animal caps with the high concentrations of activin (100 ng/ml) mentioned previously and then kept them in saline for different periods. They were later combined with uninduced ectoderm, and subsequent differentiation was monitored. Animal caps incubated for a brief period before being combined with ectoderm induced posterior (trunkhail) structures, whereas those precultured for a long time induced anterior (head) structures. Remarkably, these data correlate well with the original Spemann and Mangold (1924) observation that the early gastrula blastopore lip induces head structures and the late gastrula lip induces trunkhail structures. At the very least, these findings should offer an experimental model system for in vitro studies on the molecular mechanisms of primary embryonic induction. The activin-treated tissues represent a much simpler system than the whole, intact embryo.

VIII. Conclusion A variety of MIFs can be demonstrated to have profound effects on body patterning when either administered to whole embryos or added to the culture medium of explanted embryonic tissue. When the life histories of

FIG. 9 Activin’s role in early embryonic patterning events is depicted as being at the center of the regulatory circuits which regulate the development of various individual tissues and organs.

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several MIFs, including FGF, Vgl, and activin, are carefully reviewed, activin stands out as having the most profound effects and fulfilling most of the rigorous criteria expected of a natural (endogenous) inducer. It is of course unlikely that any single MIF-in the normal, intact embryocontrols all early programming events and differentiations. Nevertheless, activin is likely to play the key, early role. Figure 9 positions activin at the epicenter of the profusion of circuits and pathways which comprise early amphibian embryonic patterning. Activin has also been identified as playing a key role in various cell differentiation events which are separate from its role in embryogenesis, the subject of this review. In some regards, it might be considered an all-

FIG. 10 The life history of an amphibian is illustrated. Many of its most dramatic morphogenetic events are shown. The time periods during which activin is active are diagrammed as bellshaped curves around the life cycle circle. Activin involvement in developmental life cycle events “comes and goes” from oogenesis through adulthood. In this sense, activin can be conceptualized as serving as a “timekeeper” gene since it appears to play a regulatory role in each major morphogenetic transition.

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purpose signaling molecule. At the cellular level it is known to induce apoptosis in B lineage cells (Nishihara et al., 1995) and to facilitate wound repair (Hubner et al., 1996). At the subcellular level is can affect Kf and Ca2+ channels (Mogami et al., 1995) and promote insulin secretion (Furukawa et al., 1995). At the molecular level it is known to cause alterations in transcription factor (e.g., Pit-1) activity (Gaddy-Kurten and Vale, 1995). Figure 10 conceptualizes the life cycle of the vertebrate (e.g., amphibian) and illustrates the various stages during which activin is known to play roles in one or another pathway or regulatory circuit. It should therefore be presumed that studies on the role that activin plays in specifying embryonic patterns (e.g., via dorsal mesoderm induction) stand to benefit from the data being collected in subdisciplines which do not overlap with embryology/developmental biology. By collecting information from a broader range of activin discoveries, developmental biology researchers might be able to make better use of intuition for deducing the signaling pathways and regulatory circuits used in amphibian embryos. Thereby, more freedom from the current heavy dependence on Drosophila studies, which lead to an invertebrate bias in the development of hypothetical models, could be achieved.

Acknowledgments M. A., K. K., and T. A. acknowledge the financial assistance provided by a grant-in-aid from the Ministry of Education, Science and Culture of Japan and CREST (Core Research for Evolution Science and Technology) of the Japan Science and Technology Corporation. G. M. M. acknowledges the National Science Foundation for support of the Indiana University axolotl colony. Susan Duhon provided expert editing of the manuscript.

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Calcium Regulation of the Actin-Myosin Interaction of Physarum polycephalum Akio Nakamura and Kazuhiro Kohama Department of Pharmacology, Gunma University School of Medicine, Maebashi, Gunma 371-8511, Japan

Plasmodia of Physarum polycephalum show vigorous cytoplasmic streaming, the motive force of which is supported by the actin-myosin interaction. Calcium is not required for the interaction but inhibits it. This calcium inhibition, a regulatory mode first discovered in Physarum, is the overwhelming mode of regulation of cytoplasmic streaming of plant cells and lower eukaryotes, and it is diametrically opposite to calcium activation of the interaction found in muscle and nonmuscle cells of the animal kingdom. Myosin, myosin I I in myosin superfamily, is the most important protein for Ca2+action. Its essential light chain, called calcium-binding light chain, is the sole protein that binds Ca2'. Although phosphorylation and dephosphorylation of myosin modify its properties, regulation of physiological significance is shown to be Ca-binding to myosin. The actin-binding protein of Physarum amplifies calcium inhibition when Ca2' binds to calmodulin and other calcium-binding proteins. This review also includes characterization of this and other calcium-binding proteins of Physarum. KEY WORDS: Physarum polycephalum, Cytoplasmic streaming, Actin, Myosin, Actin-myosin interaction, Calcium ion, Phosphorylation, Inhibitory effect of Ca2'. 0 1999 Academic Press.

1. Introduction Concentration of intracellular Ca2+is usually kept as low as possible by extrusion through the cell membrane and by sequestration into the endoIiirernononol Review of Cviology. Vol. I Y I

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plasmic reticulum. When the cell is excited, the concentration is increased by the entry of extracellular Ca2’ and by the release of sequestered Ca2t. However, the increase is transient because Ca2+concentration ([Ca2+])will soon be reduced again. The cell utilizes such a transient increase in [Ca”] as a second messenger to regulate various intracellular reactions. Most examples of calcium regulation observed involve activation, as exemplified by muscle contraction (Ebashi and Endo 1968): Muscle is relaxed in low [Ca”], and the elevation of [Ca”] leads to the interaction of myosin with actin to induce contraction. With the subsequent decrease in Ca”, the interact is abolished to cause relaxation. An alternative mode of regulation is theoretically possible: The interaction proceeds without requiring Ca”. An increase in [Ca”] inhibits the interaction, which will be relieved when [Ca”] returns to low levels. Such a mode has been indicated for the actomyosin system of the lower eukaryote, Physarum polycephalum (Kohama et al., 1980). Physarum plasmodium has attracted the interests of scientists in the field of cell motility because of its vigorous cytoplasmic streaming. The streaming is induced as a result of the contraction of plasmodium (Kamiya, 1981). Therefore, quantitative evaluation of the motile event in plasmodium is possible by measuring the contraction by physiological methods. Biochemical methods to purify proteins are also applicable because Physarum can be cultured in a large quantity despite the cells being eukaryotic (Kohama et al., 1998). This feature enables actin, myosin, and actomyosin-related proteins to be purified by modifying the procedures for muscle proteins (Hatano, 1973). In this review, we will explain how the interaction between actin and myosin is inhibited by Ca”. The regulation by Ca2+ of Physarum has been reviewed in detail by Kohama (1987) and subsequent reviews have appeared (Kohama, 1990; Kohama et al., 1992, 1993; Nakamura and Kohama, 1995), but these do not include the results obtained by up-to-date methods, which will be included in this review. Actin and myosin are believed to be present in all eukaryotic cells and take part in their motile events (Citi and Kendrick-Jones, 1987). Typical examples are muscle contraction in animal cells and cytoplasmic streaming in plant cells. Muscle contraction occurs at high [Ca”] and cytoplasmic streaming at low [Ca”.]. Therefore, it is expected that the effect of Ca2+ on the actin-myosin interaction is diametrically different between animals and plants (Kohama, 1990), i.e., CaZt works as an activating signal for contraction but as an inhibition one for streaming (Table I). This review also addresses an inhibitory mode of calcium regulation: calcium inhibition of the actin-myosin interaction of plant and other cells.

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TABLE I Regulation of Actin-Myosin Interaction by Ca2+

Cells Plant Animal

Resting (Ca2+< phf)

Excited (Ca2+< phf)

+

-

-

+

Note. +, active interaction between actin and myosin; -, no active interaction. Ca2+concentrations in the cells of both kingdoms are increased transiently when they are excited. The interaction of Physarurn can be classified as plant type.

II. Calcium Inhibition of Motile Events Related to Actomyosin A. Calcium Inhibition of Cytoplasmic Streaming When plant cells are observed under a microscope, organelles move along actin cables that run beneath the cell membrane. The internodal cells of Chara and Niteffa are popular materials for the observation of this cytoplasmic streaming. When they are excited by the electrical stimulation, their cytoplasmic streaming is abolished transiently (Kishimoto and Akahori, 1959; Tazawa and Kishimoto, 19681, an observation that is associated with an increase in the intracellular concentration of Ca2+(Kikuyama and Tazawa, 1982; Williamson and Ashley, 1982). The inhibitory role of Ca2" was confirmed by perfusing artificial protoplasm containing various concentrations of Ca2+into the cells and by destroying the plasma membrane to soak the cytoplasm in various concentrations of Ca2+ (Williamson, 1975; Shimmen and Yano, 1984; Tominaga et af., 1983). A similar inhibitory effect of Ca2+is demonstrated with Vaflisneria cells; its cytoplasmic streaming is detectable when observed under red light and is abolished by the application of infrared light (Takagi and Nagai, 1985, 1986). Red light induced efflux of Ca2+to, and infrared light influx of Ca2+from, the medium outside the cell (Takagi, 1993). It has been suggested for some time that the organelles of the internodal cells are associated with myosin, which interacts with actin cable to causes cytoplasmic streaming. The myosin is expected to be subjected to calcium inhibition. However, myosin (or myosin-like protein) purified from Chara is not sensitive to Ca2+(Yamamoto et af., 1995). In the myosin superfamily, conventional myosin corresponds to myosin 11. Currently, the superfamily

'

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consists of more than 10 varieties (Mooseker and Cheney, 1995). However, biochemical characterization as a motor protein has been carried out only with myosins I, 11, V, and VI. This suggestion was recently confirmed by purifying myosin-like protein from the pollen tube of Lilies, in which cytoplasmic streaming has been demonstrated to be abolished by an increase in the cystoplasmic [Ca2’] (Kohno and Shimmen, 1988). The major component of the protein is a 170-kDa peptide whose actin-myosin interaction observed in vitro was inhibited by Cazt (Yokota and Shimmen, 1993). A myosin-like protein susceptible to calcium inhibition has also been purified from BY-2, a cell line established from tobacco (Yukawa et al., 1997).

B. Calcium Inhibition of the Cytoplasmic Streaming of Plasmodia of Physarum polycephalum Cytoplasmic streaming of plasmodia is observed as a vigorous shuttle movement of organelles. Unlike in plant cells, plasmodia1 actin does not form actin cables but rather forms a complex with myosin to build up “vessels,” in which cytoplasm streams passively due to the contraction of the actomyosin vessel wall (Komiya, 1981). The concentration of cytoplasmic Ca2’ changes within pM levels, as can be observed by injecting aequorin into plasmodia as a Ca2’ probe (Ridgway and Durham, 1976). When plasmodia are soaked in a solution containing caffeine, tiny drops of cytoplasm are released from plasmodia. Hatano (1970) observed the movement of organelles in these caffeine drops and found that an increase in [Ca2’] in the solution is associated with an increase in movement. A similar claim was also published in the 1970s (Ueda et al., 1978) in analogy to the activation of muscle contraction by Ca2’ (Ebashi and Endo, 1968). It must be noted that the activating effect of Ca2t is based on observations with models for cytoplasmic streaming that are furnished with intact cell membranes. The concentrations of Ca2+ are not necessarily identical between the outside and inside of the membrane. Yoshimoto et al. (1981a) and Yoshimoto and Kamiya (1984) permeabilized the membrane of a plasmodial strand to alter the cytoplasmic concentration of Ca2’ directly and related its contraction to the concentration of Ca2+.They concluded that the actin-myosin interaction, as measured by the contraction, is maximal in the absence of Ca2’ and reduced with an increase in the Ca” concentration. This inhibitory effect of Ca2+ is confirmed with similar cell-free models under various concentrations of cytoplasmic Ca2’ (Achenbach and Wohlfarth-Bottermann, 1986a,b; Pies and Wohlfarth-Bottermann, 1986) (Table 11).

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TABLE I1 Calcium Inhibition of Actin-Myosin Interaction of Physarum Plasmodium Actin-activated ATPase activity of myosin" Superprecipitation of actomyosin* In vitro motility assay Nire//a-bassl motility assay" Myosin-coated surface assay" Tension development of actomyosin threads" Contraction of cell-free model' Intact cellR ~

~~

Ogihara er a/. (1983). Kohama and Kendrick-Jones (1986). Kohama ef al. (1991a). and Ishikawa e t a / . (1991). " Kohama et a/. (1980) and Ogihara et a/. (1983). ' Kohama and Shinimen (1985). "Okagaki et al. (1989). '' Sugino and Matsumura (1983). 'Yoshimoto et rr/. (1981a). Yoshimoto and Kamiya (1984), Achenbach and Wohlfarth-Bottermann (1986a,b), and Pies and Wohfarth-Bottermann (1986). fi Ishigami ef al. (1996). o

It has been proposed that the cytoplasmic concentration of Ca2+might not be identical throughout the plasmodium (Kuroda et al., 1988). Recent technical advances have made it possible for this argument to be resolved. Using a small fragment of plasmodium that shows contraction-relaxation movement rather than the cytoplasmic streaming, Ishigami et al. (1996) were able to relate this movement to the changes in cytoplasmic concentration of Ca2+.They concluded initially that the increase in local [Ca2+]acts as a trigger to make the plasmodium relax (Table 11).

C. Calcium Inhibition of Motile Events in Vertebrate and Invertebrate Cells Ca" activates the interaction between thick and thin filaments of muscle cells. However, the effect of Ca2+on the cleavage furrow, which is one of the typical structures that are related to actomyosin system, is diametrically different. Yoshimoto and Hiramoto (1985) related changes in Ca2+levels of echinoderm and medaka eggs to the cleavage cycle. They found that the construction and destruction of the cleavage furrow were respectively associated with a decrease and increase of Ca" concentration and suggested that the actin-myosin interaction of the furrow is subject to calcium inhibi-

58

AKlO NAKAMURA AND KAZUHIRO KOHAMA

tion. This suggestion conforms with their further observation using demembranated cell models of the eggs that a [Ca”] higher than 0.1 p M inhibited construction of the furrow (Yoshimoto et al., 1985). The inhibitory effect of Ca2+ reported for contractile rings isolated from newt eggs was such that their ATP-dependent contraction was inhibited by p M levels of Ca2+ (Mabuchi et al., 1988). The inhibitory effect of Ca2’ has been documented (Kohama, 1993). Examples are accumulating not only for motility-related events [for sperm motility, see Hong et af. (1991); for secretion of renin and parathyroid hormone, see Churchill (1985) and Shoback et af. 1983, respectively] but also for various other phenomena [for adenylate kinase of cardiac muscle, see Colvin et al. (1991); for adenylyl cyclase, see Nakamura and Bannai (1997); for NO synthetase, see Mittal and Jadhav (1994); for rhodopsin kinase, see Kawamura et af. (1993); for thioredoxin reductase, see Schallreuter and Wood (1989); for regucalcin of liver, see Simokawa and Y amaguchi (1993)]. Recently, myosin-like proteins, which have calmodulin as their light chain, have been purified from vertebrate nonmuscle cells. Myosin I from brush border consists of a 119-kDa heavy chain and calmodulin. Ca2+ dissociates calmodulin from the heavy chain and prevents the interaction of myosin I with actin (Collins et aL, 1990; Swanljung-Collins and Collins, 1991). Myosin V, a myosin-like protein from brain, is another calmodulincontaining protein. The effect of Ca2+as monitored by the in vitro motility assay is inhibitory (Cheney et af., 1993). However, the actin-activated ATPase activity of the protein is stimulated by Ca2+,a discrepancy that must be solved in the near future. Some of vertebrate smooth muscles are known to maintain their tone even if they are immersed in a solution containing EGTA for a few hours. Upon returning to ordinary Ca-containing solution, they relaxed with an increase in cytoplasmic [Ca”] (Sasaki and Uchida, 1980). We speculate that acalmoddin-containing myosin is involved in the contraction in these conditions because myosin I is present in smooth muscles (Kohama et d.,1991c; Chacko et al., 1994;Hasegawa et af., 1996). Similar speculation is possible for the type of myosin associating with the cleavage furrow and contractile ring mentioned previously: It might be a calmodulin-containing myosin.

111. Calcium Inhibition of the Actin-Myosin Interaction of Physarum as Detected in Vitro A. Studies with a Crude Actomyosin Preparation

The effect of Ca2+ on actin-myosin interaction of Physarum could be examined in vitro if one prepared crude actomyosin from plasmodia. Early

P. POLYCEPHALUM CALCIUM REGULATION

59

examination of this preparation indicated that Ca2+stimulated the interaction in a similar way to the actomyosin prepared from vertebrate muscle (Nachmias and Asch, 1974; Kato and Tonomura, 1975). Concerning the movement of amebae, Taylor et al. (1973) prepared a cell-free model of Chaos charolinesis; the model contracted in a Ca2+-containing solution and relaxed in the presence of EGTA. Myosin, whose actin-activated ATPase activity is stimulated by Ca2+,has been purified from Acanthamoeba (Collins and Korn, 1981). On the other hand, the actin-activated ATPase activity of myosin purified from amebae of Physarum (see Section VIII) was inhibited by Ca2+ essentially in the same way as that of plasmodia1 myosin (Kohama et al., 1986a). Inhibition by Ca2+of the contraction of a cell-free model of Amoeba proteus (Sonobe et al., 1985) is in good accordance with Physarum myosins. We have also observed a marked elevation of the ATPase activity of the crude preparation (Table 111; Fig. 1). However, when the preparation was subjected to further purification, the effect of Ca2+ on the activity was eventually reversed, with the ATPase activity of purified native actomyosin decreasing with an increase in [Ca”]. Because a crude extract of plasmodia is abundant in calcium-activated soluble ATPase(s) (Table 111), and contains an ATP pyrophosphohydrolase (Kawamura and Nagano, 1975), the apparent activation by Ca2+of the crude preparation was due to contamination by the soluble ATPase(s). Accordingly, we have reached the conclusion that Ca2+controls the actin-myosin interaction by inhibiting the ATPase activity of actomyosin. This inhibition is also detected by inducing the

TABLE 111 Soluble and Actomyosin ATPase Activities of Physarum Plasmodia

Condition

ATPase activity (nmol/min/mg protein)

Supernatant (0.1 mM EGTA, 50 yM Ca2’) Native (0.1 mM EGTA) Actomyosin (50 yM Ca”)

11.2 57.2 15.7 4.7

~

~~

Total ATPase activity (nmol/min/100 g plasmodia)

3.1 x 16.0 x 0.14 x 0.04 x

104 104 104 104

~~

Nore. Fresh plasmoida of Physarum polycephalum were harvested, homogenized in high salt (0.5 M NaC1, 10 mM EGTA, 0.1 mM D’IT, and protease inhibitors) at pH 8.4, and centrifuged at 100,000g for 1 h. The supernatant, adjusted to pH 6.5, was dialyzed against 5 vol of water, and centrifuged at 10,OOOg for 20 min. The supernatant was then assayed for ATPase activity. The precipitate was dissolved in high salt and used as crude native actomyosin. From this, native actomyosin was purified by repeated washing. ATPase activities were determined by the pH-stat method in 0.5 mM Mg-ATP, 1.0 mM Mg2+,30 mM KCI, and 0.1 mM EGTA-Ca buffer at pH 7.50 and 25°C (Kohama and Ebashi, 1986).

60

AKlO NAKAMURA AND KAZUHIRO KOHAMA

v

I

I

I

I

6

5

4

3

FIG. 1 Relative NTPase activity of crude native actomyosin as a function of [CaZt] (Kohama and Kendrick-Jones, 1986). Mg-NTPase activities of crude native actomyosin were determined at various concentrations of CaZt.The activities were determined by the pH-stat method under 5 mM Na+/K+,5 mM Mg acetate, 1.5 mM NTP, and 0.1 rnM EGTA-Ca buffer at pH 7.50 and at 25°C. In the presence of EGTA (loo%), the ATPase (O), UTPase (A) and GTPase (0)activities were 7.2,15.8, and 32.3 nmol/min/mg actomyosin, respectively. Calcium inhibition of actomyosin i n crude actomyosin preparation can be monitored by UTPase and GTPase activities without complicating the Ca-activatable soluble ATPase(s) contaminating the preparation (see Table 11). The difference between the Ca2+concentration that causes half-maximal inhibition for UTPase and GTPase activities can he attributed to the actomyosin properties, because a similar difference can be detected with actomyosin reconstituted from skeletal muscle actin and Physnrurn myosin (see Fig. 6 in Kohama and Kendrick-Jones, 1986).

superprecipitation as shown in Fig. 2 (Kohama et al., 1980; Ogihara et aZ., 1983) (Table 11). It should be noted that the crude preparation has been prepared as quickly as possible to avoid possible artificial modifications (see legend to Fig. 1). Therefore, actomyosin in this crude preparation is as close as possible to in vivo actomyosin. The calcium inhibition can be detected by

P.POLYCEPHALUM CALCIUM REGULATION

61

A660

*Y . c) I

0

1

3

2

Time(min)

I

6

4

pCa2+ FIG. 2 Inhibitory effect of Ca” on superprecipitation of purified preparation of native actomyosin from Physunrrn plasmodia. The extent (relative value) of superprecipitation at various concentrations of CaZ-was plotted against &a2* (Kohama e t a / . . 1980). Inset shows the time course of superprecipitation after the addition of ATP in the presence of 0.1 mM EGTA (G) or 10 p M Ca” (Ca). and the increase (relative value) in the absorbance at 660 nm in 3 min was plotted against pCa2+.

measuring UTPase and GTPase activities of such an actomyosin (Fig. 1) by utilizing the property of the Ca-activated ATP pyrophosphohydrolase mentioned previously-that it hydrolyzes UTP and GTP very slowly.

B. Myosin Is the Site of Action of Ca2’: Myosin-Linked Nature of Calcium Inhibition Observed with Crude Actomyosin from Physarurn As the first step in identifying the site of action of Ca”, we purified myosin and actin from the actomyosin preparation that shows calcium inhibition (Kohama and Kendrick-Jones, 1986).

62

AKlO NAKAMURA AND KAZUHIRO KOHAMA

1. Purification of Myosin The actomyosin preparation was dissolved in 20 mM ATP containing DTT, mixed with concentrated Mg acetate to give 0.1 M in the final concentration, and centrifuged at 100,OOOg for 30 min. The supernatant was diluted with cold water by 2.5- to 3.0-fold and allowed to stand for 3 or 4 h on ice. Myosin is then recovered as a precipitate. Because this myosin preparation is often contaminated by a trace amount of actin, as monitored by SDSpolyacrylamide gel electrophoresis (PAGE), we normally repeat these steps once more (Kohama and Kendrick-Jones, 1986). To prepare myosin that shows calcium inhibition, it is important to avoid artificial modifications during the purification procedure. Unlike the procedure originally developed by Hatano and Tazawa (1968), our procedure avoids column chromatography and hour-long centrifugation, and this may contribute to obtaining intact, native myosin. This can be monitored, for example, by examining whether sulfhydryl (SH) groups of myosin analogous to reactive thiols (SH2/SH1) of skeletal myosin (Sekine and Kielly, 1964) are protected from oxidation. Physarum myosin prepared by our procedure has intact reactive thiolds, the modification of which by N-ethylmaleimide makes myosin insensitive to calcium (Kohama et al., 1987). 2. Purification of Actin Because Physarum contains inhibitors to actin polymerization such as fragmin (see Section VI,D), the procedures that include acetone treatment for purifying actin from muscles are not applicable. Hatano and Owaribe (1977) have overcome this by heat treatment, which we employed as follows. The actomyosin preparation in 0.1 M Mg acetate and 20 mM ATP (see Section 111,BJ) was centrifuged and the resulting precipitate was suspended in 0.1 M KCl containing ATP and DTT. The suspension was incubated at 55°C for 20 min and dialyzed against 5 mM NaHC03 containing ATP and DTT. From the supernatant obtained after centrifuging the dialyzate, actin was purified by polymerization-depolymerization procedures. 3. Calcium Inhibition of Hybrid Actomyosins

The site of action of Ca2+is contained neither in actin nor in myosin of skeletal muscle. To use such a property, we reconstituted actomyosins from actin and myosin of Physarum and those of skeletal muscle. The respective actomyosins were then tested for Ca sensitivity by measuring of ATPase activity (Kohama and Kendrick-Jones, 1986). As shown in Fig. 3, the activity of the actomyosin was inhibited by Ca2+when myosin was from Physarum.

63

P.POLYCEPHALUM CALCIUM REGULATION

100

50

‘I

I

I

I

6

5

4

I

3

FIG. 3 Effect of Ca” on the ATPase activity of hybrid actomyosin formed from skeletal muscle and Physarum plasmodia (Kohama and Kendrick-Jones, 1986). Specific activities (nmol/min/mg myosin) at 100%were 164.5 for actomyosin reconstituted from Physarum actin and Physarum myosin ( O ) ,118.4 for actomyosin reconstituted from skeletal muscle actin and Physarum myosin (0),128.6 for actomyosin reconstituted from Physarum actin and skeletal muscle myosin (A),and 232.1 for actomyosin reconstituted from skeletal muscle actin and skeletal muscle myosin (A). Activities were determined by the pH-stat method in 15 mM KCI, 2 mM NaCI, 3.5 m M Mg”, 1.5 mM Mg-ATP, and 0.1 mM EGTA-Ca buffer at 25°C at pH 7.50.

However, the ATPase activity of the actomyosin produced from skeletal muscle myosin was not affected by Ca2+ no matter whether actin was from Physarum or skeletal muscle. Thus, Ca2+is demonstrated to exert an inhibitory effect through myosin. In the absence of actin, the ATPase activity of Physarum myosin is also inhibited by Ca2+,although the effect is slight (Kohama and Kendrick-

64

AKlO NAKAMURA AND KAZUHIRO KOHAMA

Jones, 1986). This observation indicates that the site of action of Ca2+is myosin and suggests that the effect of Ca2+should be amplified by actin. The role of actin will be further discussed in relation to the phosphorylated state of myosin in Section V,B.

4. Calcium Inhibition of Actin-Activated ATPase Activity of Physarum Myosin under Physiological Conditions 31P-NMR magnetic resonance studies of living plasmodia1 cells (Kohama et al., 1984) revealed that the intracellular concentrations of Mg2+and ATP and the intracellular pH were about 1 mM, 0.1-0.5 mM, and about 6.9, respectively. By other methods, the intracellular conditions of ATP, Kt, and pH were estimated to be 10.5 mM (Yoshimoto et al., 1981b), 30 mM (Anderson, 1964), and 7.0-7.5 (Morisawa and Steinhardt, 1982), respectively. Therefore, we measured the actin-activated ATPase activity of Physarum myosin in 1 mM Mg-ATP, 1 mM Mg2+,and 30 mM KC1 at pH 7.0 and observed a clean inhibitory effect of Ca2+ at p M levels as shown in Fig. 4 (Kohama and Kohama, 1984). Thus, the results shown in Fig. 3 were confirmed under physiological conditions.

5. Calcium Inhibition as Detected by in Vitro Motility Assays To demonstrate the myosin-linked nature of calcium inhibition by a quite different method, small latex beads were coated with Physarum myosin. They were then allowed to move along actin cables of Nitellu internodal cells as described by Shimmen and Yano (1984). As expected from the inhibitory effect of Ca2+on the ATPase activity of myosin, the beads moved much faster in the absence of Ca2' than in its presence (Shimmen and Kohama, 1984; Kohama and Shimmen, 1985; Table IV). Calcium regulation of the ATPase activities for myosin from scallop muscle is well characterized as will be described in Section IV,B. In this case, the effect of CaZt is diametrically different, stimulating the activity (Vale et al., 1984). Accordingly, the beads with scallop myosin moved in the presence of Ca2+,and the movement was abolished in the absence of Ca2+ (Table IV). The different effect of Ca2' using the same actin cables in this Nitella-based motility assay indicates that myosin is the site for Ca2' inhibition of the actin-myosin interaction in Physarum. To try another in vitro motility assay (Kron and Spudich, 1986; Harada et af., 1987), we prepared coverslips coated with either Physarum myosin or scallop myosin. Fluorescent actin from skeletal muscle was mounted on the coverslips in the presence of ATP and was observed with a fluorescence microscope (Okagaki et al., 1989). As shown in Fig. 5, actin moved in the

P. POLYCEPHALUM CALCIUM REGULATION

65

2001 A

c

'8

P

L

L.

L.

.5 E

tc Y

P

'5

. I

c1

..

7

5

6

4

3

pCaa FIG. 4 Effect of [Ca"] on the Mg-ATPase activity of Physariim myosin in the presence ( 0 ) and absence (0)of skeletal muscle actin under physiological conditions (0.5 mM Mg-ATP. 1 mM Mg", 30 mM KCI, and 0.1 mM EGTA-Ca buffer at pH 6.90 and 25°C) as measured by the pH-stat method (Kohama and Kohama, 1984). The conditions were estimated from "P-NMR spectrum (inset) from living plasmodia (Kohama et a/., 1984).

TABLE IV Myosin Movement along Actin Cables (pmlsec)

Solution

Physaritrn

EGTA Ca"

1.40 2 0.08 ( n = 18) 0.40 2 0.08 ( n = 11)

Scallop 0 (n 1.22 ? 0.39 ( n

= =

3) 3)

Note. Beads coated with Physarum or scallop myosin were introduced into Nitella cells together with EGTA solution (30 mM PIPES, 5 mM EGTA, 2 mM MgCI2, 66 mM KOH, 1 mM ATP, 1 mM DTT. and 170 mM sorbitol, final pH = 7.0) or with Ca'f solution (30 mM PIPES, 5 mM EGTA, 5 mM CaCll, 3 m M MgCI2, 76 mM KOH, 1 mM ATP. 1 mM DTT, and 170 mM sorbitol, final pH = 7.0) and observed with a Nomarski microscope (Shimmen and Kohama, 1984; Koham and Shimmen, 1985).

66

AKlO NAKAMURA AND KAZUHIRO KOHAMA Physarum

Scallop

l o t a

s

Q w

C

5-

5.

I

0

0

1.o

2Io

0

0

1.o

2.0

pm 'sec-1 Ca2+ myosin C (active) 4

L

b myosin-ca (inactive)

G+

myosin (inactive) 4 6

myosin-Ca (active) 2

+

FIG. 5 Inhibitory (a, b) and stimulatory (c, d) effects of CaZ' as measured by the myosincoated surface assay (Okagaki et al., 1989). Actin was labeled with rhodamine-phalloidine and mounted on a coverslip coated with Physarurn myosin (a, b) or scallop myosin (c, d). ATP-dependent movement of actin was observed in the presence of 0.1 mM EGTA (a, c) or 0.1 mM Ca2+(b, d) with a fluorescent microscope equipped with a video camera. Ordinate, number of moving actin; abscissa, velocities (pmhec). Arrows indicate average velocity of the movement.

absence of Ca2+on the Physarum myosin coverslip, and the movement was inhibited by Ca2+.Conversely, the same actin moved on the surface coated with scallop muscle myosin in the presence of actin but not in the absence of Ca2+.These differences confirm the myosin-linked nature of calcium inhibition. The effects of Ca2+on the actin-myosin interaction of Physarum myosin as examined by these two motility assays are not necessarily identical, as can be seen when Physarum myosin is dephosphorylated (see Section V,B,3).

67

P. POLYCEPHALUM CALCIUM REGULATION

IV. Ca-Binding Properties of Physerum Myosin Physarum myosin was found to bind 45Ca2twith a high K D affinity at p M levels (Kohama and Kendrick-Jones, 1986) and with a binding capacity of 2 mol CaZt per mole myosin (Table V). These data are comparable with those of myosin from scallop muscle, a typical myosin that is in an active form when it binds in Ca-containing solution. Release of Ca2' from scallop myosin upon the withdrawal of Ca2+inactivates its activity (Kendrick-Jones et al., 1970). The Ca-binding properties of Physarum myosin suggest that its calcium switch resembles that of scallop myosin (see Section IV,B). However, the effect of Ca2' for the interaction is quite distinct; Physarum myosin is in an active form when it loses Ca2+and in an inactive form when it binds Ca2t (Fig. 5).

A. Calcium-Binding Light Chain as a Ca-Receptive Subunit of Physarum Myosin Physarum myosin is composed of a pair of heavy chains (230 kDa in SDS-PAGE) and two pairs of light chains (18 and 14 kDa in SDS-PAGE). The domain structure of heavy chain has been examined; the binding sites for ATP, actin, and light chains are all in the heavy chain (Kohama et al., 1988b). The Ca-binding site is localized in the 14-kDa light chain, which we named the calcium-binding light chain (CaLc). The primary structure of CaLc was determined by both peptide analysis and cDNA cloning as shown in Fig. 6 (Kobayashi et al., 1988), and its molecular weight is calculated to be 16,084 Da. The EF-hand structure, which is a consensus sequence for Ca-binding proteins (Kretsinger, 1980), is identified at one position at the N terminal. The highest homology of CaLc

TABLE V Calcium Binding to Pbysarum Myosin and Its Subunits ~

~

Myosin (phosphorylated) Myosin (dephosphorylated) Calcium-binding light chain Phosphorylatable light chain

1.27 % 0.16 mol/mol (n = 3) 1.21 2 0.07 mol/mol ( n = 3) 0.37 -C 0.092 mollmol ( n = 6 ) ND

Nore. The binding of CA" to myosin and its subunits was measured in 0.5 M KCI, 1 nM MgClz, 20 m M Tris-HCI (pH 7.5) and 30 pM Ca" containing 4'Ca by equilibrium dialysis (Kohama et al. 1991a,b). ND, not detectable.

68

AKlO NAKAMURA AND KAZUHIRO KOHAMA

Physarum CaM 10 20 30 40 50 60 A C - V D S L T E E O I A E F K E A F S L F D K D G ~ ~ ~ N I ~ B L ~ S ~ ~ ~ Q D M I N ~ ~

70

80

90

100

110

120

G T I D E ' P B E ' L T B B I R E A F K V F D K D G N G F I S ~ ~ ~ ~ S D B E

130

140

VDEMIRBADVDGDGWNYDEFVKMMLSK

Physarum CaLc 10 20 30 40 50 60 Ac-TASAWIOECFPIFDKD~KVSIBBLGSAtRSLGlWPTNAELNTIK~LWLKEFDLATF 70 80 90 100 110 KTWRKPIKTPTEQSKEbEDAPRALDKBGN~IO~LRQL

120

130 140 VSVSGDGAINYESFVDbEVTGYPLASA FIG. 6 Amino acid sequences of calcium-binding light chain (CaLc) (Gene Bank/EMBL accession number(s) 503499) of Physarum myosin (Kobayashi etal., 1988) and Physarum calmodulin (CaM) (DDBJ/Gene Bank/EMBLaccessionnumber(s)AB022702) (Todaetal., 1990).Molecular weights and statistical pIhfor CaLc are 16,080and 4.33, and for CaM 16,606 and 3.92.

is found in calmodulin of bovine brain (see Section IV,C for phylogenic considerations). On a biochemical basis, CaLc shows calmodulin-like activity in that it activates phosphodiesterase activity (Table VI; Fig. 7). Interestingly, Ca2' at TABLE VI Summary of Functional and Structural Properties of Calcium-Binding Light Chain of Physarum Myosin Binds Ca"" Interacts with actin calcium-dependentlyh Interacts with heavy chain of skeletal muscle myosin as substitute for essential light chain" Shows similarity with vertebrate calmodulin in the amino acid sequence and in activating phosphodiesterase" Confers calcium inhibition on the actin-myosin interaction by binding to actin" "Kohama e t a / . (1991a). " Kohama et al. (1988a). ' Kohama et al. (1991b). " Kobayashi ef al. (1988). Kohama et al. (1985).

f . POLYCEPHALUMCALCIUMREGULATION

69

Y

FIG.7 CaLc stimulates phosphodiesterase activity in the presence of Ca2+.Phosphodiesterase activities (continuous line) (Kohama er al., 1991b) were measured in the presence of CaLc under conditions that allowed direct comparison with the ATPase activity as shown by the broken line.

pM levels activates phosphodiesterase activity through CaLc in the reverse manner as Ca2+inhibition of the ATPase activity of Physarurn myosin via CaLc. It has been determined that CaLc is not the site of activation or inhibition by Ca”. Thus, CaLc appears to work merely as a Ca-receptive subunit in Physarum myosin (Kohama et al., 1991b). Other evidence for CaLc being a Ca-receptive subunit includes the following: (i) The mobility of CaLc in SDS-PAGE of Physarum myosin changes in the presence of Ca2+(Kessler et al., 1980), (ii) the CaLc band after SDS-PAGE of Physarum myosin binds 4sCa2+(Kohama et al., 1985, 1986 a), and (iii) biochemical measurement (Table V) shows that CaLc binds 0.4 mol Ca” per mole. This figure is too low to explain Ca binding (1.3 mol Ca2’ per mole) of parent myosin. We speculate that the Ca-binding activity of CaLc may increase when incorporated into the myosin molecule. As will be described in Section V,A,1, the 18-kDa myosin light chain is phosphorylatable and is called the phosphylatable light chain (PLc). The

70

AKlO NAKAMURA AND KAZUHIRO KOHAMA

isolated PLc binds to skeletal muscle heavy chain as a substitute for 5 3 ' dithiobis(2-nitrobenzonic acid) (DTNB) light chain (Kohama et al., 1991b), but it does not bind Ca2+(Table V).

B. Speculative Mode of Ca Binding of Physarum Myosin Based on the Knowledge of Scallop Myosin The crystal structure of the regulatory domain of scallop myosin, which is composed of the essential and regulatory light chains and the light chainbinding fragment of the heavy chain, shows that essential light chain sequesters Ca2+at its N-terminal EF-hand structure, where the regulatory light chain is closely associated (Xie et al., 1994). The isolated essential light chain is unable to bind Ca2+.However, the regulatory domain can bind Ca2+ as strongly as parent scallop myosin (Kwon et al., 1990). The association of regulatory light chain allows the regulatory domain to bind Ca2+by stabilizing the bond. Chimeras between the essential light chain of Ca-binding scallop myosin and that of non-Ca-binding cardiac myosin were produced in Escherichia coli as recombinant proteins, and it was demonstrated that the third EFhand structure of the scallop essential light chain was required for the myosin to bind Ca2+ (Jancso and Szent-Gyorgyi, 1994). Similar analysis was carried out with chimeras between regulatory light chains of scallop and skeletal muscle myosins. The crucial portion for Ca binding by scallop myosin is at the C terminal of scallop regulatory light chain (Fromherz and Szent-Gyorgyi, 1995). Analysis with amino acid replacement showed that Gly117 at the C terminal of the regulatory light chain is of primary importance to support Ca2+binding to the scallop essential light chain (Jancso and Szent-Gyorgyi, 1994). Physarum CaLc and PLc are classified into essential and regulatory light chains, respectively, as shown in Table VI (Kohama et al., 1991b). An EFhand structure is identified at the N terminal of CaLc of Physarum myosin as described previously (Fig. 6). Resembling scallop myosin, Ca binding to CaLc is too weak to explain the Ca-binding activity of Physarum myosin (Table V). We speculate that Physarum myosin binds Ca2+at its N terminal in a mode similar to that for scallop myosin and that the binding is stabilized by PLc. It would be intriguing to know whether PLc has a Gly residue analogous to Gly117 in its C-terminal portion.

C. Phylogenic Considerations of Ca-Binding Light Chain of Physarum Multiple alignment of amino acid sequences of essential light chains (ELc) including CaLc were performed with DNASIS (Version 3.6), and phyloge-

71

f . POLYCEPHALUMCALCIUMREGULATION

netic trees were constructed from the sequence alignment with the Neighbor-joining method in the PHYLIP package (Version 3 . 5 ~ )Physarum . ELc showed the highest identity with Dictyosteliiim ELc (65%). The identity with protista and other invertebrate is 19%, and the level of identity with vertebrate and invertebrate ELc is 16%. As shown in Fig. 8, the sequences were separated into two major clusters, with one cluster containing skeletal muscle and smooth muscle. Physarum and Dictyosteliiim ELc sequence form a subcluster with high bootstrap probability (100%) in the protista/ other invertebrate clusters. We speculated that Physarum ELc evolved by a different route to vertebrate ELc.

Acanthamoeba

FIG.8 A phylogenetic tree constructed from the alignment of 15 sequences of myosin essential light chains using the SEQBOOT. PROTDIST, NEIGHBOR, and CONSENSE prograins provided in PHYLIP 3 . 5 ~(Felsenstein. 1993). The numbers at each branching point show bootstrap values (100 replications).

72

AKlO NAKAMURA AND KAZUHIRO KOHAMA

V. Phosphorylated State and Calcium Inhibition of Physsrum Myosin A. Phosphorylation and Dephosphorylation of Physarum Myosin

1. The Sites of Phosphorylation in Physarum Myosin Myosins from vertebrate muscle and nonmuscle sources are purified in the unphosphorylated state. However, those from lower eukaryotes, such as Physarum, Dictyostelium (Kuczmarski and Spudich, 1980; Maruta et al., 1983a), and Acanthamoeba (Collins and Korn, 198l), are prepared in the phosphorylated form. In the case of Physarum myosin, the total phosphate content as determined after assignment was 4.0-6.8 mol Pi per mole myosin (Kohama and Kendrick-Jones, 1986). We cultured Physarum cells in the presence of H3[32P]04and then prepared crude myosin. Autoradiography after SDS-PAGE showed that the major site of phosphorylation is in the heavy chain, but PLc is also partly phosphorylated (Kohama and KendrickJones, 1986). 2. Dephosphorylation and Calcium Inhibition We dephosphorylated Physarum myosin with phosphatase (Ogihara et al., 1983) and compared its actin-activated ATPase activity with that of phosphorylated myosin (Kohama et al., 1991a). As shown in Fig. 9, the activity of phosphorylated myosin is high in the absence of Ca2' and decreased with an increase in the Ca2+ concentration. In contrast, the activity of dephosphorylated myosin was low irrespective of Ca2+concentration. We examined the Ca-binding activity of myosin following dephosphorylation. As shown in Table V, dephosphorylated myosin bound Ca2+ as strongly as the phosphorylated form. Thus, dephosphorylation of Physarum myosin minimizes its ATPase activity so that there is no activity to be inhibited by Ca2+(Kohama et al., 1991a). The next step is to identify the site(s) that is responsible for the effect of dephosphorylation of myosin by replacing phosphate groups.

B. The Role of Actin in Calcium Inhibition 1. Dephosphorylation Affects the Affinity of Physarum Myosin to Actin Dephosphorylation reduces the affinity of myosin for actin, whereas ATPase maximum activity (Vmax) remains unaffected (Kohama et al., 1991a).

a

b .

t o -* IlKm I

0

n.s

I/Acl

2 0 4 0

I I .Q

Actin (mp ml -1)

FIG. 9 (a) Actin-activated ATPase activities of control, phosphorylated Physarum myosin ( 0 ) and . dephosphorylated Physarum myosin (0)(Kohama et a[., 1991a). Dephosphorylation reduced phosphate to 1.6 mol P, per mole of 500 kDa myosin. The activity was measured by calorimetry in 0.08 rng/ml of phosphorylated or dephosphorylated Physarum myosin, 0.08 mgl ml skeletal muscle actin, 13 m M KCI, 1 mM ATP, 2 mM MgCI2 20 mM Tris-HCI (pH 7.5), 0.1 m M DTT, and 0.1 mM EGTA-Ca buffer. (b) Effect of actin on ATPase activities of phosphorylated ( 0 )and dephosphorylated (0)Physarum myosins (KohamaetaL, 1991a). Double-reciprocal plots (inset) showed that V,,, (the ATPase activity at infinite concentration of actin) was not significantlydifferent between phosphorylated and dephosphorylated myosins. K , (the actin concentration required to achieve one-half V,,,,,) was smaller for phosphorylated myosin than for dephosphorylated myosin. Assay conditions: 0.044 mg/ml myosin, 12 mM KCI, 1 mM ATP, 2 mM MgC12,20 mM Tris-HC1 (pH 7.5). 0.1 mM DTT, and 0.1 mM EGTA.

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AKlO NAKAMURA AND KAZUHIRO KOHAMA

Thus, when actin concentration is increased toward the concentration that gives Vmax, the activity of dephosphorylated myosin is similar to that of phosphorylated myosin. Thus, under actin-poor conditions, the activities of the dephosphorylated myosin were reduced to the basal level, irrespective of Ca2' concentration (Fig. lob). On the other hand, the activities of phosphorylated myosin were high in the absence of CaZt and low in the presence of CaZt (Fig. 10a). When actin concentration was elevated about 10-fold over that of myosin on a weight basis (Fig. lOd), the activities of the

Actin-Rich

Actin-Poor

0

50

100

0

I

I

I

50

100

150

--

0

so

100

0

50

100

d

I50

nmolmln-hny'

FIG. 10 Effect of phosphorylation on the actin-activated ATPase activity of Physariim myosin

(Kohama et al., 1991a). Physariim myosin was prepared in the phosphorylated form and an aliquot was dephosphorylated as described in the legend to Fig. 9. Actin-activated ATPase activities of the phosphorylated (a, c) and dephosphorylated (b, d) myosins were determined by the colorimetry in the presence of 0.1 rnM EGTA (solid bars) or 0.1 mM Ca2' (open bars). The protein concentrations were 0.072 mglml myosin (a-d), 0.056 mg/ml actin (a, b), and 0.46 mg/ml actin (c, d). Numbers on the abscissa are the ATPase activities in nmol min-' myosin, and bars are SEM ( n = 3). Under actin-rich conditions, the ATPase activity of dephosphorylated myosin shows Ca inhibition.

P.POLYCEPHALUM CALCIUM REGULATlON

75

dephosphorylated myosin were similar to those of phosphorylated myosin, i.e., high in the absence of Ca” and low in its presence.

2. Ca-Binding Mode, Rather Than Phosphorylating Mode, Is of Physiological Importance Under actin-poor conditions (see Section V,B,l), the ATPase activity of Physarum myosin is modified both by binding Ca2+ to myosin and by changing its phosphorylated state (Figs. 10a and lob). Under actin-rich conditions, only the former mode regulates the activities (Figs. 1Oc and 10d). Therefore, the crucial factor in determining which of the two modes is dominant in vivo is the concentration of actin in Physarum cells. In muscular tissues, the concentration of actin is comparable to that of myosin on a weight basis. Nonmuscle tissues, however, are expected to be in an actin-rich condition because actin greatly exceeds myosin in concentration (Pollard and Weihing, 1974). This is true in Physarum cells (Ishikawa et al., 1991), and hence the mode of Ca binding should be physiological. Change in the phosphorylated state of myosin may not play a major role in vivo.

3. In Vitro Motility Assays in Relation to Actin-Rich and Actin-Poor Conditions Superprecipitation studies with a spectrophotometer detect an ATP-dependent shrinkage of the actomyosin complex (Kohama et al., 1986b). Physarum myosin superprecipitated with actin is in the untreated, phosphorylated form. The extent of superprecipitation was higher in EGTA than in Ca2+(Ogihara el al., 1983). These observations are in agreement with the results of ATPase measurement under actin-poor conditions (Fig. 2). The myosin-coated surface assay also provides actin-poor conditions because single actin filaments move on a two-dimensional surface coated with myosin (Fig. 5). When coated with Physarum myosin in the phosphorylated form, rapid ATP-dependent movement of actin can be observed especially in the absence of Ca2+.However, actin does not move on the surface coated with dephosphorylated Physarum myosin irrespective of Ca’+ concentrations (Kohama et al., 1991a). Another in vitro motility assay detects an ATP-dependent movement of myosin along the actin cables fixed inside the cell membrane of Nitella. This Nitella-based motility assay allows myosin to interact with actin under actin-rich conditions. Accordingly, Physarum myosin moves faster in EGTA than in Ca2+irrespective of whether it is in the untreated, phosphory-

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AKlO NAKAMURA AND KAZUHIRO KOHAMA

lated form as shown in Figs. l l a and b or 11 in the dephosphorylated form as shown in Figs. l l c and l l d (Kohama et al., 1991a).

C. Dependence on Ca2+of Protein Kinases and Phosphatases That Act on Myosin Kinases specific for the myosin heavy chain and PLc have molecular weights of 76 and 55 kDa, respectively, as indicated by SDS-PAGE (Okagaki et

Phosphorylated

Dephosphorylated

a

C

1

I

loc ':LA 2o

00

20

0.2

0.4

0.6

-

o

0.8

b

0.2

0.4

0.6

0.8

d

20

10

0

o

0.2

0.4

0.6

0.a

0

I

1

F

I

0.2

0.4

0.6

0.8

pm sec-' FIG. 11 Velocities of movement of phosphorylated and dephosphorylated myosins as examined by the Nitellu-based motility assay (Kohama et al., 1991a). Actin cables in a Nitella internodal cell were exposed by internal perfusion. Small latex beads coated with phosphorylated (a, b) or dephosphorylated (c, d) myosin were introduced to the actin with EGTA solution (a, c) or with Ca2' solution (b, d). Velocities of the ATP-dependent movement of the beads were measured by observations under Nomarski microscope. Ordinate, number of moving particles; abscissa, velocities (pmlsec). Arrows indicate average velocity of the movement. With the Nitella-based assay, dephosphorylated myosin moved in the similar way to phosphorylated myosin. The movement of dephosphorylated myosin is blocked by Ca2+ almost perfectly. The data of a and b confirms those of a and b in Fig. 5 because Physarum myosin in Fig. 5 is in a phosphorylated form.

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77

al., 1991a,b). The effect of Ca2+on their activities is similar to its effect on myosin, i.e., these activities are most pronounced in the absence of Ca2' and are reduced with an increase in Ca2+concentration. A sole Ca-binding protein is involved in the inhibitory effect of Ca2+(see Section VI1,A). The phosphatase activities for myosin are much lower in Physarum cells than the kinase activities. However, native actomyosin preparation contained not only the kinase activities but also the phosphatase activities. We incubated the preparation with [32P]y-ATPto phosphorylate myosin; the phosphorylation was terminated by the addition of the kinase inhibitor staurosporine (Okagaki et al., 1991a). We observed that the radioactivity incorporated into these proteins was gradually lost by the phosphatase activity. The decrease in radioactivity was abolished by the phosphatase inhibitor okadaic acid. The phosphatase activities were low in the presence of Ca2t and increased with an increase in [Ca2+]to p M levels. Furthermore, the calmodulin inhibitor trifluoperazine also inhibited the phosphatase activities, suggesting the involvement of calmodulin in these activities (Kohama et al., 1993). Figure 12 summarizes the role of Ca2' and phosphorylation in the relationship between Ca binding and phosphorylation of Physarum myosin (Kohama el al., 1990, 1993). Step 2 is mediated by phosphatase activities, which works at high Ca2+levels. Kinase reactions are expressed by step 4, which occurs at low Ca2+concentrations. Steps 1 and 3 are mediated by direct Ca binding to myosin. The Ca-binding activity remains the same regardless of the phosphorylated state of myosin (see Table V, and Section V,A,2). Under actin-poor conditions (see Section V,B,l), the sole form of active myosin is phosphorylated and free from Ca2' (Fig. 12). As discussed in Section V,B,l, dephosphorylation of myosin reduces its affinity to actin, and an actin-rich condition rescues the reduction. Therefore, under actinrich conditions (Fig. 12), as found in Physarurn cells, myosins in both phosphorylated and dephosphorylated forms are in the active form in low [Ca2+].Ca binding to both myosins inhibits their activities. In other words, the physiological switch that determines whether Physarum myosin is active or inactive is Ca binding to myosin (see Section V,B,2).

VI. Actin-Binding Proteins of Physarum That Are Involved in Calcium Inhibition

The actin-linked mode of calcium inhibition has been indicated in an earlier stage of our studies (Kohama, 1981; Kohama et al., 1985). We currently understand that the actin-binding proteins may back up the Ca inhibition due to Ca binding to myosin.

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AKlO NAKAMURA AND KAZUHIRO KOHAMA

FIG. 12 A model of the role of Ca2’ and phosphorylation in fhysctrum myosin. Myosin is active only in the phosphorylated form at low Ca” concentrations (asterisk). Binding of Ca2+ occurs irrespective of the extent of phosphorylation when high [Ca2+](see steps 1 and 3). Myosin can be dephosphorylated at high [Ca”] (step 2) and phosphorylated at low Ca” (step 4). The Ca-binding proteins are calcium-binding light chain (CaLc) for steps 1and 3, calmodulin for step 2. and calcium-dependent inhibitory factor for step 4. The phosphorylated form of myosin is denoted by P. Under actin-poor conditions, the sole active form of myosin is shown by *, and under actin-rich condition the two active forms of myosin are shown by * and **. As described in the text, the actin-rich condition is physiological for fhysarurn plasmodium. Therefore, binding and release of Ca2+should be the physiological mode of regulation.

A. Search for a Regulatory Mode Other Than That Inhibited to Myosin When ATPase activity of native actomyosin was compared with actomyosin reconstituted from purified actin and myosin, the inhibitory effect of Ca2+ of the former was more pronounced than that of the latter (Kohama and Kohama, 1984). If the inhibition were exerted exclusively through myosin, the effect should be the same. Thus, the inhibitory factor(s) is speculated to be lost during the purification. The same has been suggested by the following experiment. When Mg2+ concentration was elevated in the assay medium, calcium inhibition of the actin-myosin interaction was obscured (Table VII), presumably by competition for the Ca-binding site of myosin. A fraction was obtained from the actomyosin preparation that shows calcium inhibition. This fraction was mixed with actin and myosin at high [Mg2’] concentration and superprecipi-

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P. POLYCEPHALUMCALCIUM REGULATION

TABLE VII Effect of Calcium-Binding Light Chain (CaLc) of Physarum Myosin and Bovine Brain Calmodulin on CaInhibition (%) of Actin-Activated ATPase Activity of Physarum Myosin ~

ATPase activity (nmol/rnin/rng myosin) Myosin (30 pgiml)

EGTA

50 p M Ca”

%“

Untreated NEM modified NEM modified

0 0 3.5

3.5 3.5 3.5

5 5 5

96.8 43.9 66.7

33.9 27.3 33.3

65.0 37.9 50.0

Untreated Untreated Untreated

0 0 3.5

3.5 8.5 8.5

20 5 5

79.3 119.5 123.2

28.0 97.6 61 .o

64.7 18.3 50.5

Nore. (Top) Desensitization by CaLc of the myosin desensitized by NEM modification (about 2 mol NEM/mol myosin) (Kohama et 01.. 1987). (Bottom) Desensitization by CaLc of the myosin apparent by desensitization in the presence of high (8.5 m M ) Mg” (Kohama et ul.. 1985). Assay conditions: 1.5 mM ATP, 0.1 m M EGTA-Ca buffer with a pH-stat method at p H 7.50 and 25°C. Actin concentrations are 100 pg/ml. ‘‘ 100 X (ATPase in EGTA-ATPase in Ca”)/(ATPase in EGTA).

tated, and the ATPase activity of the mixture was shown to be inhibited by Ca2+(Kohama, 1981). This inhibitory fraction binds to an affinity column, suggesting that Ca inhibition is exerted via actin. We speculate that the Ca-binding components in the factor(s) could be CaLc andlor calmodulin as will be described in Seclions VI,B and V1,C.

B. CaLc as an Actin-Binding Protein When CaLc was isolated from Physarunz myosin, it was found to interact with actin (Table VI). The interaction was first demonstrated by the binding to an actin affinity column (Kohama et al,, 1985) and was further confirmed by detecting the effect of CaLc on the actin polymerization (Fig. 13). The effect, as examined by the measure of viscosity and flow birefringence of actin, was inhibited by Ca*+(Kohama et al., 1988a). Such effects have never been observed with light chains isolated from vertebrate myosins, although it has been observed that essential light chains show a subtle interaction with actin (Yamamoto and Sekine, 1983; Kohama, 1987). To determine whether CaLc confers calcium inhibition to the actinmyosin interaction of Physarum, we examined its effect on the actin-activated ATPase activity of Physaritm myosin under the conditions in which the activity was not sensitive to Ca”. As shown in Table VII, Ca2+hardly

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AKlO NAKAMURA AND KAZUHIRO KOHAMA

0.2

1

o ! 0

I

I

I

30

60

90

min

FIG. 13 Interaction of calcium-binding light chain (CaLc) as monitored by Ca-dependent inhibition of actin polymerization (Kohama et al., 1988). Skeletal muscle monomeric actin, whose polymerization alone showed no Ca dependence, was mixed with KCI at a final concentration of 114 mM and allowed to polymerize. Viscosity was measured with an Ostowald capillary viscometer at 25°C. Conditions: 140 kg/ml skeletal muscle actin, 3.3 pg/ml CaLc, 1 mM MgC12,23 mM Tris-HCI (pH 7.5), and 0.1 mM EGTA-Ca buffer. 0 ,in 0.1 mM EGTA; 0, in 0.1 mM Ca2+.

affected the activity in the absence of CaLc. However, a lower activity was detected in the presence of Ca2+ when CaLc was mixed with actin and myosin. In Physarum cells, a significant amount of CaLc is present without associating myosin (Kohama et al., 1985), although CaLc is present in the cells as a myosin subunit. We speculate that the isolated CaLc may take part in such a role. The previous experiments were carried out using CaLc purified from P h y s a n m Now that recombinant CaLc is available, we need to confirm the results and to identify the sequences responsible by mutating CaLc.

C. Caldesmon-like Protein of Physarum We purified a 210-kDa, heat-stable protein from Physarum that reacts with an antibody against caldesmon, an actin-binding, regulatory protein of

81

f . POL YCEPHALUM CALCIUM REGULATION

smooth muscle (Ishikawa et al., 1991, 1992). This caldesmon-like protein binds to actin and stimulates the actin-activated ATPase activity of Physarurn myosin and the actin motility on a surface coated with Physarurn myosin (Table VIII). The stimulation is abolished when calmodulin is mixed with the caldesmon-like protein in the presence of Ca2+.Furthermore, calmodulin is found in Physarurn (Ishikawa et al., 1991). Therefore, caldesmon-like protein is able to produce calcium inhibition together with calmodulin. Such an actin-linked mode may back up the inhibitory effect of Ca2+ on Physarurn myosin by CaLc (Fig. 14). It must be noted that the stimulatory effect of caldesmon-like protein is distinct from the inhibitory effect reported for smooth muscle caldesmon (Sobue and Sellers, 1991). However, smooth muscle caldesmon is also shown to stimulate its actin-myosin interaction under the specified conditions. Because this stimulation is related to the myosin-binding property of caldesmon (Lin et al., 1994), we need to reexamine the regulatory mode of Physarurn caldesmon-like protein.

D. Other Actin-Binding Proteins of Physarum Fragmin binds to actin filaments at their barbed end and severs them in the presence of Ca2’ (Hasegawa et al., 1980). Furthermore, it forms a complex with monomeric actin, and the complex becomes a nucleus for polymerization of actin into filaments (Hasegawa et al., 1980; Hinssen, 1981a,b; Sugino and Hatano, 1982; Maruta et al., 1983b). Fragmin is considered to be an analog of vertebrate gelsolin (Yin and Stossel, 1980). Isoforms of fragmin were purified by Uyeda et al. (1988) and Furuhashi and Hatano (1989). Profilin was also identified in Physarurn as another protein that forms a complex with monomeric actin (Ozaki et al., 1983). TABLE Vlll Velocities of Movement of Actin Filaments on Coverslips Coated with Physarum Myosin

Actin filament

Velocity ( p d s e c , n = 30)

Control (0.1 mM EGTA) + Caldesmon-like protein (0.1 mM EGTA) Control (0.1 mM Ca2+) + Caldesrnon-like protein (0.1 mM Ca”) + Caldesmon-like protein + calmodulin (0.1 mM Ca”)

1.39 ? 0.49 1.95 t 0.66 0.99 ? 0.42 1.26 ? 0.60 1.08 t 0.50

Note. Caldemon-like protein was purified from Physarurn plasmodia as a heat-stable, actinbinding protein. Unlike smooth muscle caldesmon, caldesmon-like protein stimulated the velocity of movement. Because calmodulin in the presence of CaZ+abolishes binding to actin, caldesmon-like protein works cause calcium inhibition (Ishikawa et al., 1991).

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AKlO NAKAMURA AND KAZUHIRO KOHAMA

FIG. 14 Relationship between Ca2+ and the actin-activated ATPase activity of Physarum myosin (Jshikawa et al., 1991, 1992). (A) Myosin is regulated both by phosphorylation and by Ca” binding. Myosin must be phosphorylated to be active, and Ca” inhibits the activity by binding to the myosin. (B) A caldesmon-like protein of Physarum enhances the activity. The enhancement is abolished by calmodulin in the presence of Ca2’ with resulting enhancement of calcium inhibition of the activity (Ishikawa et al., 1991, 1992). According to Section V,B, the assay condition is actin poor.

The actin-binding proteins that may be involved in the organization of Physarum plasmodium are high-molecular-weight actin-binding protein (Sutoh et al., 1984), a homolog of smooth muscle filamin (Wang, 1977); connectin/titin (Ozaki and Maruyama, 1980; Gassner et al., 1985), an elastic protein originally found in muscle tissue (Maruyama et al., 1976); a 52kDa protein that bundles actin filaments (Itano and Hatano, 1991); and

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caldesmon-like protein (Ishikawa et al., 1991, 1992). lshikawa et al. (1995) purified an ATP-dependent actin-binding protein from Physarum plasmodia. The effect of actin binding has not yet been detected. As described in Section VI,C, caldesmon-like protein together with CaM takes part in the calcium inhibition of the actin-myosin interaction (Jshikawa et al., 1991). Sugino and Matsumura (1983) showed that the tension of the actomyosin thread was reduced by fragmin in the presence of Ca" (Table 11). Connectin/titin, although prepared from skeletal muscle, binds to actin in a calcium-dependent manner and inhibits the actin-myosin interaction (Kellermayer and Granzier, 1996).

VII. Ca-Binding Proteins in Physarum A. Three Ca-Binding Proteins That Support Calcium Inhibition In this review, we have described three Ca-binding proteins in Physarum as summarized in Fig. 15. The one most characterized is CaLc as shown in Table VI (Kohama et al., 1992). Not only does CaLc work as a Ca-binding subunit of Physarum myosin but also it binds to actin. Both myosin- and actin-linked properties contribute to the actin-myosin interaction as described in Sections IV,A and VI,B. The amino acid sequence of Physarum calmodulin (Fig. 7 ) is very similar to that of brain calmodulin (88% identity). Calmodulin binds to caldesmonlike protein in the presence of Ca2+to abolish the stimulatory effect of the protein on the actin-myosin interaction (see Section V1,C). The abolition allows calcium inhibition of the actin-myosin interaction. In the presence of Ca", calmodulin stimulates myosin phosphatase activity (Kohama et al., 1993). In the absence of Ca2+,myosin phosphatase remains low to keep myosin phosphorylated and thus sensitive to Ca". Therefore, calmodulin indirectly contributes to calcium inhibition of myosin. As described in Section V,C the activities of the kinases for the heavy (76 kDa in SDS-PAGE) and light (55 kDa) chains of Physarum myosin are also calcium inhibitory, i.e., at low Ca2+concentration Physarum myosin tends to be phosphorylated. Because phosphorylated myosin is active, calcium inhibition of kinase activities also contributes to the inhibition of the actin-myosin interaction. A Ca-binding protein of 38 kDa in SDS-PAGE has been shown to be involved in the calcium inhibition of kinase activities and is called calcium-dependent inhibitory factor (ClF) as shown in Fig. 15 (Okagaki et al., 1991a,b). Actin kinase of Physarum has also been shown

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AKlO NAKAMURA AND KAZUHIRO KOHAMA

CALCIUM INHIBITION

FIG.15 Three Ca-binding proteins involved in calcium inhibition of the Physarum actomyosin system. CIF, calcium-dependent inhibitory factor for kinases (Okagaki ef al., 1991a.b); CaLc, calcium-binding light chain of Physarum myosin (Kobayashi et al., 1988; Kohama et al., 1991b); CaM, Physanrm calmodulin (Toda et al., 1990); CaD, caldesmon-like protein of Physarum (Ishikawa et al., 1991). CaLc works as a sole Ca2+-receptive protein in Physarum myosin, producing calcium inhibition. When CIF binds Ca2+,CIF interacts with kinases for Physarum myosin. The myosin in the phosphorylated form interacts with actin more effectively than when dephosphorylated. Ca2+stimulates myosin phosphatase activity (Kohama el al., 1993) to dephosphorylate myosin, which is lost calcium inhibition. CaD enhances the interaction by binding to actin. The enhancement is negated by CaM binding Ca2+.CaLc binds actin and allows Ca2+to cause inhibition (Kohama et al., 1985).

to be subject to calcium inhibition (Okagaki et al., 1991a). When actin forms a complex with fragmin, the kinase is able to phosphorylate actin (Furuhashi and Hatano, 1990, 1992; Furuhashi et al., 1992; Gettemans et al., 1992, 1993).

B. Preliminary Characterization of Recombinant 40-kDa Protein A 40-kDa Ca-binding protein was purified from Physarum (Nakamura et al., 1994). The cloning of its cDNA showed that it is identical to LAV1-2 cloned by Laroche et al. (1989) as an abundant mRNA specific to Physarum plasmodia. We expected 40-kDa protein to work as the CIF described in Sections V,C and VII,A because CIF has a similar molecular mass (38 kDa) (Okagaki et al., 1991a,b). However, the 40-kDa protein obtained as a recombinant protein in E. coli failed to exert an inhibitory effect on myosin heavy and light chain kinases. We interpreted this to mean that CIF differs from 40-kDa protein. The other interpretation is that posttran-

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scriptional modifications such as glycosylation and phosphorylation may be required for 40-kDa protein to inhibit the kinases.

1. Structure of 40-kDa Protein As shown in Fig. 16, the 40-kDa protein consists of 355 amino acid residues (Nakamura et al., 1994). The calculated molecular mass and p l are 40,508 Da and 4.97, respectively. Four EF-hand Ca-binding consensus sequences (Kretsinger, 1980) were found in the C-terminal half, which also contained a consensus sequence for nuclear proteins as reported by Robbins et al. (1991). The sequence is 226 RKIDTNSNGTLSRKEFR 242. The N-terminal half contained an @-helical structure predicted by MacDNASIS Pro 1993. Residues 1-32 formed aggregates of the 40-kDa protein.

2. Ca-Binding Properties of the 40-kDa Protein Recombinant 40-kDa protein was obtained as a soluble form in low ionic strength. However, elevation to p M levels allowed the 40-kDa protein to form large aggregates (Nakamura et al., 1994; Nakamura and Kohama, 1995). The ability to form aggregates was abolished by producing a mutant that was deleted for residues 1-32. When this truncated protein was subjected to the Ca-binding assay with a flow dialysis apparatus (Womack and Colowic, 1973), 4 mol Ca/mol of 40-kDa protein was bound with halfmaximal binding at a concentration of 0.29 p M . The concentration is much lower than that of Physarum calmodulin (5.7 p M ) , which was also obtained in the recombinant form (Fig. 6). The C-terminal half of the 40-kDa protein is highly homologous to calmodulin, whereas the N-terminal half is devoid of calmodulin sequences. We speculate that the secret of the higher Cabinding activity of the 40-kDa protein is in its N-terminal half. It will be of interest to see whether a fusion protein of calmodulin with N-terminal half 40-kD protein has increased Ca-binding properties.

3. Other Properties of the 40-kDa Protein An antibody was raised against the 40-kDa protein. Immunofluorescent studies with the antibody showed staining of Physarum nuclei, although the cytoplasm was also partially stained (Fig. 17). When the staining between the plasmodial and amebal stages of Physarum was compared (see Section VIII), the staining was observed only in plasmodial stages. The different expression of the 40-kDa protein was confirmed by subjecting total lysates and total RNAs from both plasmodial and amebal cells to Western blotting and RT-PCR, respectively. When the genomic PCR product was compared with that from RT-PCR, they showed the same mobility

86

AKlO NAKAMURA AND KAZUHIRO KOHAMA

A~ - S Y ~ B A W N10P M S S L D E I20I S I ~ L K S K30T G A V K B I F4S0O E L M R B ~50K K ~ B N L B g60L Q I ( U D 70 80 90 100 110 120 B T S F A B K B D R D R C B A ~ I A O K B O B O ~ Y ~ ~ O N B F D ~ ~ R B R B ~ G D ~ K 130

140

150

160

170

180

190

200

210

220

230

240

~~LLKDLBDIU8GY~SKP~SBB~ILROL~SSAVSGSGKFSFODLKO~

K Y A D T I P B G P L K U ~ ~ G ~ S Y I r r V A V A V ~ ~ V ~ F ~ I ~ S N ~ S ~ 250 260 270 280 290 300 ~ B F J R L G P D K K S V O D A L F G R aYVeLGLCLLVLRILYAFADFDKSGQ Y ~ 310

320

330

340

350

~ O ~ ~ D A a I P S S ~ K ~ B O F S ~ ~ D S KVLLWEDD S L S Y O B ~

FIG. 16 Structure and function of the 40-kDa protein (Gene Bank/EMBL accession number(s) X14.502). (A) Amino acid sequence of the 40-kDa protein deduced from its nucleotide sequence. Molecular weight, 40,508 Da; statistical pl, 4.97. (B) Domain structures of the parent 40-kDa protein and the truncated form of the 40-kDa protein compared with that of calmodulin (CaM). (C) We expressed the 40-kDa protein, its truncated form, and Physarum calmodulin (CaM) in E. coli and purified them. The measurement of Ca binding to the 40-kDa protein was hampered by its Ca-dependent aggregation. Therefore, we used the truncated form as shown (0).We also measured the Ca-binding activities of CaM (0).Assay conditions; 100 mM NaCI, 20 mM MOPS (pH 7.0), and various concentrations of Cazt.

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87

FIG. 17 Immunostaining of Physarum plasmodium. Physarum plasmodium (strain Ng-1) was treated for fluorescent microscopy (Uyeda and Kohama, 1987) and double stained with D A P I and the antibody against 40-kDa protein. (A) Phase-contrast micrograph; (B) immunofluorescent micrograph. Arrows and arrowheads show the staining of cytoplasm and nuclei, respectively. (C) Nuclear staining with DAPI. Scale bar = 10 pm. (D) Specificity of the antibody. Total homogenate (PCL) and recombinant 40-kDa protein (40K) were subjected to Western blots using the antibody. CBB. protein staining with Coomassie brilliant blue R-250; anti 40K, immunostaining with the antibody.

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AKlO NAKAMURA AND KAZUHIRO KOHAMA

in the agarose gel, indicated that the 40-kDa protein had no introns; this has been confirmed by nucleotide sequencing of the products. 4. Immunostaining of Vertebrate Cells with the Antibody against the 40-kDa Protein CHO-k1 was one of the vertebrate cell lines to react with the antibody against the 40-kDa protein. In Western blots of the cell lysate, the antibody reacted with a 40-kDa band, suggesting the presence of a homolog in the CHO-k1 cell. Immunofluorescent studies showed staining of the nucleolus, as shown in Fig. 18. During mitosis, the dot-like staining of nucleoli moved to chromosomes. At the late stage of mitosis, the dot-like staining disappeared and the staining became homogeneous throughout the cells (Fig. 19). The other cell to react with the antibody was the 10T1/2 cell, which showed immunofluorescent staining at both the nucleolus and endoplasmic reticulum (Fig. 18).

VIII. Amebal Myosin and Amebal-Plasmodia1 Transition The life cycle of Physarum consists of two phases with different modes of motility: uninucleate haploid amebae showing slow ameboid movement and multinucleated diploid plasmodia showing rapid cytoplasmic streaming. Actomyosin was obtained from the amebae as described in Section III,A (Kohama and Takano-Ohmuro, 1984) and amebal myosin was purified from the actomyosin preparation as described in Section II1,B (Kohama et al,, 1986a). Amebal myosin resembles plasmodia1 myosin in its twoheaded, long-tailed shape and in its subunit composition of heavy chain, Ca-binding light chain and phosphorylatable light chain. The heavy chain and phosphorylatable light chain differ between the myosins as examined by peptide mapping, whereas the Ca-binding light chain of both myosins appears to be identical. The actin-activated ATPase activities of the amebal variety did not differ greatly from those of plasmodial myosin; the effect of Ca2' was also inhibitory. Therefore, the different motility between ameba and plasmodium cannot be ascribed to myosin but to other contractile proteins. The contractile proteins specific to plasmodium are the 40-kDa Ca-binding protein discussed in Section VI1,b and high-molecular-weight, actinbinding protein (HMWP) (Uyeda and Kohama, 1987). Immunofluorescent studies with an antibody against HMWP showed that it was detectable during the transition from ameba to plasmodium (Fig. 20). The transition

P. POLYCEPHALUMCALCIUM REGULATION

89

FIG. 18 Immunostaining of vertebrate cells with the antibody against Physarum 40-kDa protein. (A-F) CHO-K1 cells (A-C) and 10 T1/2 cells (D-F) were cultured on coverslips and double stained with the antibody and DAPI. ( A and D) Phase contrast micrographs; (B and E) immunofluorescent images-arrowheads and arrows indicate the staining of nucleoli and endoplasmic reticula, respectively; (C and F) nuclear staining with DAPI (scale bar = 10 pm. (G) Western blots of CHO-K1 cell extracts. The lysate of CHO-Kl cells was applied to the an affinity column conjugated with the antibody. After removing unbound materials, the bound proteins were eluted. PCL, lysate of Physaricm plasmodia (see the legend to Fig. 17); CHO, lysate of CHO-kl cells: AFP, elute from the affinity column; CBB, protein staining with CBB; anti 40K, immunostaining with the antibody.

90

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FIG. 19 Immunofluorescent images of CHO-K1 cells during mitosis. CHO-K1 cells were double stained with D A P I and the antibody against the Physururn 40-kDa protein. A-C, interphase; D-F, metaphase; G-I, anaphase. (A, D, G) phase-contrast micrographs; (B, E, H ) DAPI-fluorescent images, and (C, F, I) immunofluorescent images. As shown by the arrows in C and F, the staining of nucleoli in interphase moved to chromosomes during metaphase. In anaphase (I), the whole cytoplasm was stained with the antibody.

was also examined using antibodies specific to either amebal or plasmodial myosin heavy chain. Stage-specific expression of both proteins was confirmed. Interestingly, intermediate cells with swollen nuclei contained both heavy chains (Anderson et al., 1976). Similar observation was reported (Uyeda et al., 1988) with amebal and plasmodial fragmins, which are homolog of vertebrate gelsolin (Yin and Stossel, 1980). These observations indicate that the proteins related to the actomyosin system were switched in a coordinated manner during the intermediate stage. The switching contributes to a radical reconstruction of the cytoskeletal architecture during amebal and plasmodia1 transition.

IX. Concluding Remarks In this review, motility of Physarum is shown to be subject to calcium inhibition by various methods (Table VI). The major path for calcium

P.POLYCEPHALUM CALCIUM REGULATION

91 Plasmodia

-

Amoeba

PMHC AMHC ? PF AF CaLC

PLC d HMWP

40K 4 FIG. 20 Changes in synthesis ofcytoskeletal proteins that are related to the actomyosin system during amebal-plasmodia1 transition of a colonial strain (schematically presented) (modified from Uyeda and Kohama. 1987). PMHC, plasmodial myosin heavy chain: AMHC, amebal myosin heavy chain: PF, plasmodial fragmin: AF, amebal fragmin; CaLc, calcium-binding light chain expressed in both plasmodiai and amebal myosins; PLC, phosphorylatable light chain of plasmodial myosin: HMWP. high-molecular-weight myosin-binding protein: 40K, 40kDa protein.

inhibition is through myosin: Myosin is in an active form when Ca2+ is absent, and it is inactivated by binding Ca7+to CaLc (Fig. 5). Another route is via actin-binding proteins such as CaLc (Table VII), fragmin (Sugino and Matsumura, 1983),and caldesmon-like protein in association with calmodulin (Table VIII). Phosphorylation and dephosphorylation also modify the properties of Physariim myosin. It is purified in a phosphorylated form. Dephosphorylation procedures reduce its affinity to actin. Although dephosphorylation does not affect its Ca-binding property (Table V), dephosphorylated myosin does not show calcium inhibition. When the actin concentration is increased to the level found in living plasmodia, the calcium inhibition lost by dephosphorylation of myosin is recovered (Fig. 12). Therefore, changes in the phosphorylated state of myosin may not play a major regulatory role. More important for regulation is the binding of Ca2’ to myosin. The kinase activities responsible for phosphorylation of myosin are detectable for both PLc and heavy chain and are inhibited in a Ca-containing solution. Calcium-dependent inhibitory factor is expected to work as a Cabinding protein (Fig. 15). It is important to note that the kinases are active

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without requiring Ca”. Such enzymes are expected to be involved in general cell maintenance and thus be exempted from the regulation by Ca”. However, the discovery of calcium inhibition indicates that exemption is an erroneous idea and suggests that some of the housekeeping enzymes may be under the control of Ca2+if they are associated with novel Ca-binding proteins that should exert an inhibitory effect. We hope that our review may inspire scientists in the search for such proteins.

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Sekine, T., and Kielly, W . W . (1964). The enzymatic properties of N-ethylmaleimide modified myosin. Biochim. Biophys. Acta 81, 336-345. Shimmen, T., and Kohama, K. (1984). Ca2’-sensitive sliding of latex beads coated with Physarum or scallop myosin along actin bundles i n Characeae cells. Abstracts of the papers presented at the Third International Congress on Cell Biology, Tokyo, p. 504. Shimmen, T., and Yano, Y. (1984). Active sliding movement of latex beads coated with skeletal muscle myosin on Chara actin bundles. Protoplasma 121, 132-137. Shoback, D., Thatcher, J., Leombruno, R., and Brown, E. M. (1983). Effects of extracellular Ca2+and Mg” on cytosolic Ca2+and PTH release in dispersed bovine parathyroid cells. Endocrinology 113, 424-426. Simokawa, N., and Yamaguchi, M. (1993). Molecular cloning and sequencing of the cDNA coding for a calcium-binding protein regucalcin from rat liver. FEES Lett. 327, 251-255. Sobue, K., and Sellers, J. R. (1991). Caldesmon, a novel regulatory protein in smooth muscle and nonmuscle actomyosin system. J. Biol. Chem. 266, 12115-12118. Sonobe, S., Hatano, S., and Kuroda. K. (1985). Cytoplasmic movement in a glycerinated model of Amoeba proteus. In “Cell Motility: Mechanism and Regulation” (H. Ishikawa, S. Hatano, and H. Sato, Eds.), pp. 271-282. Univ. of Tokyo Press, Tokyo. Sugino, H., and Hatano. S. (1982). Effect of fragmin on actin polymerization: Evidence for enhancement of nucleation and capping of the barbed end. Cell Motil. Cytoskel. 2,457-470. Sugino, H., and Matsumura, F. (1983). Fragmin induces tension reduction of actomyosin threads in the presence of micromolar levels of Ca”. J. Cell B i d . 96, 199-203. Sutoh, K., Iwane, M.,Matsuzaki, F., Kikuchi, M., and Ikai, A. (1984). Isolation and characterization of a high molecular weight actin-binding protein from Physarum polycephalum plasmodia. J . Cell Bid. 98, 1611-1618. Swanljung-Collins, H., and Collins, J. H. (1991). Ca2’ stimulates the Mg2+-ATPase activity of brush border myosin I with three or four calmodulin light chains but inhibits with less than two bound. J . B i d . Chem. 266, 1312-1319. Takagi, S. (1993). Photoregulation of cytoplasmic streaming. Cell Struct. Funct. 18, 498. [Abstract] Takagi, S., and Nagai, R. (1985). Light-controlled cytoplasmic streaming in Vallisnerra mesophyll cells. Plant Cell Physiol. 26, 941-951. Takagi, S., and Nagai, R. (1986). Intracellular Ca2+concentration and cytoplasmic streaming in Valisneria mesophyll cells. Plant Cell Physiol. 27, 953-959. Taylor, D. L., Condeelis, J. S., Moore, P. L.. and Allen, R. D. (1973). The contractile basis of amoeboid movement. I. The chemical control of motility in isolated cytoplasm. J. Cell Biol. 59, 378-394. Tazawa, M., and Kishimoto, U. (1968). Cessation of cytoplasmic streaming of Chara internodes during action potential. Plant Cell Physiol. 9, 361-368. Toda, H., Okagaki, T., and Kohama, K. (1990). Amino acid sequence of calmodulin from lower eukaryote; Physarum polycephalum. In “Advances in Second Messenger and Phosphoprotein Research Vol. 24. The Biology and Medicine of Signal Transduction” (Y. Nishizuka, M. Endo, and T. Tanaka, Eds.). Raven Press, New York. [Abstract] Tominaga, Y., Shimmen, T., and Tazawa, M. (1983). Control of cytoplasmic streaming by extracellular Ca” in permeabilized Nitella cells. Protoplasma 116, 75-77. Ueda, T., Gotz von Olenhusen, K., and Ohlfarth-Bottermann, K.-E. (1978). Reaction of the contractile apparatus in Physarum to injected calcium, ATP, ADP, and 5’-AMP. Cyrobiologie 18, 76-94. Uyeda, T. Q. P., and Kohama, K. (1987). Myosin switching during amoebo-plasmodia1 differentiation of slime mold, Physarum polycephalum. Exp. Cell Res. 169, 74-84. Uyeda, T. Q. P., Hatano, S., Kohama, K., and Furuya, M. (1988). Purification of myxamoebal fragmin, and switching of myxamoebal fragmin to plasmodia1 fragmin during differentiation of Physarum polycephalum. J. Muscle Res. Cell Motil. 9, 233-240.

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4.

Characteristics of Skeletal Muscle in Mdx Mutant Mice Sabine De La Porte,* Sophie Morin,t and Jeanine Koenigt,' *Laboratoire de Neurobiologie Cellulaire et MolCculaire. CNRS UPR 9040, 91198 Gif sur Yvette Cedex, France; and tLaboratoire de Neurobiologie Cellulaire, Universite de Bordeaux 11, France

We review the extensive research conducted on the mdx mouse since 1987, when demonstration of the absence of dystrophin in mdx muscle led to X-chromosome-linked muscular dystrophy (mdx) being considered as a homolog of Duchenne muscular dystrophy. Certain results are contradictory. We consider most aspects of mdx skeletal muscle: (i) the distribution and roles of dystrophin, utrophin, and associated proteins; (ii) morphological characteristics of the skeletal muscle and hypotheses put forward to explain the regeneration characteristic of the mdx mouse; (iii) special features of the diaphragm; (iv) changes in basic fibroblast growth factor, ion flux, innervation, cytoskeleton, adhesive proteins, mastocytes, and metabolism; and (v) different lines of therapeutic research. KEY WORDS: Mdx, Skeletal muscle, Dystrophin, Utrophin, Regeneration, Basal lamina, Fibroblast, Nuclear magnetic resonance. 0 1999 Academic Press.

1. Introduction Approximately 700 neuromuscular diseases have been described in man and are dominated by those with primary muscle involvement. Of the latter, Duchenne muscular dystrophy (DMD), a recessive X chromosome-linked disease, is the most frequent (1 boy in 3500) and severe (lethal around the age of 20). DMD was first described in the 1860s by the French physiologist G. Duchenne of Boulogne. It is characterized by elevated serum levels of muscle enzymes (more than SO times higher than normal) and by progressive loss of muscular strength, perceptible from the age of 4 or 5. Death due

' Preserir address: Institut de Myologie, Groupe Hospitalier PitiC-Salpitriirre, Paris, France. Irirernurronid R ~ w i r i v(I! ('yrolo~v,Vol. lYI

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to respiratory or cardiac insufficiency generally occurs around age 20. This myopathy is sometimes associated with mental retardation. A less severe and more variable form of the disease, Becker muscular dystrophy (BMD), was described in 1955. This myopathy is 10 times less frequent than DMD. The gene responsible for these myopathies was localized on the X chromosome in the early 1980s by several research teams and was isolated in 1986 (Monaco et af., 1986). This 1800 kb gene is located on the short arm of X chromosome, at Xp21. It comprises exons of mean size 150 pb separated by introns of about 16 kb (Chelly and Kaplan, 1988). It is the largest of all the genes identified to date (Koenig et al., 1987); the second largest, which is 10 times shorter, is that of neurofibromatosis. It accounts for between 0.05% (Brown and Hoffman, 1988) and 0.1% (Hoffman and Kunkel, 1989) of the human genome and one-third of the Escherichia coli genome (Brown and Hoffman, 1988). The corresponding protein, dystrophin, was identified in 1987 (Hoffman et al., 1987a). It is produced in all types of muscle tissues (cardiac and skeletal striated muscle and smooth muscle) and in certain neurons (Hoffman et al., 1988). Dystrophin is large (Mr427kDa, i.e., six times that of hemoglobin) and shares many features with spectrin (Koenig et af., 1988), a cytoskeletal protein which contributes to the plasticity of the membranes of red blood cells. It is thought that dystrophin contributes to the stability of muscle cell membranes. The discoveries of the DMD gene and of dystrophin were followed by characterization in animal models of muscular dystrophies genetically identical to those in humans. The discovery in the mouse (Bulfield el af., 1984), dog (Cooper et al., 1988), and cat (Carpenter et al., 1989) of homologous mutants with the same genetic deficiency as DMD led to a new experimental approach to muscular dystrophies. These mutant animals, however, do present specific differences.

II. Animal Models

A. Mdx Mouse In 1984, Bulfield et af., who were seeking glycolysis metabolism mutants in mice, discovered some animals of the C57BL/10 strain that had abnormally elevated levels of serum pyruvate kinase and creatine kinase, suggesting the existence of a muscular dystrophy. Genetic observations established that the disease was linked to X chromosome, and histological and ultrastructural examinations revealed primary lesions of muscle fibers, without

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nervous system involvement. The mutant was named mdx (X-linked muscular dystrophy) and in 1987 the finding that its muscles lacked dystrophin suggested it was a homolog of DMD (Monaco et af., 1986; Brockdorff et af., 1987; Heilig et af., 1987; Hoffman et al., 1987a). The mdx mouse has a mutation in the dystrophin gene and so its muscle fibers degenerate following membrane damage, resulting in particular in calcium uptake by the cell, overcontraction of muscle fibers, and activation of intracellular proteases. Unlike the situation in DMD, in the dystrophindeficient mouse, there is minimal fibrosis and fatty tissue replacement, and cellular necrosis is long compensated by regeneration of muscle fibers. The molecular aspects of the mutation were studied by Sicinski et af. (1989): The replacement of a cytosine by a thymine at position 3185 of the sequence coding for the gene leads to the appearance of a stop codon which prematurely terminates the translation of dystrophin. The truncated protein (about 27% of the normal length) comprises the N-terminal domain and seven of the repetitive sequences of the central domain. The rest is absent, but it is difficult to know whether the residual protein product is stable and functional.

B. The GRMD Dog

In 1986 and 1988, dogs developing progressive muscular dystrophy linked to X chromosome with absence of dystrophin were described (Valentine et af.,1986; Kornegay et af., 1988). These dogs have a mutation which results in a change in reading phase and a premature stop codon in the gene (Sharp et al., 1992). The creatine kinase level is elevated from the first week onwards, but the disease, called canine X-linked muscular dystrophy or GRMD (golden retriever muscular dystrophy), is only present around the age of 8 weeks as muscular weakness, which worsens progressively over several months. As in humans, the muscular weakness of the dogs is paralleled by a progressive loss of muscle tissue and its replacement by fibrous tissue (fibrosis). The anomalies affect the entire musculature, and in particular reduce respiratory capacity. Death generally occurs around 1 year of age (Valentine et af., 1992). In GRMD, 1% of the fibers are dystrophin positive (Nudel, 1989). As in DMD muscle, basic fibroblast growth factor (bFGF) is present only in small amounts, essentially in the nuclei and sarcoplasm (Anderson et af., 1993). Likewise, levels of mastocytes involved in development of fibrosis rise sharply and pass from the perimysium to the endomysium. This change in mastocyte distribution strictly parallels the clinical stages of the disease (Gorospe et af., 1994).

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C. The FXMD Cat In 1989, Carpenter et al. described two 2-year-old cats with walking difficulties due to a myopathy, despite the absence of progressive muscular weakness. Dystrophin was lacking, but the genomic deletion could not be defined. Serum creatine kinase levels were much above normal. This disease was given the name FXMD (feline X-linked muscular dystrophy). Unlike DMD and GRMD, dystrophin deficiency in FXMD cats is characterized by muscular hypertrophy. These animals never lose muscle tissue and conserve a certain strength, Moderate fibrosis occurs but there is no fatty infiltration. To explain these between-species differences, Hoffman et al. (1987a) hypothesized that in the human and dog, development of fibrous instead of muscle tissue prevents the muscle from regenerating and remaining functional. Another hypothesis is that in affected muscles in the human and dog, constant deterioration of muscle fibers creates a great demand for myoblasts, such that the muscle gradually loses its ability to regenerate. In smaller animals (mice and cats), the muscular demands would be less and the “pressure” on the myoblasts would be lower. Nonetheless, it is clear that fibrosis plays a central role in disease progression in humans (Hoffman, 1993).

111. Dystrophin, Utrophin, and Associated Proteins

A. Dystrophin 1. Structure Dystrophin has a multidomain structure (Brown and Hoffman, 1988; Mandel, 1989). From the NH2 terminal to the COOH terminal, there is an actinbinding region, followed by repetitive spectrin-like motifs, a cysteine-rich zone suggestive of calcium-binding motifs, and a domain penetrating in the plasma membrane and interacting with glycoproteins (Fig. 1): The N-terminal domain (240 amino acids), analogous to those of aactinin (Slater, 1987; Chelly and Kaplan, 1988; Hoffman et al., 1989) and p-spectrin (Byers et al., 1989), binds actin cooperatively (Way et a/., 1992) at two binding sites (Levine et al., 1992). The central rod-shaped domains consist of 24 (or 25, depending on the authors) repetitive structural elements of variable length (about 100 amino acids), similar to repeated triple-helix elements of a-actinin and @-spectrin(Man et al., 1990), and they combine to form filaments. Four proline-rich hinges in this domain confer elasticity and flexibility on

Actin

FIG. 1 Schematic model of the dystrophin-glycoprotein complex as a transsarcolemmal linker between the subsarcolemmal cytoskeleton and the extracellular

matrix (reproduced with permission from the Association Franpise contre les Myopathies).

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dystrophin (Koenig and Kunkel, 1990). Deletions truncating this region reduce the extensibility of the molecule without affecting actin binding, which characterizes BMD (Cross et al., 1990). The cysteine-rich domain (142 amino acids) exhibits 24% homology with the C-terminal domain of a-actinin and has two calcium-binding sites. The 420-amino-acid C-terminal domain has no sequence similar to that of a known protein; it penetrates the membrane of the muscle fiber and interacts with the membrane glycoproteins (LCger et al., 1991). The monomers of dystrophin are rods 3 or 4 nm thick and 175 nm long (Pons et al., 1990). These monomers combine spontaneously in antiparallel homodimers by matching of central repetitive domains, forming a hexagonal network such as erythrocyte spectrin (Koenig, 1989). The N and C termini are therefore juxtaposed and associate with one another or with other dimers of dystrophin (Hoffman and Kunkel, 1989). The dystrophin molecules are parallel (or almost so) to the inner face of the plasma membrane. The N and C termini and the plasma membrane are each about 50 nm apart. The dystrophin molecules are linked to the most peripheral of the filaments of sarcoplasmic actin (Wakayama et al., 1993). The nucleotide sequences in humans and mice have a highly conserved size and organization and marked homology (Robertson, 1987; Beam, 1988; Chelly and Kaplan, 1988). In the chicken, the sequence encoding dystrophin is almost the same size as its human counterpart. Conservation is 80% for the N-terminal region, 75% for the spectrin-like domain, and 95% for the C-terminal domain, suggesting that this is an important region for interactions with other proteins (Lemaire et al., 1988). Dystrophin represents 0.002% of total muscle protein (Campbell and Kahl, 1989) and 5% of the membrane cytoskeleton in skeletal muscle (Ohlendieck and Campbell, 1991a). Although dystrophin is a minor muscle protein, it is a major constituent of the muscle membrane in which it plays an important structural role. 2. Expression

Dystrophin is present in fetal and adult skeletal, cardiac, and smooth muscles (Robertson, 1987). It is expressed to equal extents in skeletal and cardiac muscle cells and in the brain (Brown and Hoffman, 1988) and to a lesser degree in smooth muscle (Beam, 1988). In the normal mouse embryo, dystrophin is visible from the 13th day in skeletal muscle (Karpati, 1989). Dystrophin expression has been extensively studied in human muscle. At the embryonic stage (8 or 9 weeks), it is first localized in the sarcoplasm at the ends of the myotubes, beside the tendons. At the fetal stage, it is distributed throughout the myofiber. By the 22nd

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week, it is localized in the sarcolemma of most fibers. This suggests that dystrophin accumulates in the cytoplasm before being associated with the membrane (Van Ommen, 1989; Prelle et al., 1991; Wessels et al., 1991; Clerk et al., 1992). Some authors consider that dystrophin content depends on muscle type. It has been reported that there is more dystrophin in slowtwitch than fast-twitch fibers in the rat and mouse, although this difference is not reflected in the dystrophin mRNA (Ho-Kim and Rogers, 1992). In contrast, Koga et al. (1992) found no significant difference in the dystrophin contents of slow and fast muscles. In rat heart dystrophin is undetectable on the 15th embryonic day, the stage at which the heart is able to generate action potentials and beat spontaneously. Development of the first functions of the myocardium therefore does not require the presence of dystrophin. A small quantity is detected on the 17th embryonic day and this increases during the perinatal period (Tanaka and Ozawa, 1990a). Cardiac work increases at this stage. Because its expression rises before these changes, dystrophin may be necessary for rapid and large contractions of the myocardium. Adult levels of dystrophin are reached 2 weeks postnatally (Tanaka and Ozawa, 1990b). Dystrophin is localized at the membrane surface of the Purkinje cells (Bies et al., 1992). Dystrophin is also found in cultured human skin fibroblasts. The promoter used in the muscle tissue is responsible for the transcription observed in fibroblasts (Hugnot et al., 1993). Dystrophin is also expressed by the neurons (Hoffman and Kunkel, 1989), notably in the cerebral cortex and cerebellum (Lidov et al., 1990). Dystrophin-like immunoreactivity is uniformly expressed in several regions of the brain involved in learning (hippocampus and cerebral cortex) and in motor function (spinal medulla, cerebellum, thalamus, and substantia nigra) (Huard et al., 1992a). It is localized in the soma and dendrites, associated with the inner part of the membrane (Lidov et al., 1990; Torelli et al., 1992). Brown and Hoffman (1988) reported that dystrophin is not expressed in vivo or in vitro in tissues other than muscle and nerve, whereas Walsh et al. (1989) detected dystrophin in minute amounts in all normal tissues.

3. Localization Biochemical and electron microscopy studies established that dystrophin is abundant in membrane fractions of skeletal muscle, at the inner face of the membranes, and in T-tubules (Hoffman et al., 1987b; Watkins et al., 1988). These authors therefore hypothesized that dystrophin was associated with the triads (Hoffman etal., 1987b; Chelly and Kaplan, 1988). ZubrzyckaGaarn et al. (1988) then showed that dystrophin was associated with the sarcolemma rather than with the triad. In rat soleus muscle regenerating after damage, dystrophin-type reactivity is first apparent in the sarcolemma

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and various inner membranes and, 4 weeks after the damage, in the triads. Dystrophin therefore appears primarily cytoplasmic, and then associates with the sarcolemma and the T-tubules during maturation. In mature fibers, dystrophin levels in the T-tubules appear to be so low that they can only occasionally be seen by means of immunocytochemistry (Bornemann and Schmalbruch, 1991). It is now considered that dystrophin is essentially localized at the inner face of the sarcolemma. Dystrophin molecules are not uniformly spread over the surface of mammalian muscle fibers. Confocal microscopy reveals aligned fluorescent points or intermittent lines along the sarcolemma, with most fluorescent points corresponding to the I bands (Masuda et af., 1992; Porter et al., 1992). The dystrophin molecules form a network of dense transverse rings or costamers and fine longitudinal interconnections. The dystrophin network at the surface of the muscle fiber is organized in relation with the contractile apparatus. Likewise, vinculin, y-actin, talin, and spectrin constitute a submembrane cytoskeletal network with a costameric distribution (Porter et af., 1992; Straub et af., 1992). Dystrophin binds strongly to talin, and this binding is inhibited by vinculin because of steric hindrance. Dystrophin can therefore interact in vitro with proteins of the muscle cytoskeleton. These proteins constitute additional sites of dystrophin binding to the sarcolemma (Senter et af., 1993). This may explain why dystrophin is found at the sarcolemma even when it lacks its C terminus (Senter et al., 1993). Dystrophin is more abundant at the membrane surface of the intrafusal fibers and at the neuromuscular junction (NMJ) than at the membrane surface of skeletal and cardiac muscle fibers (Arahata and Sugita, 1989; Miyatake et of.,1989; Huard et af., 1992b). At the NMJ, dystrophin is found in the depths of junctional folds and is absent from most AChR-rich domains of the rat NMJ (Byers et af., 1991; Sealock et af.,1991; Yeadon et al., 1991; Huard et af., 1992b). Dystrophin is present in the immediately adjacent membranes, suggesting that it is not an obligatory component of the AChR domains in the muscle; its role at the NMJ may be linked to the organization of the junctional folds (Sealock et af., 1991). Dystrophin also accumulates at the myotendinous junction (Bonilla, 1989; Mandel, 1989; Arahata and Sugita, 1989; Masuda et al., 1992), where it is one of the components binding the terminal actin filaments to the cytoplasmic face of the membrane of the junctional folds of the tendon (Samitt and Bonilla, 1990). In culture, dystrophin is undetectable in myoblasts (Nudel, 1989) but is present in myotubes (Zubrzycka-Gaarn et af., 1988; Ecob-Prince et al., 1989). Dystrophin is localized at talin-positive sites, where the sarcolemma is in apposition to the substratum. The first sites at which dystrophin appears in cultured muscle cells are therefore adhesion sites, i.e., specific sites of

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interactions between the cytoskeleton and the extracellular matrix (Kramarcy and Sealock, 1990). Dystrophin is discontinuously distributed in the submembrane region of human muscle cells in culture. Such cultures do not contract spontaneously but contractions appear in cocultures with rat spinal cord. In contracting conditions, dystrophin occurs continuously along the inner face of the sarcolemma and in periodic dense aggregates. After addition of tetrodotoxin, dystrophin resumes a discontinuous distribution, as in myotubes cultured alone. The contractile activity of the muscle therefore plays an important role in the continuous distribution of dystrophin along the sarcolemma during development (Sklar et al., 1990; Park-Matsumoto et al., 1991). In mdx mice, dystrophin mRNA is of normal size (16 kb) but present in small amounts (25% of the normal level). An mRNA containing a nonsense mutation can be degraded in the nucleus (Monaco et al., 1986; Brown and Hoffman, 1988; Chamberlain et al., 1988). There may be somatic reversion or suppression of the mdx mutation, which explains the observation of some dystrophin-positive fibers by immunofluorescence (Hoffman et al., 1990). Dystrophin-like immunoreactivity is detected in the fetal muscles of mdx mice, perhaps due to alternative splicing, which is quite often specific to the different stages of development. Hence, in the fetus, the stop codon is eliminated, thereby allowing synthesis of a dystrophin-type protein, whereas in the adult the stop codon is retained, thereby blocking translation (Kahn, 1989). Other authors consider that in the mdx mouse fetus there is synthesis of an abnormal dystrophin which cannot be integrated into the membrane and is therefore degraded (Van Ommen, 1989). Mdx mice have structural defects at the myotendinous junction-notably reduced lateral associations between the membrane and the fine filaments. These anomalies are observed before the onset of necrosis and may therefore be directly correlated with the absence of dystrophin (Law and Tidball, 1993). Tissues other than skeletal muscle are also affected. The contractile properties of the myocardium are markedly altered (Sapp et al., 1996), which is consistent with the hypothesis that dystrophin deficiency affects cardiac contractile function. The total absence of dystrophin in the cerebellum is not reflected in particular clinical signs (Huard and Tremblay, 1992). Nevertheless, in vivo and in vitro H' magnetic resonance spectroscopy revealed an increase in choline compounds and myoinositol levels, indicative of gliosis or developmental abnormalities in dystrophic brain (Tracey et al., 1996b). Systemic dysfunction may occur in smooth muscles (blood vessels) and skeletal muscle fibers. This may explain certain clinical symptoms of DMD, such as respiratory and gastrointestinal disorders (Miyatake et al., 1989). The mdx mouse expresses the Dp71 isoform of the dystrophin gene in its brain and in other nonmuscular tissues (Rapaport et al., 1992).

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B. Glycoprotein Complex Associated with Dystrophin Dystrophin is associated with a complex of glycoproteins and membrane proteins respectively called dystrophin-associated glycoproteins (DAGs) and dystrophin-associated proteins (DAPs) (Fig. 1). The large oligomeric complex with dystrophin comprises three subcomplexes (Ervasti and Campbell, 1991; Ohlendieck and Campbell, 1991b; Ibraghimov-Beskrovnaya er al., 1993; Ozawa et a[., 1995):

Dystroglycan complex: a-dystroglycan (156 kDa DAG), P-dystroglycan (43 kDa DAG) Sarcoglycan complex: a-sarcoglycan (adhalin or SO kDa DAG), p-sarcoglycan (43 kDa DAG), y-sarcoglycan (35 kDa DAG), Ssarcoglycan (35 kDa DAG) (Yoshida et al., 1997), and sarcospan, a unique 25-kDa member of the complex (Crosbie et al., 1997) Syntrophin complex: a-syntrophin (59 kDa DAG), P,-syntrophin (59 kDa DAP), &-syntrophin (59 kDa DAP) Dystrophin, the syntrophin complex, a-dystroglycan, and a muscle isoform of dystrobrevin (78 kDa) (Blake et al., 1996a) are peripheral membrane proteins, whereas the sarcoglycan complex and P-dystroglycan are integral membrane proteins. The syntrophin complex links sodium channels of the membrane to the actin cytoskeleton (Gee et al., 1998). a-Dystroglycan binds to laminin, thereby forming a link between the sarcolemma and the extracellular matrix (ECM) (Ibraghimov-Beskrovnaya et al., 1992). This link is calcium dependent and inhibited by heparin. One of laminin’s heparin-binding domains is therefore involved in binding to dystroglycan. P-Dystroglycan and a syntrophin are associated with dystrophin (Jung et d., 1995). There is no interaction between the dystrophin/glycoproteins complex and fibronectin, type I and IV collagens, entactin, or heparan sulfate proteoglycan (HSPG). However, the dystrophin/glycoproteinscomplex cosediments with actin and may therefore play a part in the binding of cytoskeletal actin to the ECM (Ervasti and Campbell, 1993). a-Syntrophin is also complexed with neuronal nitric oxide synthase (nNOS) (Brenman et al., 1996) in normal muscle. NOS is not anchored to the skeletal muscle sarcolemma of mdx and DMD muscle, but it is mislocalized to the interior of the muscle fibers (Brenman et al., 1995; Chang et al., 1996). NOS has been reported as present in the sarcolemma of fast-twitch or both fast- and slow-twitch fibers (Kobzik et at., 1994; Grozdanovic et al., 1996). The hypothesis that free radical toxicity is due to the mislocalization of nNOS is now excluded: Transgenic mice devoid of both nNOS and dystrophin present the same dystrophic characteristics as mdx mice (Crosbie et al., 1998). NOS is also concentrated at the NMJ (Kusner and Kaminski,

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1996; Oliver etal., 1996), where its role is unclear, except in possible involvement in synaptic suppression (Wang et al., 1995). In the model proposed by Minetti et al. (1992), a cytoskeletal structure links a-actinin of the Z-line of the sarcomere to the plasma membrane via F-actin, dystrophin, and associated glycoproteins. Complex molecular interactions involving other cytoskeletal proteins (vinculin, talin, and integrin) maintain the structural integrity of the plasma membrane. Lastly, a dystroglycan acts as a link between the plasma membrane and the ECM (Ibraghimov-Beskrovnaya et al., 1992). In the mdx mouse, all dystrophin-associated proteins are greatly reduced (80-90%) in the muscle due to the absence of dystrophin and not to secondary effects of the degradation of muscle fibers. This loss could have secondary effects on basal lamina components (Ohlendieck and Campbell, 1991b). There is an 85% reduction in a- and 0-dystroglycan in mdx muscle (Ervasti et al., 1990), whereas their mRNA is expressed at normal levels (Ibraghimov-Beskrovnaya et a/., 1992). Similarly, a-sarcoglycan mRNA is present in mdx muscles, as in DMD muscles, but the protein is greatly reduced (Roberds et a/., 1993). Regulation of these proteins is therefore posttranslational in these muscles. DAGs are produced, but in the absence of dystrophin they are not correctly assembled and/or integrated in the sarcolemma and are degraded (Matsumura and Campbell, 1994). Following surgical or pharmacological denervation of skeletal muscles of mdx mice, the levels of adhalin and P-dystroglycan increased at the extrajunctional sarcolemma together with AChR, suggesting that their association is independent of the presence of dystrophin (Mitsui et al., 1996). C. Utrophin

A 13-kb autosomal transcript encoded by a gene of the long arm of chromosome 6 has been identified in human fetal muscle. It encodes a protein with more than 80% homology with dystrophin (395 kDa) (Love et a/., 1989); thus because of its ubiquitous localization, it is called dystrophinrelated protein (DRP) or utrophin (Fig. 2), The homology between dystrophin and utrophin extends over their whole lengths, suggesting that they derive from a common ancestral gene. Utrophin, like dystrophin, binds actin. The C-terminal domain is highly conserved (Tinsley et al., 1992). In the mouse, the utrophin locus is on chromosome 10. This gene is expressed to varying degrees in numerous tissues; the transcript is particularly abundant in several human fetal tissues (e.g., heart, placenta, and intestine). In normal human and murine muscles, utrophin is always found at the membrane surface of immature fibers (Zhao et al., 1993). It is concentrated at the NMJ of mature muscle fibers and reappears at the membrane surface

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FIG. 2 Schematic model of the utrophin-glycoprotein complex as a transsarcolemmal linker between the subsarcolemmal cytoskeleton and the basal lamina. ARIA, acetylcholine receptor-inducing activity; CGRP, calcitonin gene-related peptide; MASC, MuSK accessoryspecific component; MuSK, muscle-specific receptor tyrosine kinase; RATL, rapsyn-associated transmembrane linker (reproduced with permission from the Association FranGaise contre les Myopathies).

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of muscle fibers after denervation. Its localization is similar to that of the AChR, at the tops of the postsynaptic folds (Bewick et al., 1992; Appel and Merlie, 1995). Utrophin mRNA selectively accumulates within the postsynaptic sarcoplasm of adult muscle fibers (Gramolini et al., 1997). Utrophin may be one of the molecules of the cytoskeleton which organize and stabilize the cytoplasmic domain of the AChR (Takemitsu et al., 1991a, b). It is present at the earliest stages of the concentration of AChRs at the NMJ in the mouse embryo and is also concentrated in the large AChR clusters on myotubes of the C2 line, suggesting that it is mainly involved in the growth of AChR clusters (Phillips et al., 1993). Utrophin-deficient mice are healthy and show no signs of weakness. However, their NMJs have reduced numbers of AChR and decreased postsynaptic folding (Deconinck et al., 1997a; Grady et al., 1997a). Therefore, utrophin alone is not essential for AChR clustering at the NMJ. Utrophin is associated with laminin (Khurana et al., 1995) via a complex of sarcolemmal proteins identical to or at least antigenically similar to that of dystrophin (Fig. 2) (Matsumura et al., 1992). In the mdx mouse, various authors have shown that utrophin is expressed at the NMJ and is found in muscle extracts from this region (Anderson et al., 1990; Fardeau et al., 1990; Pons et al., 1991). Utrophin and associated proteins colocalize at the NMJ (Karpati et al., 1993a), where utrophin is overexpressed (Takemitsu et al., 1991a, b; Koga et al., 1993). Outside the NMJ, the results are discordant. Anderson et al. (1990), Fardeau et al. (1990), and Pons et al. (1991) report that utrophin is absent from extrasynaptic zones. Karpati et al. (1988) found utrophin along the whole sarcolemma of mdx muscle small-caliber fibers and of cardiac fibers. Because these muscles exhibit few pathological changes, utrophin may compensate for the absence of dystrophin by holding the DAGs at the extrajunctional sarcolemma (Matsumura et al., 1992; Tinsley et al., 1992). Takemitsu et af. (1991a, b) and Koga et al. (1993) consider that utrophin is present at the membrane surface of all mdx muscles. Contradictory results have emerged from studies of utrophin expression during development. All authors agree that utrophin is overexpressed in fetal mdx muscle (Takemitsu et al., 1991a, b; Koga et al., 1993; Zhao et al., 1993). Some authors consider that this overexpression continues after birth (Takemitsu et al., 1991a, b; Koga et al., 1993; Zhao et al., 1993) and that utrophin expression in the adult mdx animal is twice that in normal mice (Sugita et al., 1993; Law et al., 1994). Law and colleagues, however, did not find this increase in 2-week-old mice. Other authors have found that utrophin decreases in fast muscles before the third week and disappears in slow muscles between the third and fourth week. Love et al. (1991) identified utrophin in many fetal tissues, including the heart, placenta, and intestine. In the nervous system of the mdx mouse,

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utrophin is present in the vascular walls, pia mater, and choroid plexus of the brain but not in neuronal cells (Ishiura et al., 1990; Uchino et al., 1994).

D. Function of Dystrophin, Utrophin, and Associated Proteins Although dystrophin is a cytoskeletal protein located on the inner face of the plasma membrane of the muscle fiber, its function is still incompletely understood. It seems to be involved in maintenance of the morphological and functional structure of the striated muscle fiber (Petrof, 1998). Two main hypotheses concerning its role have been proposed. The first attributes a mechanical function to dystrophin, which is seen as the main element of an elastic net or sort of lattice within the membrane of the muscle fiber. The molecules “slide” over one another, thereby allowing the whole net to change shape during contraction and relaxation (Watkins, 1989; LCger et al., 1991). By connecting the cytoskeleton to the cytoplasmic face of the muscular membrane (Zubrzycka-Gaarn et al., 1988), dystrophin may help resist the stresses that develop during contraction (Beam, 1988). It may therefore play an important role in preservation of membrane stability (Park-Matsumoto et al., 1992). Indeed, dystrophin is located very close to the plasma membrane of the muscle fibers. The N terminus is linked to the cytoskeleton’s actin network and the C terminus to the plasma membrane. In terms of the glycoprotein complex, dystrophin is supposed to play an important role in transduction of the mechanical force of the contractile apparatus to the ECM (Straub et al., 1992; Petrof et al., 1993a). According to the second hypothesis, dystrophin is involved in calcium homeostasis: It binds the contractile filaments of the internal membrane system, thereby ensuring a link between the membranes which release calcium and the contractile proteins which are activated by this calcium (Slater, 1987; Hoffman et al., 1987a; Tay et af., 1989). Dystrophin may also play an important physiological and/or structural role in cell motility (Miyatake et al., 1991). At the synapse, dystrophin may participate in the formation of a submembrane cytoskeletal network which promotes AChR clustering (Jasmin et af., 1990). At the myotendinous junction, it may form lateral associations between the fine filaments and the cell membrane (Tidball and Law, 1991; Law and Tidball, 1993). Dystrophin’s localization in the central nervous system indicates a physiological function in the conduction of the nerve impulse rather than a mechanical function (Yoshioka et af., 1992). It may be involved in the structure and maintenance of the dendritic tree and neuronal survival (Huard et af., 1992a).

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Utrophin may have a function similar to that of dystrophin during development (Anderson and Kunkel, 1992). It may also play an important role in the organization of the postsynaptic membrane of the NMJ (Ohlendieck et al., 1991). a-Dystroglycan constitutes an agrin-binding site at the surface of the muscle cell. With DAG 50 kDa and utrophin, it colocalizes with agrininduced AChR clusters. It may therefore be involved in AChR clustering (Gee et al., 1994). One hypothesis is that agrin stabilizes the membrane cytoskeleton specific to the synapse, which in turn serves as a support in the concentration of synaptic molecules (Campanelli et al., 1994). The role of a-dystroglycan remains unelucidated (Sugiyama et al., 1994) since Glass et al. (1996) demonstrated that the main component of the receptor complex which mediates agrin signaling is a muscle tyrosine kinase (Musk).

IV. Mdx Muscle Cells A. Degeneration/Regeneration of t h e Muscle

1. Morphological Defects Mdx muscles exhibit early necrosis. From the first day, there is disorganization of the Z-line and hence of the contractile apparatus. Necrosis starts on Day 5 , but only in the muscles of the head, trunk, and girdle. The limbs are affected later (Torres and Duchen, 1987). In the first 10 days following birth, all the limb muscle fibers seem relatively normal. The necrotic fibers start to appear at 21 days and become numerous by Day 28. A lack of synchronization has been described in the course of the disease: a craniocaudal gradient in the appearance of necrosis followed by regeneration (Muntoni ef al., 1993). The degeneration and regeneration of the mdx muscle occurs essentially between the third and 10th week, with marked differences depending on the authors: Onset of degeneration around 2 weeks, with a peak between Weeks 3 and 8 (Karpati, 1989; Karpati et al., 1990; Nagel et al., 1990) Onset between Weeks 3 and 5 (Brown and Hoffman, 1988; Coulton et al., 1988) Peak at 6 weeks (DiMario and Strohman, 1988) Peak between Weeks 5 and 8 and complete regeneration around 10 weeks (Torres and Duchen, 1987; DiMario et al., 1989) Muscle fibers whose girth is below 20-25 p m in diameter (such as extraocular muscle fibers) are not susceptible to necrosis (Karpati et al., 1988). In

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such muscle fibers there is no significant differences in nNOS concentration and distribution compared to other mdx muscles (Wehling et al., 1998). Apoptosis precedes any detectable necrotic change in mdx muscle, and apoptotic events continue into the stage of necrosis (Tidball et al., 1995; Smith et al., 1995). Overcontracted fibers are seen from birth in mdx muscles (Torres and Duchen, 1987) and at least to the age of 18 months. They reflect an early stage of necrosis, due to the massive influx of calcium in a segment of the muscle fiber, provoking abnormal contraction of the sarcomeres. Calpains (neutral calcium-dependent thiol proteases) are autolytically cleaved during the disease process of mdx dystrophy, indicating that they may be activated and play a role in the proteolysis that occurs in muscle (Spencer et aL, 1995). The fragility of skeletal muscle fibers of mdx mice is attested by (i) a significant accumulation of Evans blue, a tracer molecule used to analyze sarcolemmal integrity (Straub et al., 1997), and (ii) a significant and transient rise in the level of creatine kinase and P-galactosidase (by using a construction with a muscle-specific promoter in trangenic mdx mice) in the serum of mice after eccentric running exercise (Vilquin et al., 1998). Fast-twitch fibers are preferentially but not exclusively involved (Carnwath and Shotton, 1987). Only 5% of the original fibers survive to 26 weeks in EDL muscle (fast). Muscle destruction is delayed in the slow-twitch soleus muscle and in the fast-twitch gastrocnemius compared to the intermediate tibialis anterior muscle (Dangain and Vrbova, 1984). The presence of intermediate fibers may result from coexpression of slow and fast myosin heavy chains, indicating a transition from fast to slow in regenerating fibers (Pastoret and SCbille, 1993b). The onset of muscular hypertrophy in the mdx mouse can be explained by three nonexclusive hypotheses: hypertrophy of the fibers (arise in fiber diameter and therefore in muscle bulk), hyperplasia (fiber neosynthesis), or an abnormal increase in connective tissue. In the adult mutant, none of these hypotheses alone can account for the increases in weight and muscle area. This hypertrophy peaks between 6 and 12 months of age and then regresses such that animals more than 18 months of age show substantial muscular atrophy (Pastoret and SCbille, 1993a). These animals exhibit marked fibrosis in some muscles, notably the largest proximal muscles (Hoffman and Kunkel, 1989). The contractile function of muscles of old mdx mice displays many similarities to that of DMD (Hayes and Williams, 1998), but the results of Bobet et al. (1998) disagree with this hypothesis. It is a moot point whether regeneration of mdx muscles is transient or continuous. Strohman’s team (Berkeley, CA) has shown that mdx muscle expresses fetal and neonatal myosin mRNA from Weeks 6 to 16, but that from Weeks 10 to 56 the frequency of the fibers expressing embryonic myosin decreases to about 1%(DiMario et al., 1991). This suggests that

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mdx muscle regeneration is transient. Conversely, other studies suggest that regeneration of mdx muscle is a continuous process: (i) Beilharz et al. (1992) have shown that the expression of MyoD and of myogenin, which are early and specific markers of regeneration, is elevated between 3 and 6 weeks. It then decreases to a constant, albeit relatively high, level; (ii) McGeachie et af. (1993) have shown that proliferation of muscle cells starts at about 3 weeks, peaks between 4 and 8 weeks, and continues at a lower level at least to Week 44; and (iii) necrotic foci are present throughout the life of the animal (MacLennan and Edwards, 1990; Pastoret and SCbille, 1995) but are compensated by regeneration up to 18-20 months. The number of fibers with central nuclei, i.e., fibers that have been necrotized and have regenerated, increases with age (Torres and Duchen, 1987; Karpati et af., 1990), and there is great variation in fiber diameter (Torres and Duchen, 1987). From 3 months, 80-90% of these fibers of the skeletal muscles of the mutant have a central nucleus (Carnwath and Shotton, 1987). However, the formation of such fibers appears to predominate during the acute phase of the disease, whereas peripheral relocalization of the nuclei becomes the major event during the chronic phase. The central localization of the nuclei in mdx muscle which has regenerated is not permanent but is more lasting than that in other studied species (McGeachie et al., 1993). The pattern of expression of various myogenic regulatory factors (MyoD, myogenin, Myf-5, and Myf-6) differs in mdx regenerating muscle fibers from that in developing muscle in embryos (Bhagwati et al., 1996). Leukemia inhibitory factor (LIF) and interleukin (IL-6) have been shown to promote the proliferation of myoblasts. Normal muscle rarely expresses mRNA for these two molecules, but mdx muscle expresses both LIF and IL-6 mRNA (Kurek et al., 1996). 2. Basal Lamina

The ECM of skeletal muscle consists of a fibrillar matrix, which essentially contains collagen I, 111, and V (Duance et al., 1977) and fibronectin (Chiquet et al., 1981), and a basal lamina, consisting particularly of type IV collagen, laminin, fibronectin, entactin, and HSPG (Kefalides et al., 1979; Hoover et al., 1980). Laminin is a heterotrimer (Vachon et al., 1996) composed of a heavy chain (a;400 kDa) and two similar but not identical light chains ( p and y ; 205-220 kDa). The classical laminin, laminin-1, is composed of an a1 chain and two p , I y I subunits. It has recently been demonstrated that laminin a1 is not found in muscle basal lamina of developing and adult mice (Patton et al., 1997; Tiger and Gullberg, 1997). The predominant laminin variants in the muscle fibers contain the a2 heavy chain (two fragments of 300 and 80 kDa) and are known as merosin. In muscle basal

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lamina, the a2chain associates predominantly with pl/yl chains as laminin2, whereas it associates with the p2/yIchains as laminin-4 at NMJs. The signal-transducing receptors for laminins, collagens, and other extracellular matrix proteins are integrins, a family of heterodimeric (ap)transmembrane proteins (Hynes, 1992; Sonnenberg, 1993). Different isoforms are specifically distributed in synaptic and extrasynaptic zones (Martin et al., 1996). The expression of laminin pI/yIchains and HSPG (by immunofluorescence staining and immunoblot quantification) is increased in mdx muscle no matter what the age of the mice and not just during the acute phase of the disease (Morin et al., 1993). This is in agreement with previous reports of a marked accumulation of collagen, reticulin, fibronectin, and laminin (Marshall et al., 1989; Goldspink et al., 1994; Prattis et al., 1994; Seixas et al., 1994; Quiricosantos et al., 1995). This increase in laminin a l l y l chains seems to be specific since Arahata et al. (1993) found that the laminin cyz chain is not affected. An increase in both these basal lamina components in mdx mice could contribute in different ways to the intense muscle regeneration observed in this mutant. Components of the extracellular matrix are thought to facilitate muscle regeneration (Vracko and Benditt, 1972;Sanes et al., 1978). The mechanisms by which the basal lamina influences regeneration may include stimulation of satellite cell division and growth (Ocalan et al., 1988) and fusion (Vachon et al., 1996). The increase in basal lamina HSPG, concomitant with the increase in bFGF in regenerating areas (DiMario et al., 1989; Anderson et al., 1991, 1993; Matsuda et al., 1992), would favor their interaction and HSPG could deliver the growth factor to high-affinity receptors that initiate the stimulation of myogenic cell proliferation and/or differentiation (Rapraeger et al., 1991;Yayon et al., 1991). This phenomenon could be amplified by the greater sensitivity of mdx satellite cells to bFGF (DiMario and Strohman, 1988). The mechanism of the persistent regeneration in mdx muscle is therefore currently unclear. It does seem reasonable that basal lamina components could be partly responsible for a successful regenerative process. The extracellular matrix protein tenascin-C (TN-C), expressed in wound healing and nerve regeneration, is undetectable in normal muscle except at the myotendinous junction but is prominent in degeneratingkegenerating areas of mdx muscle. TN-C staining declines around stable regenerated mdx myofibers (Settles et al., 1996). 3. Muscle Fibroblasts

Fibroblasts were thought by many to be structure-supporting cells with a negligible role in cellular homeostasis. However, the in vitro cultivation of these cells revealed their fundamental role and their interaction with other

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cell types. For instance, growth inhibitory factors were shown to be secreted by fibroblasts maintained in culture (Harel, 1981; Harel et al., 1978). Thirteen-kDa (Hsu and Wang, 1986) and 45-kDa proteins were purified from medium conditioned by dense cultures of 3T3 fibroblasts. The 45-kDa factor completely inhibits growth of chick embryo fibroblasts (Blat et al., 1987, 1989a) and has been shown to be the insulin-like growth factor-binding protein3 (IGF-BP-3) (Blat et al., 1989b). IGF-BP-3 also accumulated to high levels in conditioned medium (CM) of quiescent and senescent human fibroblasts (Goldstein et al., 1991). IGF-BP-4 and -5 were also shown to be synthesized and secreted by fibroblasts (Camacho-Hubner el al., 1992) and to play a role in the regulation of cell proliferation (Fowlkes and Freemark, 1992: Jones et al., 1993; Neely and Rosenfeld, 1992: Reeve et al., 1995). Macieira-Coelho and Soderberg (1993) also described the presence of a 1-kDa glycopeptide with growth inhibitory activity in extracts from normal human fibroblasts. These results suggest that changes in fibroblast functions may have a profound effect on muscle fibers, and in the case of muscle disease the fibroblast may play a role in determining the extent to which a disease phenotype is expressed, even in the presence of a dystrophic genotype. In view of the hypothesis that the regenerative process is successful at least partly because of modifications of muscle fibroblast functions in the mdx mouse, our approach has been to study the properties of mdx and DMD muscle fibroblasts by analyzing their proliferative response in vitro (Morin et al., 1995). We found that fibroblasts taken from human DMD and control muscle had a similar in vitro proliferation capacity. In mdx mice, the study was performed at various ages to determine if there were changes during the course of the disease. We observed a growth arrest of muscle fibroblasts during the acute phase of the disease: This arrest was partial at 6 and 10 weeks but total at 8 weeks (Fig. 3). This is in marked contrast to fibroblasts taken from animals at other ages, which show a normal proliferative capacity. These results suggested that 8-week-old mdx male mouse muscle fibroblasts release a factor that inhibits their own proliferation. Our hypothesis was confirmed using CM experiments. CM from 8-week-old mdx mouse muscle fibroblasts inhibited the proliferation of control fibroblast cultures. This growth inhibitory factor seems to be specific for fibroblasts and when added to control or mdx myoblast cultures it stimulated their proliferation, as did 8-week-old control fibroblast CM. It has previously been shown that proliferating bovine muscle fibroblasts produce a specific “growth factor activity,” distinct from bFGF or plateletderived growth factor, which increases proliferation of bovine myoblasts but not of bovine or 3T3 fibroblasts (Quinn et al., 1990). From our results, it seems that even quiescent 8-week-old mdx mouse muscle fibroblasts produce a myoblast growth factor. The nature of the factor remains to be

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Age (weeks) FIG. 3 Proliferation rates of control and mdx mouse muscle fibroblasts, determined as the slope of the proliferation curve, at 1, 4, 6, 8, 10, and 13 weeks of age. Data points represent the averages of triplicate determination ? SEM. Significant statistical differences are indicated as follows: ***p < 0,001, **p < 0.01, *p < 0.05. The proliferation rate of mouse muscle fibroblasts decreases during the acute phase of the disease, and inhibition is complete in fibroblasts from 8-week-old mdx mice. The proliferation capacity of control fibroblasts was very high at 1 week and decreased with the age of the mice. The reduction, however, was only significant between 1 and 4 weeks. The proliferation capacity of mdx fibroblasts was usually less than that of control fibroblasts. The ability of the fibroblasts to proliferate decreased between 6 and 10 weeks, the period corresponding to the onset of muscle regeneration in mdx mouse muscle. The 8-week-old mdx mouse muscle fibroblasts produce an inhibitor of their own proliferation and a growth factor specific for myoblasts in vitro. If these factors are secreted in vivo, they could directly and indirectly stimulate satellite cell proliferation, thus favoring muscle regeneration (reproduced with permission from Differentiation, Inhibition of proliferation in 8-week-old mouse muscle fibroblasts in vitro, Morin, S., De La Porte, S., Fiszman, M., and Koenig, J., 59, Fig. 3, copyright 0 1995 by Springer-Verlag).

determined. It could be, at least partly, responsible for the mitogenic activity detected in mdx and control muscle extracts (Chen et al., 1994). In addition to this factor, it is well known that growth factors such as FGFs are synthesized by myogenic cells and stimulate their proliferation. One hypothesis was that the growth inhibitory factor could be one of the IGF-BPs. Preliminary results indicate an exclusive expression of IGFBP-5 in muscle fibroblasts from 8-week-old mdx mice. In addition, messenger RNA is increased three-fold in mdx mice compared to controls, and

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the inhibitory effect on fibroblast proliferation is decreased by 37% when mdx fibroblast CM is previously incubated with antibodies against IGFBP-.5 (Zhang et al., 1996). Our results suggest that fibroblasts release a growth factor specific for myoblasts and that 8-week-old mdx mouse muscle fibroblasts release a growth inhibitory factor specific for fibroblasts. If these factors are released in vivo, the growth inhibitory factor may act in an autocrine way to stop fibroblast proliferation, whereas the mitogenic activity could stimulate satellite cell proliferation, thus favoring muscle regeneration. This hypothesis postulates an active role for quiescent fibroblasts in myogenesis and is supported by the three times increase of the number of cells in culture of myogenic cells accumulating around individual living fibers isolated from muscles of 8 week-old mdx mice (Bockhold et al., 1998).

6 . Diaphragm

The diaphragm of the mdx mouse is the only muscle to exhibit marked degeneration, fibrosis, and functional insufficiency similar to that seen in DMD muscles. Because there seem to be no differences in terms of utrophin expression between limb and diaphragm muscles, it is unclear why the diaphragm is severely affected and other muscles are relatively protected (Sugita et al., 1993). Gillis (1997) considers that the mdx mouse diaphragm combines three unfavorable factors: a large proportion of fast oxidative fibers with large diameter, a life-long sustained activity, and forced lengthening during each contraction. The collagen density in the diaphragm is 7 times that in the diaphragm of control mice and 10 times that in the limb muscles of the mdx mouse. These changes are rare before 2.5 days. At 30 days, there are foci of degeneration, necrosis, and regeneration, as in the limb muscles. However, unlike the limb muscles, the diaphragm exhibits progressive degeneration. At 6 months, fiber size varies greatly, necrosis is continuous, and there is substantial proliferation of the connective tissue. At 16 months, there is extensive loss of muscle fibers and marked fibrosis. These divergences between the diaphragm and limb muscles may reflect intrinsic differences in regenerative capacity. However, regeneration persists even at 16 months (embryonic myosin and central nuclei) and in vitro studies of the satellite cells of the diaphragm and different limb muscles indicate identical proliferative capacities. At 18 months, similar but less severe histological changes are seen in muscles involved in respiration (Stedman et al., 1991). As in the limb muscles, in the diaphragm of the 3or 4-month-old mdx mouse there is an increase in the number of fibers coexpressing the myosin heavy chains of slow and fast muscles. Regenerating fibers express embryonic myosin, whereas the number of fast-twitch

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fibers drops sharply. At 22-24 months, fibers with slow-type myosins are abundant, fast-twitch fibers have been eliminated, embryonic myosin is undetectable, and endurance is increased. The mdx diaphragm therefore responds to the progressive degeneration of the muscle by a transition to a slower phenotype associated with greater endurance. This preserves the contractile function and enhances survival of muscle fibers by lowering energy requirements (Petrof et al., 1993b). Some authors have hypothesized that dystrophin is a mechanical transducer which transmits growth stimuli from the skeleton to the muscle. Dystrophin controls the rate of addition of sarcomeres to the ends of the fibers by participating in the mechanism which allows the satellite cells to fuse with the muscle fibers. In the mdx mouse, the most acute phase of the disease corresponds to the stage at which, in controls, the rate of addition of sarcomeres to the ends of the muscle fibers is maximal. Likewise, in the diaphragm of elderly mdx mice, the fibers are 35% shorter, which supports the hypothesis that dystrophin plays a key role in the regulation of the longitudinal growth of the fibers (Brown and Lucy, 1993). C. bFGF

Studies in mdx mice have shown that (i) in vitro, replication of myoblasts and, to a lesser extent, of fibroblasts is stimulated by smaller amounts of bFGF than in control cells (DiMario and Strohman, 1988) and (ii) the ECM of mdx muscle is richer in bFGF (DiMario et al., 1989; Anderson et al., 1991) and in acidic F G F (Oliver et al., 1992a) than that of control muscle, principally in the regeneration zones. bFGF may participate in the degenerative and regenerative responses of the muscle. In the control muscle, bFGF is localized at the periphery and in the nuclei of the muscle fibers, in the satellite cells, and, to a lesser degree, in the cytoplasm. In mdx muscle, intact fibers are labeled in the cytoplasm and nucleus. Small regenerating fibers accumulate more bFGF than do larger adjacent fibers. The smaller the cells, the more intense the staining. Mature fibers no longer express bFGF. The decrease in the immunoreactivity of bFGF is therefore a function of the size of the regenerating cells. The intensely labeled cells are more numerous at 5 weeks than at 10 weeks. Elsewhere, injection of bFGF promotes in vivo muscle regeneration in mdx muscle by enhanced replication of muscle satellite cells (Lefaucheur and SCbille, 1995). bFGF may also be involved in the physiology of the different striated muscles: The slow muscles contain more bFGF than do the fast muscles, both in mdx and in normal muscles (Anderson et al., 1991, 1993; Matsuda et al., 1992). A recent study indicates that mdx muscle cells have elevated levels of HSPG receptors for bFGF (Crisona et al., 1998).

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D. Ionic Flux 1. Calcium

Studies of calcium content in mdx muscle fibers have yielded contradictory results. Some authors have reported that free intracellular calcium levels increase at rest and during stimulation, resulting in an increase in degradation of muscle proteins (Turner et al., 1988; MacLenna et al., 1991; Kamper and Rodemann, 1992; Hopf etal., 1996). Likewise, in vitro this concentration is twice as high in the mdx myotubes at rest as in normal myotubes (Bakker et al., 1993). The intracellular calcium concentration is unchanged in mdx smooth muscle at rest, indicating that the absence of dystrophin does not always result in perturbed calcium metabolism (Boland et al., 1993). Several hypotheses have been put forward to explain the origin of this rise in intracellular calcium in mdx muscle: In young mdx mice, mechanosensitive ion channels, i.e., open at rest and inactivated by stretching of the membrane, are highly active (Franc0 and Lansman, 1990). This activity diminishes during development but is compensated for because the density of channels remains constant in mdx fibers but decreases in normal fibers. An early stage in the dystrophic process could be an alteration in the mechanisms governing expression of functional channels (Haws and Lansman, 1991). Fong et al. (1990) observed calcium leakage at rest. In controls, channels are little active and calcium is strictly regulated, whereas in the mdx mouse channels are active, leading to poor calcium regulation. Carlson and Officer (1996) attribute part of this calcium leakage activity to unusual physical interactions between AChR and cytoskeleton in mdx mice. Proteolysis raises membrane permeability to calcium at rest, creating positive feedback which results in an additional entry of calcium and muscle fiber necrosis (Turner et al., 1993). The sarcoplasmic reticulum pump is functionally altered in dystrophic mdx muscle (Takagi et al., 1992; Kargacin and Kargacin, 1996). Contradictory observations have been reported regarding parvalbumin, a protein which links calcium and relaxing factor in fast muscles. Sano et al. (1990) have shown that the decrease in the parvalbumin content of mdx muscle may contribute to a rise in intracellular free calcium and to activation of calcium-dependent proteolysis. Gillis (1996), on the other hand, reports that levels of parvalbumin and its mRNA (Gailly et al., 1993a) are higher in fast muscles of mdx mice than in normal muscles. A deficit in the level of calmitine has been reported in young mdx mice (Lucas-Hkron et al., 1990). The calmitine level remained low in 3-,

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5, and 6-week-old mdx mice and was similar to controls in 16-weekold mdx mice. However, calmitine deficiency does not affect the main calcium-binding structures involved in the regulation of mitochondria1 calcium (Lucas-HCron et al., 1994). Reports of an increase in intracellular calcium in mdx muscle are now contested. Several authors (Head, 1993; Pressmar et al., 1994; Gillis, 1996) consider instead that the resting potential of the membrane, the resting calcium concentration, and the transient calcium currents are identical in mdx and normal muscles. Leijendekker et al. (1996) report that calcium concentration is similar in mdx and normal myotubes, but increased permeability to calcium is noted for mdx myotubes under specific stress conditions. Gailly et al. (1993b) report that the concentration of cytosolic free calcium is comparable in mdx and normal muscles, although the total calcium increases. Most studies have examined muscle from mice of only one age. Reeve et al. (1997) demonstrated large changes in muscle calcium content during the postnatal development of the mdx mouse, thereby providing one possible explanation for the contradictory data reported.

2. Sodium and Potassium There is no apparent difference between the properties of the sodium channels of mdx and control muscles (Mathes et al., 1991), but intracellular sodium concentrations are higher in mdx muscles. This may reflect a reduced flow via the Na/K-ATPase, which would lead to poor control of cell volume and cell death (Dunn et al., 1993, 1995). The absence of dystrophin in the muscle sarcolemma does not affect the main KATp(Allard and Rougier, 1997) and K' delayed rectifier (Hocherman and Bezanilla, 1996) channel properties.

E. Innervation Axonal transport is broadly normal (Yamashita et al., 1989), as are innervation and myelinization, but in degenerating zones nerve terminals fragment into subunits. Although there is no direct injury of motoneurons, the growth-associated protein B50/GAP-43, which is involved in axonal outgrowth and synaptic remodeling following neuronal injury, is increased in terminal nerve branches at motor endplates of mdx mice, particularly in the areas of degeneratinghegenerating myofibers (Verze et al., 1996). The frequency of the miniature endplate potentials, their quanta1 content, and their amplitude are normal in mdx muscle (Hollingworth et al., 1990; Lyons

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and Slater, 1991). In contrast, Nagel et al. (1990) report that the amplitude of the miniature endplate potentials decreases and the quanta1 content of the endplate potential increases. There is a decrease in the number of subneuronal folds, simplification of the postsynaptic membrane (Torres and Duchen, 1987), widening of the synaptic zone (Nagel et al., 1990), and redistribution of the postsynaptic molecules. The G4 form of AChE is deficient in certain mdx muscles, likely reflecting a secondary effect of the dystrophy (Oliver et al., 1992b). The postsynaptic membrane’s AChR population is normal, but two types of receptors are expressed in mdx muscle: the adult type, as in normal muscle, and the embryonic type, similar to that of denervated muscle, with changes in opening time and current amplitude (Koltgen and Franke, 1992). This expression of the embryonic AChR is not a characteristic of dystrophy but a consequence of muscle regeneration (Koltgen and Franke, 1994). Salpeter’s group reports that AChR in innervated muscles of mdx degrades as AChR after denervation in normal mice (t112-3-5 days) but at no time as embryonic receptors ( t I l 2-1 day) (Xu and Salpeter, 1997). Most nerve terminals are abnormally complex at the NMJs of the regenerated fibers (Kitaoka et al., 1997). The absence of dystrophin in the postsynaptic membrane therefore has little effect on the function of the neuromuscular junction, but the degeneration and regeneration of the fibers leads to remodeling of the pre- and postsynaptic components. Carbonic anhydrase activity, a marker of mouse proprioceptive neurons in dorsal root ganglia, is regulated by neuron-muscle interactions. In mdx mice, neuronal carbonic anhydrase expression stops when the period of muscular degeneration-regeneration begins, and this alteration persists during adulthood (Mayeux et al., 1996).

F. Cytoskeleton The distribution and relative abundance of vinculin, desmin, and nebulin are unchanged in mdx muscle. The absence of dystrophin therefore does not result in alterations in the structures linking the sarcolemma to the contractile apparatus (Massa et al., 1994). These results partially contradict those of Law et al. (1994), who showed that in 2-week-old mice vinculin and talin are expressed comparably in mdx and normal muscles, whereas their expression is increased approximately 200% in mdx mice aged 11 months. This increase is localized in the myotendinous junction. It is argued that the mdx mouse partly compensates for the absence of dystrophin by overexpression of molecules which also have a mechanical function. Vinculin binds to actin and to talin, which binds integrins, and this ensemble may provide the link between the cytoskeleton and the ECM.

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G. Adhesion Proteins The expression of N-CAM is comparable to that in normal muscle before the start of the degeneration and regeneration cycles, but then it increases. It seems to be related to the muscular regeneration process (Dubois et al., 1994).

H. Mastocytes In the mdx mouse, the number of mastocytes is three times the normal value by 4 weeks and then decreases beginning at week 9; however, as in normal muscles the mastocytes are localized around the major blood vessels supplying the muscle. The peak coincides with the period of massive necrodregeneration (Gorospe et al., 1994; Lefaucheur et al., 1996).

I. Metabolism Changes in the lipid composition of the skeletal muscles of mdx and control (C57BL10) mice have been studied by nuclear magnetic resonance (NMR), a powerful noninvasive technique for studying living material (Gillet et al., 1993). The COSY sequence has been used to assign larger molecules such as fatty acids (Gillet et al., 1989). All the muscle spectra of dystrophic and control mice showed scalar correlations of saturated and unsaturated fatty acids. However, an additional correlation corresponding to triunsaturated fatty acid (linolenic-like) was highly visible in the muscles of mdx mice aged between 2 and 4 weeks (Fig. 4). This signal decreased as the mice grew and was not detected once they were more than 2 months old. Because myogenesis and regeneration of muscle in vivo can be mimicked in vitro using cultures of myogenic cells, the metabolism of muscle cell cultures has been studied in order to determine the origin of this linolenic-like signal in comparison with the different steps of myogenesis. The state of fusion of C2 cells can be distinguished from that of replicating cells by the presence of high-resolution signals consistent with saturated and unsaturated lipids and especially with triunsaturated “linolenic-like” lipids. Myoblasts can fuse to form myotubes during muscle regeneration, so it is possible that the specific triunsaturated linolenic-like signal in the mdx mouse muscle comes from the fusing cells and that it is characteristic of the regeneration of muscle fibers (SCbriC et al., 1988). Brain metabolism is abnormal in mdx mice (Tracey et al., 1996a): an increase in inorganic phosphate/phosphocreatine and pH, a reduction in

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FIG. 4 hi vivo 'H COSY spectrum of the hindleg of 3-week-old mdx mice. Changes in the lipid composition of mdx and control skeletal muscles are studied by nuclear magnetic resonance (NMR). The spectrum shows characteristic correlations of saturated and unsaturated fatty acid chains. In contrast to control mice muscle, the mdx muscle shows an additional correlation corresponding to triunsaturated (linolenic-like) fatty acids: K signal. This specific signal is present in the muscles of mdx mice aged between 2 and 4 weeks, but decreases once they are over 2 months old. This K signal is also detected in the spectra of fusing C2 cells. It is possible that the specific signal in the mdx mouse muscle comes from the fusing cells and that it is characteristic of muscle fiber regeneration. If this hypothesis is confirmed, 2-D 'H NMR could be used noninvasively to follow the regeneration process (reprinted from Neuromusculur Disorders 3, B. Gillet et al., In vivo 2D 1H NMR of rndx mouse muscle and myoblast cells during fusion: Evidence for a characteristic signal of long chain fatty acids, 433-438, copyright 1993, with permission from Elsevier Science).

total creatine, and an increased extracellular and decreased intracellular volume. The mdx mutation results in a decrease in energy metabolism: Metabolic rate, food intake, and physical activity all decrease in mdx mice between

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4 and 6 weeks. This difference compared with control animals is no longer apparent in 1-year-old mice (Dupont-Versteegden et al., 1994). In vivo, protein synthesis and degradation are higher in mdx mice than in controls at all ages. This may be the specific consequence of the absence of dystrophin since in the liver, in which there is no dystrophin, protein turnover is identical in mdx and control mice (MacLennan and Edwards, 1990). The high rate of proteolysis may be due at least in part to the accumulation of damaged proteins since abnormal proteins tend to be quickly degraded. Elimination and replacement of damaged proteins may constitute an important feature of the strategy by which the mdx muscle responds to the absence of dystrophin (MacLennan et al., 1991). In mdx muscle, levels of the glucose transporter GLUT4 are raised by 55%. In the diaphragm, the levels are identical to control levels at 5 or 6 weeks and decrease by 40% at 6 or 7 months. The expression of the major glucose transporter is related to the capacity of the muscle to regenerate rather than to the absence of dystrophin (Olichon-Berthe et al., 1993).

V. Therapeutic Projects Current research on the treatment of DMD is developed and tested using the mdx mouse. Three types of treatment are envisaged: pharmacologic, myoblast transplantation, and gene therapy. Most pharmacologic studies have focused on the effects of a glucocorticoid, prednisone. In vitro, prednisone stimulates myogenesis in cultures of mdx and normal muscle: the number of myotubes and levels of AChR, dystrophin, and D R P increase (Metzinger et al., 1993; Passaquin et af., 1993). In contrast, when administered to 15- to 45-day-old mdx mice, prednisone has no effect on the course of necrosis and regeneration (Weller et af., 1991), whereas another glucocorticoid, deflazacort, has a beneficial effect on both (Anderson et al., 1996). Anabolic steroid treatment increases myofiber damage in the mdx mouse (Krahn and Anderson, 1994). NMR is a useful noninvasive method to follow the effects of treatments on skeletal muscle metabolism (McIntosh et al., 1998). Glucocorticoid therapy slows the progression of DMD (Hardiman et al., 1992). Part of the beneficial effect of prednisone in DMD patients could be attributed to a reduction in Ca2+ influx and in the size of Ca2+ pools in dystrophic muscle fibers (Metzinger et al., 1995). However, the duration of this beneficial effect and the risk of long-term use remain to be determined. In 1989, two teams showed that when myoblasts from healthy mouse muscle were injected into muscles of mdx mice, the new cells fused with the mdx muscle fibers and produced dystrophin (Karpati etaf.,1989; Partridge et

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al., 1989). Myoblast transplantation leads to fiber formation only when immunocompetent but fully histocompatible donors and recipients are used (Vilquin et al., 1995). The use of a Y-chromosome-specific probe to track the fate of donor male myoblasts injected into dystrophic muscle of female mdx mice revealed rapid and massive death of the donor myoblasts soon after myoblast injection. There is limited movement of the injected donor myoblasts and fusion into host myofiber is rare (Fan et al., 1996b). Nevertheless, a high percentage of muscle fibers of donor origin can be obtained when myoblasts grown with bFGF are injected (Kinoshita et al., 1995) or when a short-term immunosuppressive treatment of mice with FK 506 is used (Asselin et al., 1995). Acute myoblast death can be prevented by control of the inflammatory reaction (Guerette et al., 1997). The use of sliced muscle grafts as a potential alternative strategy for myoblast transfer therapy proposed by Fan et al. (1996a) seems hypothetical because of the lack of myoblast migration between transplanted and host muscles (Moens et al., 1996). The application of this technique to DMD muscle initially yielded some dystrophin-positive fibers, but it was subsequently found that the functional benefit was extremely limited or short-lived (Law et al., 1990; Gussoni et al., 1992: Huard et al., 1992c; Karpati et al., 1993b; Mendell et al., 1995; Miller et al., 1997). Gibson et al. (1995) proposed an alternative approach to myoblast transfer therapy and reported the presence of dystrophin-positive fibers after implanting cloned dermal fibroblasts from normal mice into mdx muscle. The conversion of dermal fibroblasts (not muscle fibroblasts-the two are of different embryological origin) to a myogenic lineage is induced by a soluble factor derived from myoblasts (Wise et al., 1996). It has been demonstrated that the regional expression of recombinant dystrophin in dystrophic muscle leads to regional restoration of normal muscle morphology (mice: Cox etal., 1993; Dunckley etal., 1993:Matsumura et al., 1993; Ragon et al., 1993: Inui et al., 1996; Fassati et al., 1997a; dog: Howell et al., 1998). Numerous groups, in particular that of Karpati, are developing vectors, viral or plasmid DNA, which can be injected intravenously, intramuscularly, or systemically. Mdx mouse muscle treated by injections in the neonatal period expressed more dystrophin than muscle treated during adulthood or old age (Acsadi et al., 1996). In the case of herpes simplex virus-mediated gene delivery, a possible explanation is that basal lamina would be a physical barrier to infection (Huard et al., 1996). By 2 months postinjection, there is a substantial reduction in the number of dystrophin-positive fibers. This effect appears to be due in part to the activity of CD8+ cytotoxic lymphocytes directed against the transduced cells, leading to eventual elimination (Petrof et al., 1996). This hypothesis is substantiated by two observations: (i) In immunodeficient (SCID) mice, lacking both humoral and cellular immune competence, expression of

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transfected dystrophin is maintained over a longer period (Acsadi et al., 1996) and (ii) optimization of immunosuppression permits a sustained highlevel expression of dystrophin after gene transfer of dystrophin (Lochmuller et al., 1996; Chen et al., 1997; Zhao et al., 1997; Yang et al., 1998). The dystrophin minigene seems more effective than the full-length dystrophin gene (cf. Acsadi et al., 1996; Deconinck et al., 1996; Yanagihara et al., 1996). In fact, it has been shown in transgenic mdx mice expressing dystrophin with deletions that the cysteine-rich domain of dystrophin is critical for functional activity, presumably by mediating a direct interaction with pdystroglycan, and that the COOH terminus is not required for this assembly (Rafael et al., 1996). Contractile properties of diaphragm muscle segments from old transgenic-mdx mice do not differ from those of young transgenicmdx mice or control mice (Lynch et al., 1997). Minidystrophin gene transfer in mdx diaphragm leads to rapid and significant functional improvements (Decrouy et al., 1997). This line of research is expanding considerably. Important questions regarding the safety of the vector used, the difficulties of access to certain muscles, the number of transfected muscle cells, and the reproducibility of the results remain to be solved, however, before this demonstration finds a simple application in clinical therapy. The possibility of implanting a large numbers of genetically modified primary fibroblasts massively converted to myogenesis by adenoviral delivery of MyoD ex vivo has been tested in regenerating muscle of immunodeficient mice (Lattanzi et al., 1998). Three other ways to circumvent the numerous obstacles to gene therapy have been proposed: (i) the use of engrafted macrophages as potential shuttles for delivering a therapeutic agent (Parrish et al., 1996), (ii) the transplantation of retroviral producer cells toward the long-term goal of gene therapy (Fassati et al., 1997b), and (iii) the antisense strategy to transform DMD in Becker dystrophy phenotype by inducing exon skipping (Matsuo, 1996). Mdx mice are only mildly dystrophic, and utrophin-deficient mice show only subtle neuromuscular defects (Deconinck et al., 1997a; Grady et al., 1997a). However, utrophin-dystrophin-deficient mice present skeletal and cardiac myopathy and are considered a better model for DMD that mdx mice (Deconinck et al., 1997b; Grady et al., 1997b). The sparing of extraocular muscle in mdx mice is lost in mice lacking utrophin and dystrophin (Porter et al., 1998). Another model is proposed: the mutant generated by targeted disruption of exon 52 to disrupt the expression of the four other shorter isoforms that are also expressed from the dystrophin gene (Araki et al., 1997). In this mutant, muscle degeneration is similar to that observed in DMD. An alternative approach is to compensate for dystrophin loss by utrophin. There is evidence that utrophin may be capable of performing the same cellular functions as dystrophin (Tinsley and Davies, 1993; Blake et al.,

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1996b; Campbell and Crosbie, 1996). Expression of utrophin, with localization at the sarcolemma, is increased in DMD and Becker dystrophy (Karpati et al., 1993a; Mizuno et al., 1993) and mdx mice (see Section 111, C). Nevertheless, Vainzof et al. (1995) have shown an absence of correlation between utrophin localization and quantity and the clinical severity in DMD and Becker dystrophy. By transgenic expression of high levels of utrophin in mdx mice, Tinsley et al. (1996) demonstrated that utrophin could functionally replace dystrophin: Overexpression of utrophin leads to the restoration of all the components of DAGs, and mechanical performance of muscle is improved (N. Deconinck et al., 1997). Overexpression of utrophin even rescues the deterioration of the diaphragm, the most severely affected mdx muscle. The longevity of the truncated transgene expression has to be determined. The expression of a truncated utrophin transgene in muscle of utrophin-dystrophin-deficient mice prevents death and development of any clinical phenotype (Rafael et al., 1998). Identification of molecules or drugs that could upregulate utrophin is a very important goal for therapy. Today, only muscle and neural isoforms of agrin have been found to increase utrophin expression (Gramolini et al., 1998). The main advantages of such putative agents are (i) that there would be no need for gene replacement, because patients already have a functional utrophin gene and (ii) minimization of immunological responses. These different strategies should not be viewed as exclusive but rather can be clinically complementary.

VI. Concluding Remarks The mdx mouse is the best studied and therefore best understood animal model of Duchenne myopathy. Certain results are contradictory and further work is needed. There are various characteristics in common between these two myopathies: the absence of dystrophin, a reduction in the complex of associated glycoproteins, and the presence of utrophin along the muscular membrane and not restricted to the NMJ as in healthy muscle. There is, though, a very important difference which remains partly unexplained: In the mdx muscle intense regeneration compensates for the degeneration of muscle fibers. In DMD muscle, on the other hand, the regeneration process is weakened and muscle fibers are progressively replaced by fatty and connective tissues. Our results underpin the hypothesis that the increase in the components of basal lamina in vivo and the cessation of fibroblast proliferation observed in vitro may at least in part favor the marked muscular regeneration noted in mdx muscle. Indeed, we have found that fibroblasts taken from human DMD and control muscle have similar in vitro prolifera-

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tive capacities (Morin et al., 1995), and immunocytochemical visualization with polyclonal antibodies shows that the rate of expression of two components of basal lamina (laminin and HSPG) is lower in DMD than in control human muscle (Morin et al., 1993). The observed differences in these various parameters between mdx and DMD muscles could partly explain the regenerative capabilities of these two muscles: The myoblast/fibroblast balance seems t o favor myoblasts in mdx muscle. In DMD muscle the poor capacity of regeneration of the muscle seems to be due to a poor proliferative capacity of the satellite cells: A preparation of pure muscle satellite-cell populations has shown that the potential to generate myogenic cells for myofiber growth or regeneration is severely lacking even in the youngest DMD patients (Webster and Blau, 1990). All the treatments envisaged are designed to palliate this absence of regeneration, by promoting myogenesis or by limiting degeneration through pharmacologic treatments, by compensating for the weakness of the satellite cells by injection of myoblasts, or by compensating for the absence of dystrophin directly by complementing the genome through the use of vectors or indirectly by attempting to overexpress utrophin. In parallel with attempts to alter the genetic material of the DMD muscle through gene therapy, it appears necessary to continue the approaches employing cell and pharmacologic therapies.

Acknowledgments We thank the Association Franqaise contre les Myopathies (AFM), which has supported this work through grants. Sophie Morin was a recipient of a scholarship from AFM.

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Sklar. R. M., Beggs. A. H.. Lev, A. A., Specht, L., Shapiro, F., and Brown, R. H. (1990). Defective dystrophin in Duchenne and Becker dystrophy myotubes in cell culture. Neurology 40, 1854- 1858. Slater, C. R. (1987). Muscular dystrophy: The missing link in DMD? Nature (London) 330, 693-694. Smith, J.. Fowkes, G., and Schofield, P. N. (1995). Programmed cell death in dystrophic (mdx) muscle is inhibited by IGF-11. Cell Death Dify 2, 243-251. Sonnenberg, A. (1993). Integrins and their ligands. Curr. Topics Microbiol. Immunol. 184,7-35. Spencer, M. J.. Croall, D. E., and Tidball, J. G. (1995). Calpains are activated in necrotic fibers from mdx dystrophic mice. J. B i d . Chern. 18, 10909-10914. Stedman. H. H., Sweeney, H. L., Shrager, J. B.. Maguire. H. C., Panettieri, R. A,, Petrof, B., Narusawa, M., Leferovich, J. M., Sladky, J. T.. and Kelly, A. M. (1991). The mdx mouse diaphragm reproduces the degenerative changes of Duchenne muscular dystrophy. Nature (London) 352, 536-539. Straub, V., Bittner, R. E.. Leger, J. J., and Voit, T. (1992). Direct visualization of the dystrophin network on skeletal muscle fiber membrane. J. Cell Biol. 119, 1183-1191. Straub, V., Rafael. J. A., Chamberlain, J. S., and Campbell, K. P. (1997). Animal models for muscular dystrophy show different patterns of sarcolemmal disruption. J. Cell Biol. 169, 375-385. Sugita. H., Takemitsu, M., Koga. R., Ishiura, S., and Arahata, K. (1993). The expression of utrophin in mdx mouse muscle dystrophy. Acro Cardiol. 5, 11-16. Sugiyama, J.. Bowen, D. C.. and Hall, Z. W. (1994). Dystroglycan binds nerve and muscle agrin. Neuron 13, 103-115. Takagi. A., Kojima, S., Ida, M., and Araki. M. (1992). Increased leakage of calcium ion from the sarcoplasmic reticulum of the mdx mouse. J . Neurol. Sci. 110, 160-164. Takemitsu, M., Ishuira. S., Koga, R., Arahata, K., Nonaka, I., and Sugita, H. (1991a). A dystrophin homologue on the surface membrane of embryonic and denervated mdx mouse muscle fibers. Proc. Jpn. Acad. 67, 125-128. Takemitsu. M., Ishiura, S.. Koga. R., Kmamkura. K., Arahata, K., Nonaka, I., and Sugita. H. (1991b). Dystrophin-related protein in the fetal and denervated skeletal muscles of normal and mdx mice. Biockem. Biophys. Res. Comrnitn. 180, 1179-1186. Tanaka, H.. and Ozawa. E. (1990a). Expression of dystrophin mRNA and the protein in the developing rat heart. Biochrm. Biophys. Res. Commun. 172, 824-829. Tanaka, H., and Ozawa, E. (1990b). Developmental expression of dystrophin on the rat myocardial cell membrane. Histochemistry 94, 449-453. Tay, J. S. H., Low, P. S.. Lee, W. L., Lai, P. S., and Gan, G. C . (1989). Dystrophin function: Calcium-related rather than mechanical. Lancer 335, 983. Tidball, J. G., and Law, D. J. (1991). Dystrophin is required for normal thin filament-membrane associations at myotendinous junctions. Am, J . Pofhol. 138, 17-21. Tidball, J. G., Albrecht, D. E.. Lokensgard. B. E., and Spencer, M. J. (1995). Apoptosis precedes necrosis of dystrophin-deficient muscle. J. Cell Sci. 108, 2197-2204. Tiger, C.-F., and Gullberg. D. (1997). Absence of laminin a1 chain in the skeletal muscle of dystrophic dyldy mice. Muscle Nerve 20, 1515-1524. Tinsley, J. M., and Davies, K. E. (1993). Utrophin: A potential replacement for dystrophin'? Neuromusc. Disord. 3, 537-539. Tinsley, J. M., Blake, D. J.. Roche, A., Fairbrother, U., Riss. J., Byth, B. C.. Knight. A. E., Kendrick-Jones, J., Suthers, G. K., Love, D. R.. Edwards, Y . H., and Davies, K. E. (1992). Primary structure of dystrophin-related protein. Nature (London) 360, 591-593. Tinsley, J. M., Potter. A. C., Phelps. S. T., Fisher, R.. Trickett, J. I., and Davies, K. E. (1996). Amelioration of the dystrophic phenotype of mdx mice using a truncated transgene. Nurure (London) 384,349-353.

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Torelli, S., Sogos, V., Ennas, M. G., Muntoni, F., Clerk, A,, Strong, P. N., and Gremo, F. (1992). Dystrophin immunoreactivity in normal and Duchenne fetal neurons in culture. J . Neurosci. Res. 32, 116-125. Torres, L. F. B., and Duchen, L. W. (1987). The mutant mdx: Inherited myopathy in the mouse. Morphological studies of nerves, muscles and end-plates. Brain 110, 269-299. Tracey, I., Dunn, J. F., and Radda, G. K. (1996a). Brain metabolism is abnormal in the mdx model of Duchenne muscular dystrophy. Brain 119,1039-1044. Tracey, I., Dunn, J. F., Parkes, H. G., and Radda, G. K. (1996b). An in vivo and in vitro H1-magnetic resonance spectroscopy study of mdx mouse brain: Abnormal development or neural necrosis? J . Neurol. Sci. 141, 13-18. Turner, P. R., Schultz, R., Ganguly, B., and Steinhardt, R. A. (1993). Proteolysis in altered leak channel kinetics and elevated free calcium in mdx muscle. J. Membrane Biol. 133,243-251. Turner, P. R., Westwood, T., Regen, C. M., and Steinhardt, R. A. (1988). Increased protein degradation results from elevated free calcium levels found in muscle from mdx mice. Nature (London) 335, 735-738. Uchino, M., Yoshioka, K., Miike, T., Tokunaga, M., Uyama, E., Teramoto, H., Naoe, H., and Ando, M. (1994). Dystrophin and dystrophin-related protein in the brains of normal and mdx mice. Muscle Nerve 17, 533-538. Vachon, P. A., Loechel, F., Xu, H., Wewer, U. M., and Engvall, E. (1996). Merosin and laminin in myogenesis; Specific requirement for merosin in myotube stability and survival. J. Cell Biol. 134, 1483-1497. Vainzof, M., Passosbueno, M. R., Man, N. T., and Zatz, M. (1995). Absence of correlation between utrophin localization and quantity and the clinical severity in Duchenne/Becker dystrophies. Am. J. Med. Genet. 58, 305-309. Valentine, B. A., Cooper, B. J., Cummings, J. F., and De Lahunta, A. (1986). Progressive muscular dystrophy in a golden retriever dog: Light microscope and ultrastructural features at 4 and 8 months. Acta Neuropathol. 71, 301-310. Valentine, B. A., Winand, N. J., Pradhan, D., Moise, N. S., De Lahunta, A., Kornegay, J. N., and Cooper, B. J. (1992). Canine X-linked muscular dystrophy as an animal model of Duchenne muscular dystrophy: A review. Am. J. Med. Genet. 42, 352-356. Van Ommen, G. J. (1989). Communication. “Dystrophin” Colloquium, April 2-5, Cold Spring Harbor, NY. Verze, L., Buffo, A., Rossi, F., Oestreicher, A. B., Gispen, W. H., and Strata, P. (1996). Increase of B50/GAP-43 immunoreactivity in injured muscle nerves of mice. Neuroscience 70,807-815. Vilquin, J. T., Wagner, E., Kinoshita, I., Roy, R., and Tremblay, J. P. (1995). Successful histocompatible rnyoblast transplantation in dystrophin-deficient mdx mice despite the production of antibodies against dystrophin. J. Cell Eiol. 131, 975-988. Vilquin, J. T., Brusse, V., Asselin, I., Kinoshita, I., Gingras, M., and Tremblay, J. P. (1998). Evidence of mdx mouse skeletal muscle fragility in vivo by eccentric running exercise. Muscle Nerve 21, 567-576. Vracko, R., and Benditt, E. P. (1972). Basal lamina, the scaffold for orderly cell replacement. Observations on regeneration of injured skeletal fibers and capillaries. 1. Cell Biol. 55, 406-419. Wakayama, Y., Shibuya, S., Jimi, T., Takeda, A., and Oniki, H. (1993). Size and localization of dystrophin molecule: Immunoelectron microscopic and freeze etching studies of muscle plasma membranes of murine skeletal myofibers. Acta Neuropathol. 86, 567-577. Walsh, F. S., Pizzey, J. A., and Dickson, G. (1989). Tissue-specific isoforms of dystrophin. TINS 12,235-238. Wang, T. I., Xie, Z., and Lu, B. (1995). Nitric oxide mediates activity-dependent synaptic suppression at developing neuromuscular junction. Nature (London) 274,262-266.

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Watkins, S. C. (1989). Communication. “Dystrophin” Colloquium, April 2-5, Cold Spring Harbor, NY. Watkins. S. C., Hoffman, E. P., Slayter, H. S., and Kunkel, L. M. (1988). Immunoelectron microscopic localization of dystrophin in myofibres. Nature (London) 333, 863-866. Way, M., Pope, B., Cross, R. A,, Kendrick-Jones, J., and Weeds, A. G. (1992). Expression of the N-terminal domain of dystrophin in E. coli and demonstration of binding to F-actin. FEBS Lett. 301,243-245. Webster, C., and Blau, H. M. (1990). Accelerated age-related decline in replicative life-span of Duchenne dystrophy myoblasts: Implications for cell and gene therapy. Somar. Cell Mol. Genet. 16, 557-565. Wehling, M., Stull, J. T.. McCabe, T. J., and Tidball, J. G. (1998). Sparing of mdx extraocular muscles from dystrophic pathology is not attributable to normalized concentration or distribution of neuronal nitric oxide synthase. Neuromusc. Disord. 8, 22-29. Weller, B., Massa, R., Karpati, G., and Carpenter, S. (1991). Glucocorticoids and immunosuppressants do not change the prevalence of necrosis and regeneration in mdx skeletal muscles. Muscle Nerve 14, 771-774. Wessels, A,, Ginjaar, I. B.. Moorman, A. F. M., and Van Ommen, G. J. B. (1991). Different localization of dystrophin in developing and adult human skeletal muscle. Muscle Nerve 14, 1-7. Wise, C. J., Watt, D. J., and Jones, G. E. (1996). Conversion of dermal fibroblasts to a myogenic lineage is induced by a soluble factor derived from myoblasts. J. Cell. Biochem. 61, 363-374. Xu, R.. and Salpeter, M. M. (1997). Acetylcholine receptors in innervated muscles of dystrophic mdx degrade as after denervation. J. Neurosci. 17, 8194-8200. Yamashita, S., Takenaka, H., Sugimoto, S., Chihara, E., Sawada, A,, Matsukura, S., and Hamada, M. (1989). Axonal transport in mdx mouse sciatic nerve. J. Neurol. Sci. 92, 267279. Yanagihara, I., Inui, K., Dickson, G., Turner, G.. Piper, T., Kaneda, Y., and Okada. S. (1996). Expression of full-length human dystrophin in mdx mouse muscle by HVJ-liposome injection. Gene Ther. 3, 549-553. Yang, L., Lochmuller, H., Luo, J.. Massie, B., Nalbantoglu, J., Karpati, G . , and Petrof, B. J. (1998). Adenovirus-mediated dystrophin minigene transfer improves muscle strength in adult dystrophic (mdx) mice. Gene Ther. 5, 369-379. Yayon, A,, Klasbrun, M., Esko, J. D., Leder, P., and Ornitz, D. M. (1991). Cell surface, heparin-like molecules are required for binding of basic fibroblast growth factor to its high affinity receptor. Cell 64, 841-848. Yeadon, J. E., Lin, H., Dyer, S. M., and Burden, S. J. (1991). Dystrophin is a component of the subsynaptic membrane. J. Cell Biol. 115, 1069-1076. Yoshida, M., Noguchi, S., Wakabayashi, E., Piluso, G., Belsito, A,, Nigro, V., and Ozawa, E. (1997). The fourth component of the sarcoglycan complex. FEBS Lett. 2, 143-148. Yoshioka, K., Zhao, J.. Uchino, M., and Miike, T. (1992). Dystrophin isoforms and/or crossreactive proteins on neurons and glial cells in control and mdx central nervous system. J. Neurol. Sci. 108, 214-220. Zhang, K. X., Petry, K., Cournu, I., Morin, S., De La Porte, S., and Koenig, J. (1996). Inhibition of the DNA synthesis by insulin-like growth factor-binding protein-5 (IGF-BP5) in fibroblasts from hindlimb muscle of the 8-week-old male mdx mouse. Paper presented at the sixth Colloquium on Neuromuscular Diseases, Versailles. [Abstract] Zhao, J. E., Yoshioka, K., Miike, T., and Miyatake, M. (1993). Developmental studies of dystrophin-positive fibers in mdx, and DRP localization. J. Neurol. Sci. 114, 104-108. Zhao, J. E., Lochmuller, H., Nalbantoglu, J., Allen, C., Prescott, S., Massie, B., and Karpati, G. (1997). Study of adenovirus-mediated dystrophin minigene transfer to skeletal muscle

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by combined microscopic display of adenoviral DNA and dystrophin. H u m Gene Ther. 13, 1565-1573. Zubrzycka-Gaarn, E. E., Bulman, D. E., Karpati, G., Burghes, A. H. M., Belfall, B., Klamut, H. J.. Talbot, J., Hodges, R. S., Ray, P. N., and Worton, R. G. (1988). The Duchenne muscular dystrophy gene product is localized in sarcolemma of human skeletal muscle. Nature (London) 33, 466-469.

Regulation of Phosphate Transport and Homeostasis in Plant Cells Tetsuro Mimura Biological Laboratory, Hitotsubashi University, Naka 2-1, Kunitachi, Tokyo 186-8601, Japan

The inorganic phosphate (Pi) status of plants was reviewed based on the current knowledge of membrane transport systems and ion homeostasis. The Pi content and Pi distribution in plant cells were first considered in relation to experimental procedures used in their measurement. In order to understand the mechanisms which contribute to the Pi status, transport systems for Pi across the plasma membrane and the tonoplast were examined in detail. Recent progress in molecular biological approaches is discussed, especially for Pi transport across the plasma membrane. The molecular basis for Pi efflux across the plasma membrane and for Pi movements across the tonoplast still remains to be resolved. The involvement of Pi transport in Pi homeostasis is discussed from both the cellular and the whole plant perspectives. KEY WORDS: Apoplast, Inorganic phosphate (Pi), Membrane transport, Pi homeostasis, Plasma membrane, Plant cell, Tonoplast, Vacuole 0 1999 Academic Press.

1. Introduction Phosphorus is one of the most essential elements for biological organisms. There are many compounds in cells which contain phosphorus. The genetic apparatus is based on phosphate groups in the polynucleotides of DNA and RNA. Phospholipids are fundamental building blocks for cellular membranes, whereas energy transduction revolves around phosphate in ATP and in numerous other metabolic compounds. Recently, it has been established that protein phosphorylation-dephosphorylation by phosphate transfer is a major reaction in the regulation of various cellular functions (Ranjeva and Boudet, 1987). lnrernnriunal Rrvrrw of Cyrologs, Vol. 191

0074-7hYhiYY $30.(!4)

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Copyright ri3 1999 hy Academic Press. All rights 01 reproduction in any form rrherved.

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Phosphorus was one of the first elements to be recognized, being isolated from human urine in 1669. Isolation of elements from organic material is quite rare and phosphorus may therefore be considered an element symbolic of living organisms. Of course, carbon, nitrogen, oxygen, and hydrogen may be more important elements than phosphorus, but the position of phosphorus as an essential element in living organisms is unique. First, the total phosphorus in the earth which is utilizable by living organisms is limited differently from that of other major elements. All phosphorus in organisms originates in phosphate molecules which are taken up from the soil by plants. Free phosphate concentrations in the soil, however, are very low-usually <10 p M (Marschner, 1995; Schachtman et al., 1998). Plants must take up large amounts of phosphorus from this poor supply-to the benefit of all living things-and to achieve this their uptake systems must maintain a high level of activity capable of exploiting this resource. Stated simply, the high activity of phosphate uptake systems in plants is supporting all life on the earth. In this review, I focus on phosphate transport mechanisms and their regulation in plants. The phosphate transport systems of intracellular membranes, especially in the tonoplast, will also be considered. The second important characteristic of phosphate is that it exists either as inorganic ions or as organic derivatives such as sugar phosphates in the cell. Pi is therefore situated at a branch point between inorganic and organic metabolism. For this role, the concentration of phosphate in vivo must be strictly regulated in the various compartments, i.e., there must be phosphate homeostasis. Phosphate (orthophosphoric acid) homeostasis is achieved via many cellular activities, including membrane transport, metabolic conversion between inorganic ions and organic molecules, and chemical reactions with other inorganic ions (e.g., Ca). In this review, I discuss Pi homeostasis from the standpoint of how plants recognize the Pi status in the cell and how plants then control the transport activities which depend on the cellular Pi status. Bieleski (1973) published an excellent review on phosphate status in plants. It dealt with most of the Pi-related phenomena in plant tissues and cells. Later reviews covered similar subjects (Bieleski and Ferguson, 1983; Clarkson and Gringnon, 1991; Mimura, 1995a; Schachtman et al., 1998). Recent experiments using molecular biological techniques are clarifying some of the mechanisms of Pi transport and Pi homeostasis supposed by physiological investigations. I deal with the relationships between past and curent physiological efforts and modern molecular investigations into plant Pi-related phenomena. Lastly, I define phosphate in the context of this review. Inorganic phosphates in living organisms are usually in the form of orthophosphoric (monophosphoric) acid, pyrophosphoric (diphosphoric) acid, and polyphos-

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phoric acid. Throughout this review, the term phosphate (or inorganic phosphate), abbreviated as Pi, will be used to denote orthophosphoric (monophosphoric) acid only and not pyrophosphate or polyphosphate, which will be referred to explicitly.

II. Measurements of Inorganic Phosphate All research concerning Pi-related phenomena must ultimately be based on the measurements of Pi concentrations in plant cells and tissues. Without such data on Pi levels, it is difficult to judge what really occurs in situ. The most popular method for detecting phosphorus is the colorimetric measurement based on the complex formed between phosphorus and molybdic acid (molybdophosphoric acid). There are many molybdophosphoric acid methods, although they are basically variations of the original method of Fiske and Subbarow (1925),which is still useful for measuringphosphorus concentrations. The most sensitive of these methods can detect less than approximately 10 p M Pi solution in normal cuvettes. Orthophosphate can also be measured by enzymatic reactions based on a sequence of three reactions: (i) glycogen + Pi 4 glucose-1-P by phosphorylase, (ii) glucose1-P + glucose-6-P by phosphoglucomutase, and (iii) glucose-6-P + NADP + 6-P-gluconolactone + NADPH by glucose-6-P dehydrogenase (Lowry and Passonneau, 1972). Recent progress in ion chromatography has made the measurement of Pi much easier. Ion chromatograms equipped with an anion suppressor can detect <1 p M Pi in solution. Although colorimetry and chromatography are the easiest ways to measure phosphorus or phosphoric acid, phosphorus must be extracted and therefore cells and tissues must be destroyed. Pi is usually extracted by hot water or TCA or PCA solutions. For colorimetric measurements, acid extraction is widely used, although it has the disadvantage that some of the esterified phosphate, such as in DHAP, is also released as Pi. When Pi is measured with ion chromatography, it is difficult to use acids such as TCA or PCA because the background sample conductance becomes too high. In this case, hot water extraction is used, and there may be some alteration in Pi levels due to the activity of enzymes which are not rapidly denatured by the hot water treatment. The extraction of Pi from tissues and cells has some essential difficulties. To avoid the problems caused by extraction, in vivo measurements are required. In vivo measurement of some ions (e.g., Ca2+,K+, Na+, and Cl-) has been possible using either ion-selective intracellular microelectrodes or penetrating ion-sensitive fluorescent dyes. Attempts to use similar techniques for Pi have proved unsuccessful. Perhaps the most promising area

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of research is the development of Pi-dependent biosensors based on enzyme reactions. Watanabe et al. (1988) used nucleoside phosphorylase to detect a few hundred p M Pi. D'Urso and Coulet (1990) improved this sensor to detect a few pM Pi, whereas Nakamura et al. (1997) showed that with a combination of pyruvate oxidase and peroxidase <1 pM Pi could be detected. These sensors are still being improved and it is difficult to measure intracellular Pi levels using these sensors in microelectrodes. However, currently it is possible to monitor the extracellular Pi level with the previously mentioned sensors in real time. The development of an intracellular Pi monitoring system would greatly enhance our understanding of the relationship between plant growth and Pi uptake. Although currently there is no convenient method, such as ion-selective electrodes or fluorescent dyes, for Pi, it is possible to measure the concentrations of Pi and many other P compounds in vivo using nuclear magnetic resonance (NMR) spectrometry (Lee and Ratcliffe, 1983; Rebeille et al., 1983; Roberts, 1984). The NMR signal of 31Pis weaker than 'H but much stronger than other major elements in living organisms. When NMR measurement was applied to living materials 25-30 years ago, the magnetic fields of the available instruments were too low to detect anything but the strongest P signals. This still required long signal accumulation times and/ or a large amount of materials to distinguish the signal from the noise (Mimura and Kirino, 1984). Modern NMR spectometers can detect in vivo phosphorus within a few seconds and in small samples. A further advantage of 3'P-NMR is that it is possible not only to measure Pi in vivo but also to distinguish Pi pools in different compartments, mostly because of the different p H in these compartments. p H affects the degree of dissociation of Pi and thereby alters the chemical shift of Pi peaks. In plant cells the vacuole is a large compartment with a p H of approximately 5 compared to the cytoplasm which has a pH of approximately 7.5. The Pi signals from these two pools are therefore quite different, as shown in Fig. 1 for Catharanthus suspension-cultured cells in which Pi in the cytoplasm and vacuole are clearly distinguishable. Also, we can measure other Pi-containing substances using 31P-NMR signals. Regarding NMR measurements, it is noted that when the pH of one compartment changed to a similar value of the other compartment, it may be judged that Pi moved from the former to the latter compartment.

111. Distribution of Inorganic Phosphate Measurements of total phosphorus in whole plants or tissues give values between about 0.3 and 0.5% of dry matter. The value is strongly dependent

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PHOSPHATE TRANSPORT AND HOMEOSTASIS IN PLANT CELLS

3

MDP

0

-10

-20

-30

-40

Chemical shift (ppm) FIG. 1 An example of "P-NMR measurements. "P-NMR spectrum of Catharanthus suspension cultured cells. MDP, methylene diphosphonic acid; chemical shift standard. Peak assignments: 1, G6P; 2, cytoplasmic Pi; 3, vacuolar Pi; 4, y-nucleotide triphosphates; 5 , a-nucleotide phosphates; 6 , and 7, UDPG; 8, P-nucleotide phosphates. Cytoplasmic and vacuolar pH values are calculated from Pi resonances of peak 2 and 3. respectively. Pi content of each compartment is calculated from the area under each peak (from Sakano er al., 1992, by courtesy of the authors and kind permission from the American Society of Plant Physiologists).

on the nutrient Pi supply. In most cases, unless the plant is under Pi deficiency, the phosphorus content of organic compounds is relatively constant and independent of the Pi supply; only the Pi fraction changes. Table I summarizes some of these values for the Pi content of various tissues from a range of plant species. Pi is often expressed on a dry weight basis, but the most important value when considering biological reactions should be the concentration derived from the fresh weight. Henceforth, I will discuss Pi levels in terms of concentration. When plants are subjected to Pi deficiency, Pi is retranslocated from older tissues (leaves) to younger tissues. In this case, Pi concentrations of the older tissues and the younger tissues change with growth. The dynamics of Pi distributions between tissues via xylem and phloem is one of the most important phenomena in plants (Jeschke et al., 1997). Currently, there is little information on the mechanisms which lead to differences in Pi distribution between different cell types. For example, in barley leaves, Pi levels of mesophyll cells were found to be much higher than those in epidermal cells (Dietz et al., 1992). This difference seems reasonable since Pi is one of the key substrates of photosynthesis, which is more active in the mesophyll cells. However, Outlaw et al. (1984) derived the opposite results (Table I).

TABLE I Pi Distribution in Cells and Tissues

Pi level

Material

Method of Pi measurement

Culture condition

Original value

Estimated level (mM)

Root Hordeum vulgare

Colorimetry

Trifoliunz repens

Colorimetry

-Pi (8 days) +Pi -Pi (14 days) +Pi

Original value

Estimated level (mM)

Reference

Shoot

43 pmol P/g DW 150 pmol P/g DW

6.1 P" 21.4 P"

5.1 mg Plg DW 8.9 pmol Plg DW

23.5 Pa 41.0 P"

49 pmol P/g DW

7 P"

P

205 pmol P/g DW

29.3

4.5 mg P/g DW

20.7 P" 35.5 P"

7.7 pmol P/g DW

Clarkson and Scattergood (1982)

Dunlop and Gardiner (1993)

Leaf Spbzacia oleracea

Colorimetry

-Pi (19 days) +Pi

0.5 pmol/mg Chl 5.0 pmollmg Chl

2.5' 25.0b

Hordeunz vulgare

Colorimetry

-Pi (24 days) +Pi

0.4 pmol/mg Chl 13.0 pmol/mg Chl

2.0' 65.0h

Dietz and Foyer (1986)

Hordeum vulgare

Colorimetry

-Pi +Pi (1 mM) +Pi (25 mM)

Hordeum vulgare

3'P-NMR

+Pi

0.83 pmol/mg Chl 5.32 pmol/mg Chl 11.07 pmol/mg Chl

4.2' 26.6' 55.4'

7.9 pmol/mg Chl

39Sb

-I

Stitt et al. (1985)

rn --I m x= z

ffl

'0

::

Leaf tissue

-i

Epidermal protoplast Hordeum vulgare

Anion chromatograph

+Pi (SO mM)

5.3 p m

D

z

Mesophyll protoplast

0

I

0

Dietz et al. (1992)

135 mM

$j cn

Leaf tissue Epidermal cell Vicia faba

Enzymatic measurement

+Pi

60 mM

Note. Abbreviations used: DW, dry weight; FW, fresh weight P level (not Pi) was estimated based on 1 g DW/7 g FW. * Pi level was estimated based on 0.2 gFWlmg Chl.

Palisade cell 5mM

5

@

z '0

Spongy cell 4mM

5 z

Gurd cell 100-200 mM

-4 0

Outlaw el al. (1984)

rn

r r ffl

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TETSURO MIMURA

Cells have many intracellular compartments. In plant cells, there is a nucleus (sometimes several), mitochondria, chloroplasts, golgi apparatus, cytosol, vacuole, etc. We do not yet know the Pi level in all these compartments. Most research on the subcellular distribution of Pi in plant cells has concentrated on the cytoplasm, the vacuole, and plastids (Table 11). Pi levels in cytoplasm have been estimated by various methods, including NMR and X-ray microanalysis, and also by subtraction of the vacuolar content from the whole cell Pi content. There are large differences in these estimates, ranging from a few mM to higher than 20 mM (Table 11). Methods that involve mechanical extraction are likely to overestimate Pi levels because of the unavoidable release of some Pi from organic P by enzymatic hydrolysis. On the other hand, 3LP-NMRcan detect only active forms of Pi in cellular compartments and not the NMR-invisible Pi in the cell, such as Pi in precipitated salts. These insoluble fractions are in equilibrium with metabolic Pi pools and may be important buffers of intracellular Pi. Not surprisingly, the levels of Pi in the cytoplasm measured by NMR are usually lower than levels measured by other methods, usually < l o mM). In many cases, it is most difficult to estimate the compartment volume, since methods for this quantify the amount of Pi in a given compartment and in order for this to be converted to a concentration, the volume of the compartment also must be known. Winter et al. (1993,1994) estimated the cytosolic volume of mesophyll and epidermal cells of spinach and barley leaves by stereological analysis of light and electron micrographs. They showed that in mesophyll cells, the vacuole occupied about 60-80%, the chloroplasts about 20-30%, and the cytosol 5-10% of total cell volume. These values were similar to those formerly supposed. We have measured the cytoplasmic Pi concentration of characean cells in which cytoplasmic and vacuolar volumes can be determined relatively directly (Mimura and Kirino, 1984;Takeshige etal., 1992) and the vacuole can easily be separated. Characean internodal cells have a simple cylindrical form so that total cell volume can be measured accurately. The cytoplasmic volume can be calculated from the thickness of the cytoplasmic layer measured under a microscope or by centrifuging the cytoplasm to one end of the cell. Because of the large size of these cells, the cytoplasmic fraction and the vacuolar contents are easily separated by the intracellular perfusion technique (Tazawa et d., 1987). We estimated that the cytoplasmic Pi concentration was approximately 15 mM (Mimura and Kirino, 1984; Takeshige et al., 1992). This value may be overestimated because of some Pi released from the organic P fractions during extraction, but the normal cytoplasmic Pi level is unlikely to be <10 mM. In green cells, it is known that the chloroplasts sometimes become Pi deficient in the light (Sivak and Walker, 1986; Walker and Sivak, 1986). This suggests that photosynthesis can be limited by the supply of Pi from

PHOSPHATE TRANSPORT AND HOMEOSTASIS IN PLANT CELLS

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the cytoplasm. In spinach, under these conditions, increasing Pi by feeding through the transpiration stream increased the photosynthetic rate (Dietz and Foyer, 1986). Pi is supplied from the cytosol through a Pi translocator across the inner envelope (Fliigge and Heldt, 1991) and the results from these experiments indicate that the biochemically active cytoplasmic Pi may be much smaller than the total cytoplasmic Pi estimated following extraction. The vacuolar Pi level fluctuated greatly depending on the nutritional status. When barley plants were grown in the presence of 40 mM Pi, the vacuolar Pi level reached about 120 mM (Mimura et al., 1990), although this was an unusual situation. On the other hand, under Pi deficiency, the vacuolar Pi pool was virtually exhausted in many plants (Lee et al., 1990; Mimura et al., 1996). Thus, the vacuolar Pi level can vary over a wide range. The Pi levels in other organelles are not known with any degree of confidence. Although the vacuole, cytoplasm, and plastids occupy more than 95% of the total cell volume, the smaller organelles may make a disproportionate contribution to the overall P metabolism. To discover whether this is the case, it will be necessary to measure Pi levels in these small compartments. Pi concentrations in the extracellular phases have been estimated for the xylem and cell wall (Marschner, 1995). Although these values were also dependent on the Pi nutrition, they existed in a narrow range from a few hundred p M to a few mM. Lastly, we must consider the relationship between the levels in plants and in the environment from which plants must obtain Pi. As mentioned previously, the fraction of the total Pi in the earth which is accessible to living organisms is limited (Marschner, 1995; Schachtman et al., 1998). Pi levels are low not only in soil but also in lakes and seas (Bieleski, 1973), often <1 p M . Thus, most wild plants in nature are usually subjected to Pi deficiency. In order to understand the Pi status of plants, it is necessary to understand how plants can absorb Pi from such low environmental concentrations.

IV. Membrane Transport of Inorganic Phosphate Plants communicate with their environment through the plasma membrane. Pi transport across the plasma membrane is therefore of fundamental importance to other processes involved in the subsequent redistribution of Pi into various organelles and to other plant parts. I discuss what is known about plasma membrane Pi transport before examining the role of the vacuole and transport across the tonoplast in maintaining the Pi status of

TABLE I1 Estimation of Subcellular Levels of Pi

Pi level Cytoplasm

Material Hordeum vulgare Leaf Protoplast Triticum sp. Leaf Protoplast Zea mays root

Zea mays Root rips

Method of Pi measurement

Culture condition

Original value

Vacuole Estimated level (mM)

Estimated level

Original value

(mM)

Reference Stitt er al. (1985)

31P-NMR

+ Pi

35 mM

35

75% of 7.9 mmol/mg Chl 89% of 7.3 mmol/mg Chl

37" 41"

31P-NMR

+Pi

28 mM 18 mM

28 18

78% of 5.1 mmol/mg Chl 82% of 5.0 mmollmg ChI

25" 26"

31P-NMR

-Pi (7 days) 1pM Pi (7 days) 10 p M Pi (7 days) 500 p M Pi (7 days)

0.44 pmol/ml 0.41 pmol/ml 0.43 pmollml 0.49 pmollml

Stitt et al. (1985)

root vol. root vol. root vol. root vol.

2.2' 2.1' 2.2' 2.5'

0.6 pmollml 1.7 pmollml 3.9 pmol/ml 6.2 pmollml

root root root root

vol. vol. vol. vol.

0.75' 2.1' 4.9b 7.8'

Lee and Ratcliffe (1993)

rri -I

v)

c D

0

Lee et al. (1990) 31P-NMR

6mM

6

2

2c D D

Pisurn sativurn Root tips

31P-NMR

4.2 mM

4.2 11.5 21.5

Zea mays

Leaf Acer pseudoplatarnus

3'P-NMR

-P (9 days) +P (9 days)

11.5 mM 21.5 mM

"P-NMR

0.3 mM Pi (8 h)

1 pmol/g FW

5'

5.1 mM 38mM 2.3 pmollg

5.1 38

FW

2.9'

Cultured cells 14.7 mM

14.7

5.7 mM

5.7

Mimura and Kirino (1984)

Vacuolar sap isolation

Hordeum vulgare

Leaf protoplasts .~

Rebeille er al.

(1983)

Nitellopsis obtusa

Internode

Loughman et al. (1989)

Vacuole isolation

5

a

-I

z 71

-Pi 1.5 mM Pi 40 mM Pi

26 mM 24.8 mM 35.1 mM

26 24.8 35.1

5.2 mM 40.5 mM 144.7 mM

5.2 40.5 144.7

Mimura et al. (1990)

Pi level of the cell was estimated based on 0.2 g FW/mg Chl. Then, the vacuolar Pi level was estimated on the assumption that volume of vacuole was 80% of total cell volume. The cytoplasmic volume was estimated as 20% of total cell volume.

'

3

5 z

-I 0

rn r FY

160

TETSURO MIMURA

the cell. I only briefly mention the Pi transport mechanisms related to photosynthesis and respiration since there are many excellent reviews on Pi transport across the chloroplast envelope and the inner membrane of the mitochondrion (chloroplasts: Fliigge and Heldt, 1991; mitochondria of yeast and animal cells: Ferreira and Pedersen, 1993). A. Plasma Membrane

Pi transport across the plasma membrane has been measured using various cells and tissues. Physiological research has concentrated on root cells, but there have also been some good studies on cultured cells, algae, and yeast.

1. Measurement of Pi Influx Net uptake of Pi is usually determined by measuring the increase of Pi content in the plant over a given period of time or from the decrease of the Pi content in the extracellular solution. To measure unidirectional influx it is usually necessary to use radioisotopic tracers (32Phalf-life of 14 days; 33P half-life of 25 days) and short uptake times. Uptake rates of Pi for various plant material are summarized in Table 111. The Pi uptake rates vary considerably between species, between different tissues of the same plant, with P nutritional status, and with the method of measurement. P nutritional status is one of the most important determinants of Pi uptake rate; under Pi deficiency, the capacity for Pi uptake increases, in some cases quite dramatically. This phenomenon has been observed not only in higher plants but also in bacteria and blue-green algae (Rao and Torriani, 1990; Wagner et al., 1995). Furthermore, in higher plants, Pi deficiency affects the Pi uptake rate not only in the roots but also in leaves. Mimura et al. (1990) noted that mesophyll cells increased their Pi uptake rate following P starvation. Table 111 shows kinetic parameters of K,, and V,,, measured in various plants. It is clear that plant cells have a variety of Pi uptake systems with different Krr,’s(Clarkson and Luiittge, 1991). Beever and Burns (1977) using Neurospora, found that K , and V,,,, vary independently when exposed to different levels of Pi in the growth medium (see also Bieleski and Ferguson, 1983). This suggested the existence of multiple Pi transport systems and a shift from one transport system to another dependent on the Pi nutritional status or level of Pi supply. In recent studies, influx isotherms for Pi have been commonly interpreted in terms of two uptake systems, one having a low K , (high affinity) and the other a high K,, (low affinity), both differing in their V,,,, In Lemna (Ullrich-Eberius et at., 1984), Catharanthus (Furihata et al., 1992; Schmidt et al., 1992), and Chara (Mimura et a[., 1998), concentra-

PHOSPHATE TRANSPORT AND HOMEOSTASIS IN PLANT CELLS

161

tion-dependent Pi uptake appeared to be composed of two saturating systems (Fig. 2a). On the other hand, barley mesophyll protoplasts showed a combination of saturation kinetics and a linear relationship between Pi uptake and concentration prevailed at higher concentrations (Mimura et al., 1990) (Fig. 2b). In barley mesophyll cells Mimura et al. were unable to find a high-affinity uptake system (K,,, <10 p M ) even under Pi-deficient conditions. Either the activity was lost during enzymatic treatment to make the protoplasts or, alternatively, mesophyll cells might not have such a high-affinity system because the apoplastic Pi level is usually maintained at a higher level (Mimura et al., 1992). In Catharanthus (Sakano et al., 1995) and tobacco (Shimogawara and Usuda, 1995) suspension-cultured cells, only one kind of Pi transporter was found irrespective of Pi conditions. Pi deficiency changed the V,,, but not K,,, in tobacco. 2. Mechanisms of Transport

It is important to note that under normal physiological conditions, uptake of Pi requires an input of energy because it moves against both electrical and concentration gradients. Near neutral pH, Pi carries either one or two negative charges and hence will be repelled by the negative potential of the cytoplasm (- 150 to -200 mV). In addition, the concentration of Pi in the cytoplasm is usually approximately 10 mM, compared to the external solution in which the concentration may be as low as 1 p M . Thus, passive movement of Pi will always be in the direction of efflux.

a. H+-Dependent Uptake It is widely believed that Pi is transported into the cell across the plasma membrane via cotransport with H'. There are three independent lines of evidence that support H'/Pi cotransport. First, when Pi is taken up into the cell, the extracellular pH increases. Sakano (1990) showed a linear increase in pH in the medium containing Catharanthus roseus cultured cells when Pi was added, and this was mirrored by a decrease in cytoplasmic pH (Ullrich and Novacky, 1990; Mimura et aZ., 1992; Sakano et al., 1992). Figure 3 shows an example of simultaneous measurements of extracellular and intracellular pH during Pi uptake of Catharanthus cells. After addition of phosphate, the extracellular p H increased until the exhaustion of added Pi. At the same time, the cytoplasmic pH first decreased and then maintained a lower value (cytoplasmic pH homeostasis; Sakano et al., 1998). After the exhaustion of extracellular Pi, both the extracellular and the intracellular p H recovered to the original values (Sakano et al., 1992). Similar acidification of the cytoplasm induced by Pi transport was measured in vivo in leaves of barley (Mimura et al., 1992).

TABLE 111 Pi Uptake Activities and Kinetic Parameters (K, and VM) of Various Materials

Material Catharanthus roseus cultured ceIls

Nicotiana glutinosa Cultured

Culture condition

K, (pM)

High affinity

Low affinity

vmax

V*,X

Original value

Estimated (nmol/ min/gFW)

-Pi

7

20 pmol/hr/g FW

330

-Pi

5.6

36 nmollminig FW

36

+Pi -Pi

3.5 3.0

11 nmol/min/g F W 0.31 nmol/h/l@ prot.

11 17"

+Pi

n.d.

+Pi

cells Nicotinana tabacum Cultured cells

-Pi +Pi

2.5 2.5

64 nmol/min/ml p.c. 12 nmol/min/ml p.c.

170' 3.2'

K* (pM)

900 46.3

Original value

110 nmol/min/g FW 1.41 nmol/h/l@ prot.

Estimated (nmol/ min/g FW)

Reference Sakano et al. (1995) Schmidt et al. (1992)

110 7.8"

46.7

1.26 nmol/h/l@ prot.

7.0"

58

5.9 nmol/h/mg prot.

46

Furihata et al. (1992) Mettler and Leonard (1979)

Lemma gibba

-Pi +Pi

7.9

0.9 pmol/h/g FW

15

Zea mays Root

+Pi

0.49 1.8

0.71 pmol/h/g FW 0.73 pmollhlg FW

12 12

Chara corallina Internode

-Pi +Pi

7 4

2.7 nmol/s/m2

0.8d

25

Nitella translucens Internode

1.8 pmol/slcm*

Neurospora crassa

SO p M Pi 10 mM Pi

2.4 2.9

2.3 pmol/min/g DW 0.3 pmol/min/g DW

Hordeum vulgare

-Pi +Pi

4.9 6.6

480 nmol/h/g FW 260 nmol/h/g FW

76

1.4 pmol/h/g FW 0.12 pmol/h/g FW

23 2

-u I I

5

Sentenac and Grignon (1985) J;: P

z

190

30 nmol/s/m2

9d

Mimura et al.

v)

220

17 nmol/s/m2

5d

(1998)

8

Sd

330E 43

Ullrich-Eberius et al. (1984)

Smith (1966)

370 1029

6.3 pmol/min/g DW 3.4 pmol/min/g DW

900' 490'

Burns and Beever (1977a)

3 P

2

v)

Pisum sativurn

Root protoplast

z 9

8 4

2 Lefebvre and Clarkson (1987)

+Pi

7.7 9.9

184 nmol/h/g FW 41.7 nmol/h/106 prot.

3 46r ~~

Note. V,, V,, ' V,, V,,, V,, V,,,

'

Abbreviations used. DW, dry weight; FW, fresh weight; P.c., packed cells. was estimated based on 0.3 pg/104 protoplasts. was estimated based on 1 ml p.c.10.37 g FW (Shimogawara and Usuda, 1995). was estimated based on 1 mg protJ7.6 X 105 protoplasts (Mettler and Leonard, 1979) was estimated based on 0.005 m2/g FW was estimated based on 1 g DW17 g F W was estimated based on 6.6 X lo7 protoplastslg FW

-I 0

rn r

G;

164

TETSURO MIMURA

b

a

I

h

1 . -

T

a300

30

0 U

v1

c

E . 220

N

5

:;": U

r-

2 200

a

0

5

v

v

x

e'E 1 0

.-

B

e

100

.-

b

E

0

0 '

0

0.2

0.4

0.6

0.8

Pi concentration (mM)

1

I

0

I

I

2

4

I

I

6

8

I

1

0

Pi concentration (mM)

FIG. 2 Typical examples of Pi influx across the plasma membrane. (a) Concentration-dependent Pi influx of Cham corallina internode shows two Michaelis-Menten-type parameters (from Mimura, T., Reid, R. J., and Smith, F. A,, 1998, Control of phosphate transport across the plasma membrane of Churn corallina, J. Exp. Bot. 49, 13-19, by permission of Oxford University Press). (b) Concentration-dependent Pi influx of barley mesophyll protoplasts shows a combination of one Michaelis-Menten-type kinetics at lower Pi concentrations and linear increase at higher Pi concentrations (from Planta, Phosphate transport across biomembranes and cytosolic phosphate homeostasis in barley leaves, Mimura, T., Dietz, K.-J., Kaiser, W., Schramm. M. J., Kaiser, G., and Heber, U., 180, 139-146, Fig. 4,1990,O Springer-Verlag).

FIG. 3 Pi-induced changes in pH of external medium (continuous line) and fluorescence intensity (noisy trace) of BCECF loaded into the suspension-cultured Catharanthus cells. A decrease in fluorescence intensity means acidification of the cytoplasm. Fluorescence intensity began to increase as soon as the external medium pH reached its peak, when exhaustion of medium Pi by cells was completed (from Sakano r? al., 1992, by courtesy of the authors and kind permission of the American Society of Plant Physiologists).

PHOSPHATE TRANSPORT AND HOMEOSTASIS IN PLANT CELLS

165

Second, accumulation of Pi is usually associated with membrane depolarization and not hyperpolarization, which would be expected if Pi was transported alone. It seems reasonable to presume, on the basis of the p H changes mentioned previously, that the positive charges transported with Pi are protons. Ullrich-Eberius et al. (1981) demonstrated transient depolarization of the membrane potential in Lemna (Fig. 4). The amplitude of the depolarization was dependent on the membrane potential before Pi treatment. Furthermore, they demonstrated that the concentration-dependent changes in membrane potential could be fitted by two MichaelisMenten functions (Ullrich-Eberius et al., 1984). Pi-induced membrane depolarization was also observed in Pi-deficient Trifolium (Dunlop and Gardiner, 1993). The third line of evidence in support of H'/Pi cotransport comes from the observation that inhibitors that collapse the electrochemical potential gradient for H' inhibit Pi uptake across the membrane (Lin, 1979). There are two different observations on the p H dependency of Pi transport. One is that the Pi uptake rate has a peak around p H 5 or 6, sometimes more acidic (Guardiola and Sutcliffe, 1971; Ullrich-Eberius, 1973; Lin, 1979). Authors often argued that this was evidence of H+/Pi cotransport. However, the use of strong buffers to fix the medium pH inhibited H' cotransport systems. Sakano et al. (1992), in fact, showed that the addition of Good buffers in the medium decreased Pi uptake. The same phenomenon was also observed in corn roots (Thibaud et al., 1988). Sentenac and Grignon (1985) showed that the rate of Pi uptake into corn roots was correlated with the pH of the cell wall and not of the medium. They found that under

Time

(min)

FIG. 4 Pi-induced transient membrane depolarization of Lemna gibba. Membrane depolarization suggests that influx of Pi cotransported with H ' carries a positive charge(s) into the cell (from Ullrich-Eberius et al.. 1981, by courtesy of the authors and kind permission from the American Society of Plant Physiologists).

166

TETSURO MIMURA

some conditions the pH in the wall was quite different from that of the medium and this altered the degree of dissociation of Pi (i.e., H2P04- or HP04*-). Thus, the pH dependence of Pi uptake based on the medium pH might not always reflect the true pH dependence of the transport system (Clarkson and Grignon, 1991). The other observation is that there is no distinct pH dependence for Pi uptake. In Catharanthus cultured cells, when the medium pH was not buffered, Pi uptake was constant between p H 3 and 6 (Schmidt et al., 1992). Between these pH values, the charge on the Pi molecule changes from 0 to - 1.We do not know which ionic species of Pi is transported across the plasma membrane. Like the Pi-binding protein of Escherichia coli, Pi transporters of the higher plant plasma membrane may be able to bind Pi molecules having different charges (Luecke and Quiocho, 1991). The absence of a significant dependence of transport on pH, despite the change in ionic species, may be a consequence of a pH-dependent change in binding that compensates for the variations in the relative proportions of each species. Most experimental analyses of Pi transport mechanisms with H' have been conducted on the high-affinity transport system. However, in some studies the low-affinity transport system has also been shown to be inhibited by H + uncouplers (Burns and Beever, 1977a; Schmidt et af., 1992). This suggests that both uptake systems in plant cells (low K, and high K,) may be H+/Picotransporters. Our work with barley mesophyll protoplasts (Mimura et al., 1990) has revealed that the low-affinity system operating in the range 1-10 mM Pi is a nonsaturating linear system (Fig. 2) and we are uncertain whether this is a cotransport system or some other transporter such as a channel. It will be necessary to measure the electrochemical gradients for Pi to determine if this is feasible. In order to understand the energetics of Pi transport, it is necessary to find the stoichiometry between H' and Pi transported. There has been much debate over the stoichiometry of the plasma membrane transporter, with numbers ranging between two and four (Ullrich and Novacky, 1992). The other important task is the identification of the Pi species transported since this influences the observed stoichiometry. Sakano (1990) suggested that the lower stoichiometry was a result of H+ extrusion by the plasma membrane H+-ATPase activated during Pi uptake.

b. Nu+-Dependent Uptake Recently, evidence has been found for cotransport of Pi with Na+ instead of H' in Pi-deficient Chara internodal cells (Reid et al., 1997). There have been a number of reports of Na+ stimulation of Pi uptake in algae, and Ullrich and Glaser (1982) suggested that Pi transport in Ankistrodesmus may be driven by a Na' cotransport system. In Chara, we were able to demonstrate both Na+ dependence of Pi uptake and the reciprocal Pi dependence of Na+ uptake (Fig. 5). The Na'-depen-

167

PHOSPHATE TRANSPORT AND HOMEOSTASIS IN PLANT CELLS

dent Pi uptake closely followed the pH dependence of the Pi dissociation curve, with uptake closely correlated with the monovalent H2P04- species (Reid et al., unpublished data). This is evidence that only the monovalent Pi species is involved in cotransport with Na'. Consideration of the electrochemical gradients for H2P04-and Nat across the plasma membrane indicated that the stoichiometry would need to be greater than 4Na+ for each Pi (Reid et al., unpublished data). In animal cells including humans, it is well known that not only Pi but also many other metabolites are transported with Na'. It has been suggested that one of the major differences between plants and animals is that animal cells use Na+-dependent transport systems, whereas plants use Ht-dependent transport systems. Certainly, Na' is not an essential element for growth of most plants. Some C4 plants and halophytes do require Na+, and in these plants there may well be Na+-dependent cotransport of Pi or other essential nutrients. However, in the Na+-dependent Amaranthus tricolor we were unable to detect any effect of Na' on Pi uptake (Reid et al., unpublished results). Na+-dependent Pi uptake has been reported in yeast (Roomans et al., 1977). The K , for Pi was 0.6 rnM and binding analysis showed that the a

b 4

30

N

E

2

. -

20

2

0

E

v

X

5

15

$

5

v

I

e

.& O

4 0 0

20

40

60

80

Pi concentration (pM)

100

0

0.1

0.3

0.2

Pi concentration

(mM)

FIG. 5 Na'-dependent Pi uptake of Chara corallina. Pi uptake was measured in Pi-starved internodal cells. (a) Concentration dependence of Pi influx with and without Na'. (b) Effect of Pi on Na' influx [from "Plant Nutrition-For Sustainable Food Production and Environment" (T. Ando et al., Eds.), 1997, A high affinity Na-dependent phosphate uptake induced by P starvation in Chara, Reid, R. J., Mimura. T., and Smith, F. A,. Figs. 2 and 4,0 Kluwer Academic Publisher, by courtesy of the authors and kind permission from Kluwer Academic Publishers].

168

TETSURO MIMURA

transport system had two Na+ binding sites. Unfortunately, there is no further information on the characteristics of the transporter. Genetic studies of yeast suggest that the YBR296C gene encodes for the Na+-dependent Pi transporter (Andre, 1995), whereas in Neurospora the P H 0 4 gene encodes the Na+-dependent Pi transporter (Versaw and Metzenberg, 1995). Pi uptake by cyanobacterium was also dependent on extracellular Na+ (Ritchie et al., 1997). Recently, a putative Na+-dependent Pi transporter gene was isolated from the ice plant, Mesernbryanthernum crystallinurn, after 30 h of NaCl treatment (Cushman, 1998).

c. Cell Wall Protein The phosphate transporter of bacteria is known to be one of the ABC transporters (Rao and Torriani, 1990). The extracellular Pi molecule first binds with periplasmic protein (Pi-binding protein) to be transported through a Pi transporter. In earlier work, the role of extracellular protein was also investigated in higher plant cells. Maas et al. (1979) showed that Pi transport activity was reduced by osmotic shock and they proposed that the extracellular protein involved in Pi transport was lost into the medium by osmotic shock. Unfortunately, this work appears not to have been pursued further. Lefebvre and Clarkson (1987) showed that protoplasts had the same Pi transport as intact plants, which suggests that the cell wall has little effect on transport. In higher plants, it is likely that Pi transport is directly carried out by the plasma membrane Pi transporter without extracellular support.

d Inhibitors Finding a specific inhibitor can often facilitate understanding of a transport process. Pi influx across the plasma membrane is sensitive to many inhibitors but none is very specific in its action. Vanadate and DES inhibit the H+-extruding pump of the plasma membrane and also inhibit Pi uptake (Lin, 1979; Poder and Penot, 1992). Likewise, uncouplers such as CCCP, FCCP, and DNP, which collapse the H’ electrochemical gradient across the membrane, inhibit Pi uptake. Arsenate is used as an analog of the Pi molecule and competes with Pi for uptake (Ullrich-Eberius et al., 1989). However, arsenate might also inhibit Pi uptake by inhibiting metabolic ATP synthesis. Phosphonate (HPO,”), another analog of Pi, is also known to inhibit Pi transport (Carswell et al., 1997). Substances that directly affect Pi cotransporters have not yet been found. SH reagents such as HgC12 and PCMBS inhibit H’ cotransport systems and also inhibit Pi uptake (Lin, 1979). Okihara et al. (1995) surveyed the reagents that inhibit Pi transport in an attempt to find one that did not also affect the H+ electrochemical potential gradient. They first found that an anion channel blocker, A9C, inhibited Pi uptake, but it also inhibited the H+ pump. Then, they found that furosemide, which is a structurally similar substance, inhibited Pi transport without inhibition of proton pump-

PHOSPHATE TRANSPORT AND HOMEOSTASIS IN PLANT CELLS

169

ing. It inhibited Pi uptake in a dose-dependent manner with a K j of inhibition of approximately 50 p M . However, it is unknown whether it reacts with the Pi transporter of the plasma membrane in situ. 3. Molecular Structure The first plasma membrane Pi transporter to be identified in eukaryotic cells was identified in yeast (Tamai et al., 1985; Bun-ya el al., 1991). A Pi uptake-defective mutant that was unable to grow under Pi-deficient conditions was discovered and this led to the recognition of P H 0 8 4 as the gene encoding the Pi transporter. The PH084 encoding protein is induced when yeast cells are subjected to Pi deficiency. This protein was later expressed in E. coli and purified, and the Pi transport activity was studiedfollowing incorporation into proteoliposomes (Berhe etal., 199.5).It was found that Pi was cotransported with H' by the PH084 encoding protein with a K , for Pi of about 8 p M . PH084 was cloned and sequenced (Bun-ya et al., 1991). The PH084protein has 596 amino acids and 12 membrane spanning domains which are separated into two sections of 6 domains by 74 amino acid residues. This protein is classified in a group of glucose transporters. Further progress has pointed to PH086, PH087, PH088, and G T R l in addition to PH084 as being involved in Pi transport in yeast (Oshima, 1997). PH086, PH087, and PH088 proteins are believed to be membrane proteins associated with PH084 transporter protein, whereas G T R l protein is a putative GTP-binding protein. The mechanisms of interaction among these proteins are still unknown (Oshima, 1997). Other Pi transporter genes to be cloned from fungi are from the mycorrhizal fungus Glomus (Harrison and van Buure, 1995) and P H 0 4 and P H 0 5 from Neurospora (Versaw, 1995). The P H 0 4 protein appears to be a Na'-dependent Pi transporter and P H 0 5 protein a H'-dependent transporter (Versaw and Mezenberg, 199.5). The double mutant of P H 0 4 and PH0.5 could not grow under Pi deficiency. In higher plants, genes of Pi transporters were first isolated from Arabidopsis using EST clones that have a similarity to yeast P H 0 8 4 (AtPT1 and AtPT2; Muchhal et al., 1996). Similar genes were subsequently isolated from potato (StPTI and StPT2; Leggewie et al., 1997), Catharanthus (PITI; Kai et al., 1997), and tomato (LePTI and LePT2; Liu et al., 1998). For Arabidopsis, two other studies reported phosphate transporter genes ( APTI and APT2: Smith et al., 1997; PHTI, PHT2, and PHT3: Mitsukawa et al., 1997a,b). Kai et al. (1997) succeeded in cloning a Pi transporter gene from Catharanthus suspension-cultured cells by a simple PCR method using the yeast P H 0 8 4 sequence. As shown in Table 111, suspension-cultured cells of Catharanthus have extremely high Pi uptake activities and this may be the result of high expression of the Pi transporter gene in these cells.

170

TETSURO MIMURA

Apart from Catharanthus, these plants have multiple Pi transporter genes. Characteristics of these genes are summarized in Table IV. In all cases, the Pi transporter has 12 membrane spanning domains as in the yeast P H 0 8 4 protein. Figure 6 shows a typical molecular structure of the Arabidopsis Pi transporter of by Smith et al. (1997). Similarities of amino acid sequences are shown in Table V. From amino acid sequences, it is proposed that there is an N-linked glycosylation site, a site phosphorylated by casein kinase 11, and a site phosphorylated by protein kinase C. Currently, no experimental information links the structures with physiological regulation phenomena. Pi transporter proteins from higher plants have not yet been purified. Pi-transport activities of the previous genes were identified by complementation of the yeast PH084-defective mutant. Introduction of the Arabidopsis AtPTl and AtPT2 genes, potato StPTl and StPT2, and Catharanthus PIT1 into the PHOWdefective mutant restored Pi uptake and phosphatase induction under Pi deficiency. Mitsukawa et al. (1997b) isolated the PHTl (AtPTZ) gene from Arabidopsis, but they were unable to complement their gene with the yeast mutant. Instead, they introduced P H T l into tobacco suspension-cultured cells BY2 and succeeded in increasing Pi uptake activity of those cells. Complementation against PH084-defective mutant of yeast by potato StPTl and StPT2 did not show the high affinity for Pi supposed from the physiological characteristics of the plant cells in situ (Leggewie et al., 1997). The previous results may suggest that Pi transport across the plasma membrane is catalyzed by a combination of proteins. In yeast it appears that many membrane proteins are involved directly in Pi transport (Oshima, 1997) and perhaps the proteins produced by the transporter gene of higher plants might not be able to interact with yeast proteins effectively. 4. Regulation of Transport

Transport activities for many nutrients change depending on nutritional status and various environmental factors. In this section, I focus on how plants control Pi transport activities. Activation or inactivation processes can be divided into short term (fine control) or long term (coarse control). The short-term processes relating to Pi uptake occur over periods ranging from a few minutes to several hours, whereas long-term changes in Pi transport activity might be observable over days or weeks. In a shortterm process there may be no change in the numbers and/or species of transporter proteins. a. Short-Term Regulation Short-term regulation of Pi uptake is usually caused by changes in various extracellular conditions.

PHOSPHATE TRANSPORT AND HOMEOSTASIS IN PLANT CELLS

171

i. Light In algae or green cells, Pi uptake was activated by light (Smith, 1966; Raven, 1974). Since the photosynthetic electron flow inhibitor, DCMU, inhibited this activation, it was concluded that Pi uptake involved photosynthesis-dependent activation. There are two possible mechanisms for the effect of light. First, the plasma membrane H' pump is activated by light. It is well known that the plasma membrane H' pump in green cells is activated by light (Mimura, 1995b). Activation of the H' pump results in an increase in the H* electrochemical potential gradient which would drive cotransport of Pi uptake with Hi. The plasma membrane H' pump of plant cells is also electrogenic so that activation of the electrogenic pump would hyperpolarize the plasma membrane and increase the inward driving force for cotransport reactions for which there is a net transfer of positive charge (e.g., 2H'/H,P04-). This would also apply to Na' cotransport systems. In fact, many substances that are cotransported with H' are transported more in light than in dark. The photosynthesis-induced activation mechanism of the H' pump is still under discussion (Mimura, 1995b). The second possible mechanism by which light rapidly activates Pi uptake is via a change in the cytoplasmic Pi level. During photosynthesis, Pi is metabolically converted to organic phosphates in chloroplasts and this consumption leads to a decrease in the cytoplasmic Pi level. Lefebvre and Glass (1982) suggested that the cytoplasmic Pi level may affect Pi transporter activity. They showed that addition of Pi to Pi-deficient barley reduced Pi influx within 1 h and proposed that the rapidity of the reduction of Pi influx resulted from allosteric inhibition of influx by intracellular Pi. The other possibility is that Pi is acting as a noncompetitive inhibitor of the plasma membrane H' pump (Gonzalez and Medina, 1988; Takeshige et af.,1992). Reduction in the cytoplasmic Pi level increased the H' pump activity. Although I have mentioned that the photosynthesis-induced activation mechanism of the H' pump is still under discussion, a decrease in the cytoplasmic Pi level may be the major factor for the photosynthesis-induced H' pump activation. Pi can work as a noncompetitive inhibitor not only for the plasma membrane H' pump but also for the tonoplast H+-ATPase and H'-PPase (Takeshige et al., 1992). ii. Temperature Carter and Lathwell (1967) measured effects of temperature on Pi uptake by excised corn roots between 20 and 40°C. Increasing temperature affected both high-affinity and low-affinity systems equally with a Qloof approximately 2. McPharlin and Bieleski (1989) showed that a decrease in temperature decreased Pi uptake between 5 and 25°C. Low temperature inevitably causes an inhibition of energy metabolism and decreases the electrogenic H' pump activity and H' motive force. The decrease in Pi uptake should be at least partly explained by these changes. Interestingly, efflux was greatly enhanced. Since a depolarization of the

TABLE IV Characteristics of Pi Transporter Genes

Material

Gene

Saccharomyces cerevisiae

PH084

Neitrospora crassa

PHO-4

Chromo- Amino some acids No. residues

+Pi

-Pi

Characteristics

Na/Pi transporter 569

Glomus versiforme

GvPT

521

A ra bidopsis thariana

phol

3

pho2

2

5

524 534 524

Reference Bun-ya et al. (1991)

596

PHO-5

APT1 AtPT2 APT1

Expression pattern in tissues

Versaw and Metzenberg (1995) Versaw (1995) Harrison and van Buure (1995)

Root (+) Root (+/-) Root (+)

Leaf (-) Leaf (-) Leaf (-) Stem (-) Flower (-)

Root (++) Root (+) Root (++)

Defective in a xylem Poirier et al. (1991) loading Pi accumulation in Delhaize and Randall shoot (1995) Muchhal et a/. (1996)

-I

rn

$

APT2 PHTl PH T2 PH T3

5 5 5 5

524 524 524 521

Leaf (+/-)

Leaf (-)

PIT1

542

Root, stem, young plant

Solanurn IU berosicrn

StPTl

540

Root (+ ) Sink leaf (-). Source leaf (+) stem (-) Flower bud (+) Tuber ( + / - ) Flower (+/-) N o expression

Lycopersicon esculenrurn

527

LePTl

538

LePT2

528

I

Mitsukawa et al. (1997a) Mitsukawa el al. (1997b)

Catharanthus roseus

SIP72

T I

Leaf (-)

Root (+)

Leaf (-) Root (-)

I

3 rn

Kai et al. (1997)

Root (++) Source leaf (+ + ) Tuber (+ +) Stem (++) Stolon (++)

Leggewie er al. (1997) 0

z

v)

Root ( + + ) Leaf (+) Root (++)

Liu el a/. (1998)

-u r D

z -I 0

rn r cn r

174

TETSURO MIMURA

OUT

in kinase I I

OOH

IN

V

FIG. 6 Membrane spanning topology and putative sites of posttranslational modifications of Pi transporter (APT1 and APT2) of Arabidopsis lhaliana (from Smith, F. W., Ealing, P. M., Dong, B., and Delhaize, E., The cloning of two Arabidopsis genes belonging to a phosphate transporter family, Plant J., by courtesy of the authors and kind permission from Blackwell Science).

membrane under low temperature would actually make passive efflux less likely, the increase in Pi efflux may involve energy-dependent mechanisms. iii. Zntracellular Phosphate Pi uptake sometimes decreases within 1 h of addition of Pi to the external medium (Lefebvre and Glass, 1982). Since this period is too short to change the overall cytoplasmic Pi concentration or to result from genetically mediated changes in Pi transporter activity, it is proposed that Pi transported from outside to the cytoplasm may directly affect the cytoplasmic face of plasma membrane Pi transporter. Of course, this is only speculation. As mentioned previously, since Pi is an inhibitor of the H' pump, transported Pi may act against the Ht pump and not directly against the Pi transporter. In blue-green algae, a pulse addition of Pi changes the characteristics of Pi uptake for a longer time (Wagner et al., 1995) and this may be caused by only transient changes in cytoplasmic Pi levels. iv. Intracellular p H There are few reports dealing with the effects of intracellular pH on Pi influx across the plasma membrane. Recently, we treated Chara cells with butyric acid (acid load) or NH4Cl (alkali load) and measured the Pi influx (Mimura et al., 1998). In both cases, the Pi influx was slightly inhibited compared with control cells. The changes were relatively small compared to intracellular p H effects on C1- transport (Smith, 1980). Based on these results it seems unlikely that intracellular pH plays a major role in the regulation of Pi influx across the plasma membrane.

b. Long-Term Regulation i. Pi Starvation When plants are subjected t o Pi deficiency, Pi uptake activity dramatically increases. In the most rapid case, it occurs within a

TABLE V Percent Similarities among Phosphate Transporter Genes

a D

Materials

Gene

Saccharomyces cerevisiae Neicrospora crassa Glotnus versiforme A rabidopsis thariana

Caiharanihus rooseus Solanum ticberosum Lycoperiscon esculentum

PHO-5

GvPT

AtPTl

AiPT2

APTI

APT2

PHOR4

47

46

34

34

34

34

PHO-5

100

43

34

31

33

I00

42

39

41

PHTI

PHT2

PHT3

PIT1

SiPTI

34

34

34

34

34

34

34

34

32

33

42

42

42

42

40

SiPT2

LePTl

LePT2

33

34

33

31

32

31

31

40

41

40

40

5 W

5> z

0

GvPT

5

E: --I

AiPTl A tP T2 APT1 APT2 PHTI PHT2 PHT3 PI TI

100

78 100

99 78 100

100" 78 99 100

SiPT1 StPT2 LePTl

" AtPTl and PHTI are named for the same gene. AiPTI ( P H T I ) and APT2 have one difference in amino acid sequences. ' A P T I and P H R have one difference in amino acid sequences.

100" 78 99 100'' 100

99 78

low 99 99 100

94 79 94 94 94 94 100

79 83 79 79 79 79 79 100

80 83 80 80 80 80 81 86

75 75 75 75 75 75 76 76

79 82 78 79 79 78 81 86

78 77 78 78 78 78 79 78

100

79 100

96 79 I00

81 94 81

II 5

5

176

TETSURO MIMURA

day, but generally it takes 2 or 3 days to detect a distinct activation. Kinetic analysis of the activation of Pi uptake showed that K , and/or V,,, changed during Pi starvation. In experiments using Neurospora, Burns and Beever (1977a,b) showed that the K,,, of the low-affinity transport system decreased from 0.9 to 0.3 mM when the Pi levels in the culture medium changed from 0.05 to 10 mM. On the other hand, the K,, of the high-affinity transport system (around 3 p M ) did not change. In higher plants, there are two phases of changes. After Pi starvation, V,,,, first increases and then K,, decreases. Sometimes we can see only one of these responses. If we assume that the K,,, is a characteristic feature of one protein, then a change in K , for Pi implies that the molecular species has also changed. On the other hand, changes in V,,, may occur either by changes in the nature of the molecular species or by changes in the number of the same species. Although it was known that Pi deficiency increased Pi uptake, it remained uncertain whether in higher plants there were really different transporters with different affinities for Pi. Molecular biological research on Pi transporters finally concluded that Pi transporters belonged to a multiple gene family. Muchhal et al. (1996) and Leggewie et al. (1997) have shown that there are more than one gene that complements the yeast PH084 mutant. Mitsukawa et al. (1997b) isolated four different genes from Arabidopsis. Among these genes, some were expressed only in Pi-starved conditions (Table IV). Thus, there is no doubt that Pi deficiency can induce different kinds of transporters. Pi transport activities of these genes were analyzed by complementation of the yeast Pi transport mutant. K,, values found in these experiments do not always agree with affinities for Pi obtained from in vivo flux measurements in higher plants (Leggewie et al., 1997). Mitsukawa et al. (1997b) have also succeeded in introducing a Pi transporter gene from Arabidopsis into tobacco suspension-cultured cells. These transgenic tobacco cells showed higher Pi uptake activity than before, indicating that at least some of the genes for Pi transporters must have a high affinity for Pi. There is evidence from split-root experiments that Pi uptake activity is determined not by localized Pi status but at the tissue or whole plant level. Drew and Saker (1984) divided barley roots into two parts, one part remaining Pi deficient and the other part supplied with Pi. Pi uptake of the Pi-deficient part was found to be affected by the Pi-supplied part. This was confirmed at the molecular level by Liu et al. (1998), who showed that expression of the Pi transporter in the Pi-deficient part changed when Pi was supplied to the other part. Drew and Saker suggested that the internal Pi level of cells in the Pi-deficient part might change by Pi movement from the Pi-supplied part. However, there is no evidence that the cytoplasmic Pi level of the Pi-deficient part really changes when the other part is supplied with Pi. We need t o determine how the Pi level of the apoplast in the Pideficient part is influenced by Pi supply through the xylem and phloem. It

PHOSPHATE TRANSPORT AND HOMEOSTASIS IN PLANT CELLS

177

would be interesting to determine whether Pi transport activity can respond to internal signals even when there is no Pi in the extracellular medium. ii. Growth and Differentiation There have been many studies on the relationship between plant growth and Pi nutrition. Lefebvre and Glass (1982) traced growth-dependent changes in Pi influx in barley. After germination, Pi influx based on the root fresh weight gradually decreased in the presence of an adequate Pi. Under Pi starvation, it dramatically increased after 12 days and then returned to the original activity after 16 days. It is likely that Pi uptake in the root is regulated by the demand for Pi for shoot growth. Pi uptake of growing plants has been measured by various methods (Breeze et af., 1984, 1985; Ericsson and Ingestad, 198S), with the general finding that growth rate and Pi uptake rate are strongly correlated. Clarkson and Saker (1988) concluded that under low levels of external Pi (the normal condition in nature), growth and Pi uptake rates were constant between a wide range of external Pi concentrations. Plants seem to control their Pi uptake activity to keep the growth rate constant, independently of the extracellular Pi concentration. Experiments in which Pi uptake is studied using suspension-cultured cells under a continuous flow of culture medium may help to clarify the relationship between Pi uptake rate and cell division (cell cycle) at the cellular level. Molecular analyses of Pi transporter genes have demonstrated that the expression of each gene is dependent on not only the Pi status but also the type of plant (Table IV). Liu et al. (1998) showed that the expression of Pi transporters was differently regulated in the genes (LePTI and LePT2) in tomato tissues. Both were expressed in the root epidermis. Under Pi starvation, LePTI transcripts were also detected in the root central cylinder and in the leaf palisade parenchyma and phloem cells, whereas LePT2 was not detected in the leaf at all.

c. Pho Regulon and Two-Component Systems A high-affinity Pi transport system of prokaryote cells has been shown to be composed of outer membrane protein, a Pi-binding protein in the periplasm, and a phosphate transporter belonging to the ABC transporter group. Although I have not dealt with Pi transport in procaryotes in this review (see Nakata et al., 1987; Rao and Torriani, 1990), I draw attention to its regulation system. The high-affinity Pi transport system is under the control of the phosphate regulon. Induction of the Pho regulon is mediated by a two-component system. Two-component signaling systems have been identified as important receptors of environmental signals, e.g., osmolarity, chemotaxis, and nitrogen source (Wurgler-Murphy and Saito, 1997; Chang and Stewart, 1998). The level of extracellular phosphate is also an environmental signal. Pho R and Pho B proteins function to transmit information about the Pi level to the Pho regulon. The Pho R protein is known to be a sensor protein of

178

TETSURO MIMURA

the cell membrane and is believed to detect the extracellular Pi concentration and not the cytoplasmic Pi level. Blue-green algae also respond to the extracellular Pi conditions via a similar two-component system (Aiba et al., 1993). In higher plants, two-component systems have recently been reported to work as ethylene receptors and cytokinin receptors (Wurgler-Murphy and Saito, 1997; Chang and Stewart, 1998). Currently, there is no experimental evidence that they may operate for the detection of nutritional status in higher plant cells, as they do in procaryotes.

5. Measurement of Pi Efflux and Xylem Loading Pi efflux from cells through the plasma membrane is an important aspect of Pi transport. When Pi moves from root to shoot, Pi must be released from root cells into the xylem (xylem loading). Movement into the apoplast will normally require the expenditure of metabolic energy for export across the plasma membrane. There are few reports on Pi efflux compared to those on the influx measurements. In order to measure the efflux accurately, it is necessary to use tissues loaded with radioisotopes of phosphorus. The most popular method is the time-dependent efflux analysis from labeled cells. The efflux of "Pi usually shows three different time-constant curves, as is observed for many other ions. The fastest efflux is interpreted as release from cell wall, the second fastest as efflux from the cytoplasm across the plasma membrane, and the slow phase comes from the vacuole across the tonoplast (Lefebvre and Clarkson, 1984; Woodrow et al., 1984). However, smaller phases with time constants for exchange similar to those of the larger pools will not be detected by this method. McPharlin and Bieleski (1989) reported the following characteristics of Pi efflux in Spirodela or Lemna. In Pi-adequate plants, Pi efflux was about 10% of the influx at moderate Pi concentrations. In Pi-deficient plants, it decreased to approximately 1%. A t very low external concentrations, efflux can be similar to influx (i.e., no net uptake). This is known as the equilibrium concentration point. In Pi-deficient plants, this was < 0.1 p M . Pi-adequate plants showed three or four times higher values than did Pi-starved ones. This was confirmed by Bieleski and Lauchli (1992). Low temperature and metabolic inhibitors such as CCCP, NaN3, and KCN increased Pi efflux. Such experimental treatments usually cause membrane depolarization. Membrane depolarization, however, is favorable for Pi influx, not Pi efflux. Thus, we do not know the reason why metabolic inhibition increases Pi efflux. Table VI summarizes the characteristics of Pi efflux. McPharlin and Bieleski (1989) observed that both Pi efflux and Pi influx increased as the extracellular Pi concentration increased. A rapid increase

-I

*

TABLE VI Comparison of Pi Influx and Efflux

33

z

U

Material

Pi concentration of external medium ( p M )

Influx Original value 0.11 pmolldcm’

Hydrodictyon africanum

n

Efflux Estimated (nmol/min/g FW) 0.31“

Original value 0.02 pmol/s/crn’

-I

D

Estimated (nmol/minlg FW) 0.06‘

z 0

Reference Raven (1974)

I 0

5

g

--I

!%

Hordeum vulgare Zea maiz

0.2 2.0

836 nrnol/h/g FW

13.9

206 nmol/h/g FW

3.43

Lefebvre and Clarkson (1984)

0.27 nmol/min/g FW 4.91 nmol/min/g FW

0.27 4.91

0.24 nmol/min/g FW 0.62 nmollminlg FW

0.24 0.62

Elliot ef al. (1984) McPharlin and Bieleski (1989)

Lemma major

1 10

21 nmol/h/g FW 109 nmollhlg FW

0.35 1.82

15 nmollhlg FW 25 nmollhlg F W

0.25 0.42

Spirodela oligorrhiza

1 10

22 nmollhlg FW 130 nmol/h/g FW

0.37 2.17

17 nmol/h/g FW 30 nmol/h/g FW

0.28 0.5

Azolla mexicana

1 10

22 nmol/h/g FW 101 nmollh/g FW

0.37 1.68

11.7 nmol/h/g FW 13.8 nmol/hr/g FW

0.20 0.23

Flux was estimated based on 0.005 mz/g FW.

0

z

9

b z -I

0

Bieleski and Lauchli (1992)

rn r

cn

180

TETSURO MIMURA

in Pi efflux from Chara cells was also found when Pi was added to the extracellular medium during the measurement of efflux (Mimura et al., 1998). Thus, cells appear to have an ability to sense changes in the extracellular Pi supply and to respond through the Pi efflux system. It is well known that symbiotic mycorrhizal fungi play an important role in Pi uptake from soil in some plants (Schachtman el al., 1998). In phosphate transfer from mycorrhizae to plant root cells, Pi efflux from fungal cells to the plant host is an important step. Unfortunately, the factors that determine the rate of efflux are not understood. Currently there is no information on the molecular mechanisms which mediate Pi efflux across the plasma membrane. Since the electrochemical potential gradient of Pi is usually outward, it is feasible for Pi to efflux through ion channels. I have not encountered any reports of studies in which the Pi permeability of anion channels in plant plasma membrane has been examined. Some mutants of Arabidopsis relate to Pi efflux. The Phol mutant may be a Pi efflux mutant (Poirier et al., 1991; see Table IV) since it was not able to accumulate Pi in the shoot, even though uptake by the root was normal. The most likely explanation for this observation is that there is a defect in the loading of Pi into the xylem in the root. The Phol gene may code the transporter contributing Pi efflux. 6 . Tonoplast Pi taken up into the cytoplasm across the plasma membrane is partly metabolized and most of the rest is transported into the vacuole (Bieleski and Laties, 1963) which occupies more than 80% of the cell volume. Pi accumulated in the vacuole is used to buffer the cytoplasmic Pi level against fluctuations caused by variable external supply. Thus, Pi transport across the tonoplast (the vacuolar membrane) becomes an important factor in Pi metabolism of plant cells.

1. Measurement of Pi Influx There are many methods for measuring Pi transport across the tonoplast. The in vivo measurement using "P-NMR can simultaneously measure both the cytoplasmic and the vacuolar Pi contents. Time-dependent changes in the vacuolar Pi content reflect the net flux of Pi. In sycamore suspensioncultured cells, when Pi was depleted from the culture medium, the vacuolar Pi level gradually decreased over 50 h until it coincided with the cytoplasmic Pi level. When Pi was added to the Pi-deficient cells, the Pi level of the cytoplasm increased within 2 h, and then the vacuolar Pi level gradually

PHOSPHATE TRANSPORT AND HOMEOSTASIS IN PLANT CELLS

181

increased (Rebeille et al., 1983). More detailed analyses were obtained in Cutharunthus suspension-cultured cells which were almost Pi deficient (Sakano et al., 1995). Pi added to the culture medium induced a twofold increase in the cytoplasmic level, after which it remained steady. Afterwards, the vacuolar Pi level dramatically increased, although the rate of increase was slower than that in the cytoplasm. Pi influx has also been estimated from direct measurement of Pi concentrations in vacuolar sap. In C h r u cells, it is easy to isolate the sap because of the large size of the internodal cells. Smith (1966) showed that labeled Pi added to the medium first appeared in the cytoplasm and then in the vacuole. The level of radioactivity in the cytoplasm increased only for the first few hours, but in the vacuole it continued to increase for more than 10 h. In barley mesophyll cells, the intact vacuoles of protoplasts, were isolated every 30 min after an addition of '2Pi and the radioactivity in the vacuoles was measured (Mimura etal., 1990). "Pi was detected in the vacuole within 30 min in Pi-deficient protoplasts but it took longer than 1 h in Pi-rich protoplasts (Fig. 7a). This suggests that Pi deficiency may induce new Pi transport activity

a

T

I

0

I

I

I

20 40 60 Time (min)

80

FIG. 7 Pi uptake in vacuoles of Barley mesophyll cells. (a) I n vivo Pi uptake into vacuoles. Mesophyll protoplasts were incubated with "Pi, and then the vacuoles were isolated and 33P in the vacuole was counted. (b) Pi uptake by the isolated vacuoles. After isolation of vacuoles, they were incubated with "Pi. 0 and A. Pi-adequate barley; 0 and A, Pi-deficient barley. In b. solid lines indicate that the isolated vacuoles were incubated with A T P (broken line, without ATP) (from Planla, Phosphate transport across biomembranes and cytosolic phosphate homeostasis in barley leaves, Mimura. T.. Dietz. K.-J., Kaiser, w., Schramm, M. J.. Kaiser, G . . and Heber. U., 180, 139-146. Fig. 6, 1990, 0 Springer-Verlag).

182

TETSURO MIMURA

not only in the plasma membrane but also in the tonoplast. This was confirmed by isolating vacuoles from both Pi-rich or Pi-deficient barley protoplasts and comparing Pi uptake rates. Pi uptake rates into the vacuole of Pideficient cells were higher than those of Pi-rich cells (Fig. 7b). However, the increase in Pi uptake under Pi deficiency was only observed in the presence of ATP. Without ATP, Pi influxes in both vacuoles were the same. Thus, Pi transport into the vacuole may depend on the cellular energy status, at least in the Pi-deficient cells. This experiment also indicated that Pi influx to the vacuoles of Pi-rich cells may be very slow. Measurement of transport activities across the tonoplast can also be performed using tonoplast membrane vesicles. If a transport process is energy dependent, the membrane vesicles can be energized by addition of ATP or PPi to drive Hi pumps, p H jumps of incubation media to create pH gradients, or generation of membrane potential differences with K t and ionophore. Kaestner and Sze (1987) measured potential-dependent anion transport using tonoplast vesicles from oat. They showed that the permeability of Pi was very low compared with that of other anions. However, because Pi may also have been acting as an inhibitor of both H+ATPase and PPase (Gonzalez and Medina, 1988; Takeshige et al., 1992), their results were ambiguous. 2. Mechanism and Regulation of Transport Few reports deal with the mechanism of Pi transport across the tonoplast. Mimura et al. (1990) measured the concentration dependence of Pi influx into vacuoles isolated from Pi-rich barley mesophyll cells. Pi influx did not saturate until 20 mM Pi in the extravacuolar medium, which is at the upper end of measured cytoplasmic Pi concentrations. These results help establish the electrochemical conditions under which Pi transport to the vacuole operates and allow educated guesses regarding the likely molecular mechanism. It is difficult to estimate accurately the electrochemical potential gradient of Pi across the tonoplast because it is unknown which molecular species of Pi is transported and the vacuolar Pi level varies greatly depending on the Pi nutrient status. At pH 7.5 in the cytoplasm, most of the Pi is in the divalent HP04’- form, but in the vacuole, where the pH is approximately 5, most of the Pi is present as the monovalent H2P04-. The electrical potential difference across the tonoplast is usually slightly positive with respect to the cytoplasm. If we consider only HPO?-, both concentration and electrical potential gradients are inward into the vacuole. HP04*- can therefore be passively transported via either carrier or through ion channels. On the other hand, if we consider H2P04-,the electrochemical potential gradient changes its direction dependent on the vacuolar content and it

PHOSPHATE TRANSPORT AND HOMEOSTASIS IN PLANT CELLS

183

may therefore be necessary to invoke an active transport mechanism such as Pi-ATPase, Pi-PPase, or H' antiport system under some conditions. In Pi-deficient vacuoles, the presence of extravacuolar ATP accelerated Pi influx (Mimura et al., 1990).Eligny et ul. (1 997), using "P-NMR, measured the effects of anoxia on cellular Pi-containing substances. In their measurements, when cells were exposed to low-oxygen conditions, the cellular ATP level decreased and Pi moved from the vacuole to the cytoplasm. On recovering to normoxia, Pi in the cytoplasm returned to the vacuole. They suggested that ATP played an important role in Pi uptake to, and keeping Pi in, the vacuole. A possible complication with these NMR experiments arises from the fact that if anoxia causes the pH of cytoplasmic organelles to fall to a similar pH to that of the vacuole, then the apparent vacuolar pool will seem to increase (see Section 11), thereby causing an underestimation of the efflux across the tonoplast. There are three possible roles of ATP. ATP may work as a direct energy source for Pi transport. ABC transporters such as the glutation-S-conjugate transporter are known to operate in the tonoplast (Martinoia et al., 1993; Lu et al., 1998). Pi uptake into the vacuole may be mediated by one of these transporters. Second, V-type H+-ATPases may be involved in the transport of Pi. Activation of the H'-ATPase produces a vacuole-inside positive potential. If Pi transport is driven by the electrical potential gradient, addition of ATP may increase Pi influx. In preliminary experiments, specific inhibitors of H'-ATPase, bafilomycin and folimycin, partly inhibited Pi uptake into the isolated vacuole. Very recently, we have confirmed that not only ATP but PPi is also effectual to Pi uptake of Pi-deficient vacuoles isolated from Cutharunthus roseus cell (Mimura et al., unpublished data). Lastly, ATP is known to be a transport regulator of the tonoplast. Dietz et al. (1990) showed that ATP activated amino acid transport into the vacuole without hydrolysis of ATP. Such a role for ATP in Pi transport is possible but there is no evidence to support such a mechanism. Klughammer et al. (1992) measured the ion channel activities of the tonoplast integrated into planer lipid bilayers. They detected anion-dependent currents with a range of anions including Pi, although the conductance of Pi was very low compared to that of some other anions. Two reports have recently dealt with Pi transport across the tonoplast in yeast (Booth and Guidotti, 1997; Kulakovskaya and Kulaev, 1997). Booth and Guidotti suggested the existence of a bidirectional Pi transporter in the tonoplast. This transporter had a relatively high affinity for Pi (K,,, approx 0.4 mM) and had a higher activity at low pH. Kulakovskaya and Kulaev described Pi uptake through a channel-like transporter, which was independent of the electrochemical proton gradient.

184

TETSURO MIMURA

There is currently no information on the molecular structure of the tonoplast Pi transporter(s) in plants. However, as shown in Table IV, there are many Pi transporter genes that have been identified in Arabidopsis, and some of them may well be tonoplast transporters. 3. Measurement of Pi Efflux from the Vacuole The most important function of the vacuole is to store excess Pi and buffer the cytoplasmic Pi level. Mobilization of vacuolar Pi under conditions of deficiency involves controlled efflux of Pi across the tonoplast. As with influx, efflux of Pi has also been measured by "P-NMR by following the changes in the vacuolar and the cytoplasmic Pi contents. Since the cytoplasmic Pi may be immediately metabolized, the changes in vacuolar Pi are a better indication of the efflux of Pi. When sycamore suspension-cultured cells were deprived of sucrose, most of the Pi accumulated in the vacuole. Addition of sucrose immediately changed the cytoplasmic Pi level but did not affect the vacuolar pool (Roby er al., 1987). This suggests that efflux of Pi from the vacuole in sitic may be very slow. The Pi efflux from isolated vacuoles has also been investigated (Martinoia et al., 1986). Incubation of isolated barley vacuoles in Pi-free medium did not result in a Pi release compared to other anions (C1- and NO,-), which is consistent with the view that Pi movement out of the vacuole is slow. Observations with NMR of pea leaves under anoxia, however, showed that Pi rapidly moved between the vacuole and the cytoplasm (Bligny et al., 1997). In yeast, the addition of Pi to the extravacuolar medium induced rapid efflux of Pi (Booth and Guidotti, 1997). The apparent conflict between these studies remains to be resolved. Figure 8 shows a schematic diagram of membrane transport of Pi in plant cells. Molecular analyses of Pi transporters have concentrated only on Pi uptake across the plasma membrane. Studies on the other transporters and on the mechanisms of regulation of Pi transport are important subjects that should be studied in the near future.

V. Homeostasis and Detection of Pi Status in Plant Cells Homeostasis of Pi is an overall phenomenon resulting from the orchestration of many individual processes, including membrane transport, binding to the membrane, sequestration as Ca or Mg precipitate, and metabolic conversion between inorganic and organic phosphates. Homeostasis means the control of physiological status and the maintenance of a certain steady state. Feedback regulation is used to counter any perturbation. For this, the system has to be able to constantly monitor the prevailing conditions.

PHOSPHATE TRANSPORT AND HOMEOSTASIS IN PLANT CELLS

185

FIG. 8 Schematic diagram of Pi transport systems in plant cells. Transporters drawn with broken lines are putative molecules.

In Pi homeostasis, this may require monitoring of the Pi concentration in each compartment. Here, I focus only on the relationship between Pi homeostasis and Pi transport activities. Regarding metabolic processes, there are excellent reviews (Theodorou and Plaxton, 1993: Duff era/., 1994). A. Cytoplasm and Vacuole Bieleski and Laties (1963) made it clear with radioisotopes that there are two different pools of Pi in plant cells; one in which the Pi is actively

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metabolized and one in which Pi metabolism is static. It is evident that the former is the cytoplasm and the latter is the vacuole. In all experiments in which Pi influxes to the cytoplasm and to the vacuole have been measured separately, the transported Pi accumulated first in the cytoplasm and then in the vacuole. The cytoplasmic Pi level eventually reached a steady level, whereas the vacuolar Pi level continued to increase. These studies suggested that the cytoplasmic Pi level might be regulated at a certain value but that the vacuolar Pi level might not. 'lP-NMR measurements showed that under Pi deficiency, the vacuole was acting as a Pi reservoir to maintain the cytoplasmic Pi pool constant (Rebeille et al., 1982, 1983; Lee and Ratcliffe, 1983; Lee et al., 1990). In 1990, Mimura el al. isolated protoplasts and vacuoles from barley leaves under Pi deficiency and measured changes in subcellular distribution. Mimura et al. (1992, 1996) have analyzed changes in Pi concentrations in subcellular compartments during the growth of barley. These studies showed that Pi concentrations in the cytoplasm were always constant and were independent of the Pi supply. On the other hand, the vacuolar Pi changed in order to keep the cytoplasmic Pi concentration constant. Although the exact cytoplasmic level of Pi is still unknown (see Section 111), it is believed to be from a few to approximately 10 mM. Maintenance of this level of Pi in the cytoplasm is essential to keep the Pirelated metabolism at a normal state, i.e., Pi homeostasis in the cytoplasm. Lee and Ratcliffe (1983, 1993) found that Pi homeostasis was achieved under moderate Pi deficiency but once the vacuolar Pi pool was completely exhausted, the cytoplasmic Pi concentration also began to decrease. Cytoplasmic Pi homeostasis is mainly achieved via a combination of membrane transport and metabolic conversion from organic phosphates to inorganic phosphates. When the cell increases in volume or divides into two daughter cells, the cytoplasmic Pi content will be diluted because a part of Pi is consumed for structural materials of new cells. To compensate, Pi is taken up from the external medium and from the vacuole. Under Pistarved conditions, recovery of the cytoplasmic Pi concentration can only be achieved by transport from the vacuole. The use of the vacuole as a reservoir for cytoplasmic Pi also applies in yeast (Shirahama et al., 1996). It is known that yeast cells accumulate large amounts of phosphate as polyphosphate which is stored in the vacuole. Cytoplasmic Pi homeostasis in yeast and other polyphosphate-accumulating fungi and algae is dependent on the synthesis and the degradation of polyphosphate and may therefore be a more complicated process than that in higher plant cells. Metabolism of Pi has a greater effect on the cytoplasmic Pi level than on the total cytoplasmic phosphorus content, which is the sum of inorganic and organic phosphates. In green cells, light activates photosynthesis and some of the Pi present in the dark is esterified into organic compounds in

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chloroplasts. Takeshige et al. (1992) measured the cytoplasmic Pi level of Chara cells between light and dark conditions and found that 20 min after the beginning of the light period, the cytoplasmic Pi level decreased to almost 50-60% of that in the dark. In suspension-cultured cells of Acer, it was found that sucrose starvation induced a large accumulation of Pi in the vacuole and a small increase in the cytoplasmic Pi concentration (Rebeille et al., 1985) as a result of the degradation of organic phosphate. Thus, although cytoplasmic Pi levels measured over long periods may appear to be constant, there may nevertheless be shorter term fluctuations resulting from variations in metabolic activity. Intracellular depletion of Pi can lead to changes in many processes, for example, activation of Pi uptake, induction of phosphatase, and RNase. How do these processes detect changes in Pi levels in the different compartments in which they operate? Unfortunately, this question cannot be answered for plant cells. Currently, there is only knowledge of the bacterial two-component systems which detect osmotic or nutritional conditions (Wurgler-Murphy and Saito, 1997). The main problem in plant cells is that many of these changes occur in the absence of, or before, measurable changes in the Pi level of the cytoplasm. It is known however, that the vacuolar Pi concentration undergoes marked changes and this might therefore be a suitable level to monitor. Mimura et al. (1998) analyzed the activation of Pi transport under Pi deficiency using mature Chara cells which do not grow or divide (k, intracellular Pi stores are not diluted by growth or division). In these cells, changes in Pi transport resulting from Pi starvation were completely independent of the vacuolar Pi level. Thus, the assumption that the vacuolar Pi level may act as the signal for Pi status is excluded, at least in these cells. The second difficulty is that the cytoplasmic Pi level, especially that of photosynthetic cells, changes greatly between light and dark within a few minutes. If the cell responds to the cytoplasmic Pi level, deficiency-induced responses should also occur in the light (the stimulation of Pi uptake may be consistent with this). Another possibility is that the plant cell may detect the extracellular Pi concentration. In prokaryotes and yeast. the extracellular Pi is likely to be monitored by the cell. It has been proposed that the two-component system membrane receptors detect extracellular Pi in E. coli and Syrzecococcus (Torriani, 1990; Aiba et al., 1993). In yeast, some of the transporter proteins may be involved in the detection of extracellular Pi (Oshima, 1997) but further evidence is needed. Recently, in an ingenious experiment using tomato suspension-cultured cells, Kock etal. (1998) showed that the coexistence of extracellular Pi and Pisequestering substances such as mannose induced RNase, which was usually induced under Pi-deficient conditions. They suggested that the application

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of these substances sequestered intracellular Pi and transiently decreased the cytoplasmic Pi level before recovery as a result of release of Pi from the vacuole. This experiment provides support for the proposal that the cell detects the cytoplasmic Pi level and not the extracellular Pi level. Moreover, since the changes in Pi were expected to be transient (they did not actually measure changes in cytoplasmic Pi), it implies that responses can be rapid. In addition to detecting intracellular Pi or extracellular Pi, it is also possible that plant cells are able to detect Pi fluxes directly, but this suggestion remains entirely speculative. There is clearly a need to find a receptor, a process, or a level that can be used to predict a plant’s response to changes in Pi status.

B. Apoplast The “symplast” is the intracellular phase of a plant. Its counterpart, the “apoplast,” comprises those parts within the plant that are extracellular. The main components of the apoplast are the cell walls and extracellular spaces and the conductive tissue of the xylem. I previously discussed how intracellular phases of plant cells are subject to homeostasis. This is also true of animals, but in animal tissues homeostasis of the extracellular fluids is of a similar importance to the maintenance of a controlled intracellular phase. In plants, there is evidence for homeostasis of the apoplast (Canny, 1995), albeit limited by the constraints imposed by widely varying environmental conditions. Measurements of Pi levels in the xylem (Bieleski, 1973; Bieleski and Ferguson, 1983; Marschner, 1995) yield values much higher than those in the external medium. There must therefore be steps between the external medium and the xylem in which Pi becomes concentrated. Except in the small region near the root tip where the endodermis is immature, there is no direct connection between the external medium and the xylem. The increase in concentration of Pi in the xylem must therefore originate in processes involving the symplast, either uptake into the root cells or unloading into the xylem. The Arubidopsis Phol mutant, which appears to be defective in xylem loading (Poirier et uf., 1991),may be useful in this respect. . measured the growth-dependent changes in xylem Mimura et ~ l (1996) Pi levels. When plants were subjected to Pi deficiency, the Pi levels in the xylem decrease to approximately 1 m M compared to 5-7 mM in the nondeficient control plants. As the plant became more P deficient and the whole plant Pi concentration gradually declined, the Pi level in the xylem remained constant at approximately 1 mM. It seems likely that the xylem Pi concentration is a function of both the supply of Pi from the soil and the demand for Pi by the shoot.

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There is also evidence that Pi levels in the cell walls are regulated. Mimura etal. (1992,1996) measured Pi in the apoplastic fluid collected by infiltration and centrifugation of barley leaves. They found that the concentration of Pi in the apoplast paralleled that of the whole leaf, which in turn was dependent on growth and Pi nutrition. However, under Pi deficiency, in which the whole leaf Pi concentration dropped to approximately 15% of the control, the apoplastic Pi level fell to only 30% of the control (Mimura et al., 1990, 1992, 1996). In experiments in which Pi was fed to detached leaves via the transpiration stream, the level of Pi in the apoplast was independent of the supply concentration up to approximately 10 mM (Fig. 9). When the 10 mM Pi solution was replaced by pure water, the apoplastic Pi concentration remained constant. These results suggest that the apoplastic Pi concentration can be kept quasi-constant and it seems likely that this occurs via exchange with vacuolar Pi, a process which requires the participation of both the tonoplast and the plasma membrane. C. Whole Plants Pi is one of the most mobile molecules in plants (Bieleski and Ferguson, 1983). Pi is known to be retranslocated from older to younger tissues, from

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Pi concentration fed to the leaves (mM) FIG. 9 Changes in the apoplastic Pi Concentration of barley leaves as a function of the Pi concentrations fed through the transpiration stream (modified from Planfa, Phosphate transport across hiomembranes and cytosolic phosphate homeostasis in barley leaves, Mimura, T., Dietz. K.-J.. Kaiser, W.. Schramm, M. J., Kaiser. G., and Heber. U.. 180, 139-146. Fig. 2. 1990, 0 Springer-Verlag).

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Pi-supplied to Pi-demanding tissues, or back to the root from the shoot (Clarkson et al., 1978; Mimura et al., 1996; Jeschke et d., 1997). These are well-known phenomena which reflect an apparent homeostasis of Pi at the whole plant level (Bieleski and Ferguson, 1983). Biddulph et al. (1958) first showed retranslocation of Pi from old leaves to fresh leaves using radioautograms. Furthermore, the following studies provide more detail on Pi retranslocation (Greenway and Gunn, 1966; Smith er al., 1990). The split root experiments of Drew and Saker (1984) clearly showed that Pi taken up by Pi-supplied roots moved to Pi-deleted ones. Recently, Liu et al. (1998) reinvestigated Drew and Saker's work and found that Pi transporter genes were expressed not only in the Pi-deficient roots but also in the Pi-supplied roots in the divided root system. Pi depletion in one part of roots affected the other part of roots. They suggested that the internal signal (possibly the cytoplasmic Pi level) induced an expression of Pi transporter in both parts. Mimura et al. (1996) visualized and quantified the Pi movement between barley leaves with radioisotopes (Fig. 10). Using a highly sensitive imaging plate (Fuji Film, BAS2000), Mimura et al., analyzed the redistribution of Pi in the same individual plantlet (Mimura, 1995a; Mimura et al., 1996). After pulse labeling of the Pi-deficient barley plant, 32Pmoved sequentially from the first to the second and from the second to the third leaves (Fig. lo), confirming the results of Biddulph et al. (1958). Also using the imaging

FIG. 10 An example of autoradiograms obtained using an imaging plate. Barley grown in Pideficient conditions was pulse labeled with '*Pi for 30 min and then radioactivity was chased (from Mimura, 1995, with kind permission of Japanese Society of Plant Physiologists).

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plate, Mimura et al. could quantitatively estimate amounts of 32Pin each part (Fig. 11). Interestingly, the Pi-adequate plant (Fig. l l a ) behaved like the Pi-deficient plant (Fig. l l b ) after the plant was exposed to Pi-depleted medium. Since the Pi-adequate plant was grown in the presence of Pi for 10 days, it should have enough Pi in tissues. The imaging plate technique should prove useful for investigating the time-dependent and space-dependent changes in 32Pdistribution in whole tissue. Retranslocation of Pi between tissues is believed to occur via phloem (Bieleski, 1973). However, little is known about the mechanism of Pi movement from cells to phloem and from phloem to cells. There are two pathways from cells to phloem, symplastic and apoplastic. For symplastic movement, a concentration gradient of Pi must be formed between the cytoplasm of cells and phloem through the plasmodesmata. Such a concentration gradient of Pi between cells has not been detected. For the apoplastic pathway, the Pi status of the external phases must be known. Katou and Enomoto (1991) mathematically demonstrated the existence of concentration gradients in the apoplast in order to explain a driving force for the water transport in growing tissues. They named a small and thin space of the cell wall con-

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FIG. 11 Numerical analysis of 32P distribution in autoradiograms. Diamonds. whole plants;

inverted triangles. root: circles, the first leaf; triangles. the second leaf, squares, the third leaf. In (a), radioactivity of Pi-adequate barley was chased in the absence of Pi. In (b), radioactivity of Pi-deficient barley was chased in the absence of Pi (from Mimura, T., Sakano, K., and Shimmen, T., Studies on distribution, re-translocalion, and homeostasis of inorganic phosphate in barley leaves, Planr Cell Envirnn., with kind permission from Blackwell Science).

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nected to the xylem the “apoplast canal.” In these canals, the extracellular concentrations of solutes decrease proportional to the distance from the xylem as these solutes are taken up into cells (Katou and Okamoto, 1992). A similar situation occurs in solute retranslocation from cells to the phloem through the apoplast. Pi effluxed creates a concentration gradient toward the phloem. The Pi level in the phloem is a major factor in determining and controlling Pi translocation. We have recently measured the Pi level in the phloem of rice using the aphid method (Mimura et al., unpublished data). Under Pi deficiency the Pi level of the phloem decreased to one-fifth its original value, in contrast to root or mesophyll cells. This was also confirmed by Jeschke et al. (1997). The phloem appears to lack a homeostatic mechanism for Pi, but the large fluctuation in phloem Pi content may be a necessary part of the regulation of long-distance Pi transport as suggested by Clarkson and Saker (1988). In Arabidopsis, the Pho2 mutant (Delhaize and Randall, 1995) accumulates Pi in the shoot more than the wild type. Dong et al. (1998) suggested that the Pi accumulation in shoot might result from a defect in Pi uptake to the phloem in Ph02, i.e., the Pi accumulation in the shoot was due to the depression of retranslocation from shoot to root through the phloem. They proposed an alternative explanation-that the shoot cells lacked the ability to regulate intracellular Pi concentration. The latter could arise if there was a defect in the mechanism for export of Pi from the vacuole. To accomplish Pi homeostasis, a complex system is necessary-detection of Pi levels in each compartment, changes in the distribution of Pi between organelles in the cell or between cells in a whole plant, or changes in metabolic processes. After attainment of a new Pi state, the new level of Pi might be referred back to the Pi detection system (Fig. 12)-a conventional feedback system. In a simpler alternative model, the strongest Pi sink assumes a central role. If the cytoplasm in the cell or the youngest tissues in the whole plant absorbs Pi according to demand, the other parts may respond dependent on their individual Pi status. In this system Pi levels are driven by the demand from the strongest sink and no feedback system is needed. I believe that the former system operates at the cellular level, but the latter may be sufficient between cells and tissues.

VI. Concluding Remarks Phosphorus is one of the most essential elements in not only plants but also animals. All phosphorus in living organisms is derived from Pi absorbed

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// FIG. 12 A hypothetical diagram of Pi homeostasis in a plant cell. Putative sensor seems t o respond to the following factors; 1, the cytoplasmic Pi (andlor organic-P) level; 2. the extracellular Pi level: or 3. Pi flux across the membrane. Afterwards, the sensor would influence the membrane transport systems of both plasma membrane and tonoplast. and the Pi metabolisms directly or indirectly. There are also interactions between transport systems and metabolisms, for example. an inhibition of H+-ATPase by Pi.

by plants. The total amount of phosphorus easily accessible by plants in the environment is quite limited. Because of limited phosphorus, plants have developed various adaptive systems, i.e., high-affinity Pi uptake, storage of Pi in the vacuole, retranslocation of Pi between tissues, and secretion of phosphatases or organic substances. I have undoubtedly concentrated on those aspects of phosphorus which are closest to my own research interest, perhaps at the expense of other, equally important aspects such as the metabolic processes involving phosphorus. Owing to the importance of phosphorus, there is a multitude of published papers, and it was impossible for me to refer to them all. I hope that I have not overlooked any of the important studies. There is clearly a need to unify all knowledge about Pi in plants, i.e., Pi transport and Pi-related metabolism. The recent molecular approaches of both fields are certainly making clear the molecular networks for Pi in the cell, especially mechanisms for Pi uptake across the plasma membrane. However, there is still a large gap between physiological and molecular research which needs to be bridged. Furthermore, we may need to consider more carefully the relationship between global Pi circulation

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and plant availability in order to maximize agricultural production while protecting natural environments.

Acknowledgments I express my best gratitude to Prof. M. Tazawa (Fukui Institute of Technology), Prof. T. Shimmen (Himeji Institute of Technology), Prof. U. Heber (University of Wuurzburg), Dr. K. Sakano (National Institute of Agrobiological Resources), Prof. K.-J. Dietz (University of Bielefeld), Prof. E. Martinoia (University of Neuchatel), Prof. F. A. Smith (University of Adelaid), Dr. R. J. Reid (University of Adelaide), and all my colleagues for their kind and critical discussions. Especially, 1 express my sincere appreciation to Dr. Reid and Prof. Shimmen for their kind reading and many suggestions for the manuscript. I also thank Yokogawa Analytical Systems, Inc., and Dr. Y. Inoue for their continuing support to measure phosphates.

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Synaptic-like Microvesicles in Mammalian Pinealocytes Peter Redecker Medizinische Hochschule Hannover, D-30625 Hannover, Germany

The recent deciphering of the protein composition of the synaptic vesicle membrane has led to the unexpected identification of a compartment of electron-lucent microvesicles in neuroendocrine cells which resemble neuronal synaptic vesicles in terms of molecular structure and function. These vesicles are generally referred to as synaptic-like microvesicles (SLMVs) and have been most intensively studied in pancreatic pcells, chromaffin cells of the adrenal medulla, and pinealocytes of the pineal gland. This chapter focuses on the present knowledge of SLMVs as now well-established constituents of mammalian pinealocytes. I review the results of morphological, immunocytochemical, and biochemical studies that were important for the characterization of this novel population of secretory vesicles in the pineal organ. The emerging concept that SLMVs serve as a device for intercellular communication within the pineal gland is outlined, and unanswered questions such as those pertaining to the physiological function and regulation of pineal SLMVs are discussed. KEY WORDS: Pineal gland, Secretory vesicles, Synaptic membrane proteins, Paracrine communication, Neuroactive amino acids, Neuroendocrine cells, Melatonin. 0 1999 Academic Press.

1. Introduction Many endocrine cells share common structural and functional features with neurons and have therefore been classified as members of the diffuse neuroendocrine system or of the family of paraneurons (Pearse and Takor Takor, 1979; Fujita et al., 1988). Importantly, these similarities also manifest themselves in the regulated secretory pathways of neurons and neuroendocrine cells. Thus, it has been known for some time that the peptide hormone/~ltc'r?l
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storing secretory granules of neuroendocrine cells can be regarded as equivalents of dense-core vesicles of nerve cells. In addition, recent studies have disclosed that neuroendocrine cells also contain organelles which are now generally considered as counterparts of neuronal synaptic vesicles. The electron-lucent synaptic vesicles, once thought to be specific for neurons, are characteristic components of both presynaptic nerve endings and vascular nerve terminals at neurohemal contact zones (Fig. 1). As organelles which take up, store, and release nonpeptide neurotransmitters, synaptic

FIG. 1 Electron micrograph of axon terminals bordering the perivascular space which surrounds capillaries of the primary portal plexus in the neurohypophyseal median eminence. The nerve terminals of this neurohemal contact zone contain numerous electron-lucent microvesicles (arrowheads) in addition to dense-core vesicles (for details, see Redecker, 1991). L, capillary lumen. Scale bar = 500 nm.

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vesicles are key players in the process of fast synaptic communication. The comprehensive molecular analysis of the membrane composition of synaptic vesicles over the past 15 years has sparked a series of studies in which it could be demonstrated that the major proteins of the synaptic vesicle membrane are also expressed by neuroendocrine cells. Interestingly, in the latter cells these proteins have been localized to a population of clear microvesicles which are morphologically similar to synaptic vesicles and, hence, are designated as synaptic-like microvesicles (SLMVs). A considerable body of evidence has accumulated justifying the classification of SLMVs as previously unknown secretory organelles of neuroendocrine cells distinct from classical secretory granules. To date, SLMVs have been most intensively studied in islet cells of the endocrine pancreas, in chromaffin cells of the adrenal medulla, and in pinealocytes of the pineal gland. This review will provide an account of our current knowledge of SLMVs in the mammalian pinealocyte, a cell type which is a very suitable model for investigations of this novel secretory pathway in neuroendocrine cells.

II. The Mammalian Pineal Organ: A Mediator of Darkness With regard to the pineal gland of mammals, darkness appears on center stage. For a long time scientists have considered the pineal gland to be like a black box, an organ which resisted efforts to unravel its function or even a functionless evolutionary relic (Reiter, 1992). Thus, in a figurative sense, the prevailing scientific view of these earlier days anticipated the now wellestablished key role of darkness for pineal function. Today, it is no longer questionable that the pineal organ is an endocrine gland in its own right which has a significant impact on a variety of body functions. In contrast to other endocrine tissues, hormone synthesis and release in the pineal gland only occur during the night (Reiter, 1981; Vollrath, 1981, 1984; Korf, 1994; Korf et al., 1998). The mammalian pineal organ, which develops as an evagination from the epithalamus, is characterized by a compact and seemingly simple architecture in which two principal parenchymal cell types can be distinguished, i.e., pinealocytes and interstitial cells. Whereas endocrine secretion is a hallmark of pinealocytes, interstitial cells are regarded as nonendocrine components of the pineal which resemble glial cells and may play important roles surpassing a merely supportive function (Redecker et al., 1996a; Redecker, 1998).

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Remarkably, the pineal organ has undergone profound morphological and functional changes in the course of evolution (Collin and Oksche, 1981). Briefly, whereas classical photoreceptors predominate in pineal organs of anamniotes, and pineal organs of reptiles and birds additionally contain varying numbers of modified photoreceptors, the secretory pinealocytes of mammals lack direct photosensitivity. According to recent findings, these three categories of cells seem to have evolved in parallel during phylogeny (Korf, 1994; Korf et al., 1998). The close phylogenetic relationship between mammalian pinealocytes and pineal photoreceptors of poikilothermic vertebrates is highlighted by the demonstration of various proteins of the phototransduction cascade in pinealocytes (Korf, 1994; Schomerus et al., 1994; Korf et al., 1998). It is now commonly known that the principal hormone produced in the pineal glands of virtually all vertebrates is the methoxyindole melatonin, the “chemical expression of darkness” (Reiter, 1991a), which was originally isolated from the bovine pineal gland (Lerner et al., 1958). The rhythmic synthesis and secretion of melatonin according to the environmental lighting conditions is a typical feature of the vertebrate pineal. Being no longer a mainly photoreceptive organ as in lower vertebrates, the mammalian pineal gland receives photic information via an intricate multisynaptic pathway which begins in the retina and ends with intrapineal sympathetic nerve fibers originating from the superior cervical ganglia (Kappers, 1965). The latter nerve fibers release noradrenaline during the dark period and thus drastically enhance the synthesis of melatonin in pinealocytes by induction of the rate-limiting enzyme of its synthesis, serotonin N-acetyltransferase [also designated arylalkylamine N-acetyltransferase (AA-NAT)], whose cDNA has recently been cloned (Coon et al., 1995). Hence, pinealocytes have been classified as photoneuroendocrine cells which convert photic information and signals from endogenous oscillators of the brain into a rhythmic endocrine response (Oksche et al., 1987), i.e., the secretion of melatonin. Melatonin is required for physiological and behavioral adjustments of the organism to daily and annual changes of the environment (Reiter, 1991b; Arendt, 1995). It is beyond dispute that the timekeeping hormone melatonin can synchronize circadian rhythms of mammals and mediates the effects of seasonally changing day lengths, i.e., photoperiodic effects, on species which exhibit pronounced seasonal reproductive cycles (Steinlechner and Niklowitz, 1992). Accordingly, in seasonally breeding mammals melatonin is involved in the regulation of events such as reproduction, hibernation, or pelage coloration. Several additional functions of melatonin have been advocated in recent years, including antioxidant and antiaging properties as well as effects on the immune system. However, corresponding studies have evoked a highly controversial scientific debate (Reiter, 1995; Reppert and Weaver, 1995).

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111. Ultrastructural Observations A. Morphological Correlates of Secretory Function in Pinealocytes

Electron micrographs of pinealocytes are often hard to reconcile with the high degree of secretory activity of these cells (Vollrath, 1981, 1984; Karasek, 1992; Karasek and Reiter, 1992). This apparent discrepancy becomes less surprising if one adopts the view that melatonin leaves the pinealocyte simply by diffusion soon after its synthesis (Reiter, 1981,1991a,b). Neuroendocrine cells normally make use of secretory granules for hormonal storage and hormone release via exocytosis. In contrast, such granules (which are commonly referred to as dense-core vesicles in the pineal literature) are rare in most mammalian pinealocytes (Welsh et al., 1979; Vollrath, 1981, 1984; Karasek, 1992; Redecker and Bargsten, 1993), except for some species such as the mouse (Benson and Krasovich, 1977; McNulty et al., 1987) and the hamster (Sheridan and Reiter, 1968). Dense-core vesicles of pinealocytes are believed to store peptidergiclproteinaceous compounds for secretory release (McNulty et al., 1987; Karasek and Reiter, 1992). There are also reports of indoleamine storage within dense-core vesicles (Juillard and Collin, 1980), but this is probably of little physiological importance (McNulty et al., 1987). Further structures which have been related to the secretory activity of pinealocytes include so-called dense bodies (Przybylska er al., 1991) and vacuoles that are believed to be directly derived from the cisternae of the rough endoplasmic reticulum (PCvet, 1977,1979). However, the molecular identity of the presumptive secretory contents of all these organelles still remains to be elucidated. Compared with other neuroendocrine cells, the ultrastructure of the pinealocyte does not give the immediate impression that this cell type is specialized for the storage of larger quantities of secretory products (Vollrath, 1981,1984).

6. Clear Microvesicles Are Common Constituents of Pinealocytes Although secretory granules are rare in most mammalian pinealocytes, somata and processes of these cells are endowed with often considerable amounts of small electron-lucent vesicles whose secretory nature has only recently been disclosed with various modern techniques, as will be detailed later. It must be emphasized that in many ultrastructural studies of mammalian pinealocytes clear microvesicles were described decades ago when their secretory function could not yet be unequivocally demonstrated. Obviously,

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the clear microvesicles mentioned in these earlier electron-microscopical investigations largely correspond to the small vesicles which are now referred to as SLMVs. SLMVs have been observed to accumulate in bulbous endfeet of pinealocyte processes in various mammalian species, e.g., in rat (De Robertis and D e Iraldi, 1961; Arstila, 1967), mouse (It0 and Matsushima, 1968), gerbil (Welsh and Reiter, 1978), hamster (Sheridan and Reiter, 1968), guinea pig (Lues, 1971), rabbit (Leonhardt, 1967; Romijn, 1973), ground squirrel (Povlishock et al., 1975), cat (Wartenberg, 1968), and monkey (Wartenberg, 1968). According to these studies, the vesicle-rich dilated endfeet often are connected with the pinealocyte soma or process shaft by a thin, neck-like segment and occupy perivascular or intraparenchymal locations within the pineal tissue. There is a general lack of data on daynight differences of pineal SLMVs, apart from a report in which the volume density of SLMVs in gerbil pinealocytes was found to exhibit a diurnal rhythm (Welsh et af., 1979). Whereas in electron micrographs of rat pinealocytes clear microvesicles are not very conspicuous, in other species such as the Mongolian gerbil pinealocytes contain intriguingly high amounts of SLMVs of variable size ranging from 30-40 nm to sometimes more than 100 nm in diameter (Redecker et af., 1990; Redecker and Bargsten, 1993). Redecker and Bargsten (1993) showed that bulbous process endings of gerbil pinealocytes may indeed be filled with thousands or even tens of thousands of SLMVs. The endings are frequently situated close to or even within the pericapillary spaces (Fig. 2a), sometimes being separated from the perivascular basal lamina by processes of interstitial cells. These vascular process endfeet are reminiscent of neurosecretory axon terminals (see Section I). Other vesiclefilled process endings are totally embedded in the pineal parenchyma where they lie directly apposed to adjacent pinealocytes or interstitial cells from which they are separated by a small gap of approximately 20-30 nm (Figs. 2b and 2c). The width of this intercellular gap thus matches the size of the synaptic cleft. However, no membrane specializations comparable to preand postsynaptic thickenings are established at the sites of contact between the process endings and their neighbor cells. Some profiles of the intraparenchymal bulbous endings are completely enclosed by pinealocyte cytoplasm (see Fig. 15 in Redecker and Bargsten, 1993), showing that the endings can indent other pineal cells and thereby establish particularly intimate contacts. It should be noted that the enrichment of SLMVs in pinealocyte process terminals is unique for neuroendocrine cells in situ. Similar concentrations of microvesicles in process swellings of neuroendocrine cells can usually only be observed in cultured cells extending neurite-like processes and in all likelihood reflect local exo-/endocytotic recycling of SLMVs comparable to the recycling of synaptic vesicles inside the presynaptic nerve terminal (Reetz et af., 1991).

FIG. 2 Electron micrographs showing accumulations of SLMVs within process swellings of gerbil pinealocytes. (a) Pinealocyte process terminals with numerous SLMVs are demonstrated which border (arrow) or lie close to (arrowhead) the pericapillary space. L, capillary lumen. (b) Two dilated pinealocyte process endings (asterisks) are closely apposed to an adjacent, only partly visible pinealocyte soma (P). The process endings contain abundant SLMVs of variable size. N, pinealocyte nucleus.

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FIG. 2. (continued) (c) Two vesicle-filled pinealocyte process terminals (asterisks) establish tight contacts with both the soma of a pinealocyte (P) as well as with the process of an interstitial cell (I) which is characterized by conspicuous arrays of intermediate filaments. Pinealocyte process swellings sometimes contain a few larger vacuoles (V). (d) An electrondense “dark” pinealocyte process ending (asterisk) is filled with very densely arranged SLMVs. Note the adjacent vesicle-rich process ending of an electron-lucent pinealocyte (arrow). Short synaptic ribbons are discernible within the dark process (arrowheads). Scale bars = 500 nm.

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As a peculiarity, in the gerbil pineal gland electron-dense “dark” variants of pinealocytes, which are characterized by unusually dense arrangements of SLMVs throughout their processes (Redecker, 1993a; Fig. 2d), can be found as early as the second postnatal week (P. Redecker, unpublished observation). The significance of this ultrastructural feature is unclear, but the dense accumulation of SLMVs could indicate a principal functional heterogeneity of the microvesicular compartment among different types of pinealocytes. Alternatively, it may just reflect different secretory or metabolic states of one pinealocyte cell type.

C. Clear Microvesicles as Components of Synaptic Ribbons In pinealocytes, clear microvesicles are well-known components of synaptic ribbons (SRs). The latter are highly specialized synaptic structures which can be found in certain neurons and sensory cells such as retinal bipolar and photoreceptor cells (Vollrath and Spiwoks-Becker, 1996; Wagner, 1997). Moreover, the occurrence of SRs has been described in pinealocytes of several species (Vollrath, 1981; McNulty and Fox, 1992). In SRs, electronlucent vesicles surround trilaminar electron-dense plates which often appear rod-like in thin sections but can also acquire other shapes. The rods can measure up to several micrometers in length, are linked to surrounding vesicles by tiny electron-dense projections, and usually are arranged perpendicular to the plasmalemma. The plasmalemmal domains to which SRs of pinealocytes are attached may either face adjacent cell bodies and processes or border the perivascular spaces. In addition to other intracellular locations, SRs can also be encountered in process terminals which are filled with SLMVs (Fig. 3). However, our extensive ultrastructural investigations, including serial thin-section analyses, have revealed that several vesiclerich process terminals of adult pinealocytes definitely lack SRs. Various functions have been proposed for SRs of pinealocytes, especially an involvement in intercellular communication (Vollrath, 1981, 1984) or in endocytotic turnover of membrane components (McNulty and Fox, 1992). Importantly, SRs of pinealocytes and photoreceptors have been shown to be dynamic structures which are subject to morphological and numerical changes (Vollrath and Huss, 1973; Vollrath and Seidel, 1989; Schmitz and Drenckhahn, 1993). Consequently, it is thought that SRs are functionally related to the metabolic state of pinealocytes. Although the plasticity of SRs cannot be directly linked to melatoriin synthesis, because of diurnal fluctuations of SR frequency and the close correlation of the onset of these fluctuations with the nighttime rise of AA-NAT activity during postnatal development it can be hypothesized that SRs are somehow related to the endocrine activity of pinealocytes (McNulty and Fox, 1992).

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FIG. 3 Electron micrograph depicting two small processes (arrowheads) of gerbil pinealocytes which contain synaptic ribbons (arrows). The larger of the two process profiles is filled with abundant SLMVs. Scale bar = 500 nm.

Synaptic transmission in ribbon synapses of sensory cells and neurons differs from neurotransmission in conventional synapses. Thus, ribbon synapses are specialized for tonic transmitter release which is driven by graded potential changes rather than by action potentials (Juusola et al., 1996), although phasic release of neurotransmitter may also occur in particular ribbon-type synaptic terminals (von Gersdorff and Matthews, 1997). It is conceivable that the peculiar architecture of SRs reflects the unique functional properties of ribbon synapses. Recent membrane capacitance measurements and correlating ultrastructural analyses of the giant bipolar ribbon synapse in goldfish retina have revealed an unusually high rate of vesicle exocytosis (von Gersdorff et al., 1996). Hence, it could be a main function of SRs to recruit synaptic vesicles to plasmalemmal release zones and to provide a steady pool of rapidly releasable vesicles (for possible alternative functions, see Vollrath and Spiwoks-Becker, 1996). By analogy, SRs of pinealocytes may serve to “capture” vesicles and to direct them to the plasma membrane, thereby ensuring an ongoing high rate of exocytotic release. It can be envisaged, then, that pinealocyte SRs are randomly loaded with vesicles from the large pool of vesicles to which we refer to as SLMVs,

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comparable to the mechanism of vesicle recruitment discussed for ribbon synapses of photoreceptors and retinal bipolar neurons (Schaeffer and Raviola, 1978; von Gersdorff and Matthews, 1997). However, since the precise relationship between SLMVs and SRs of pinealocytes has not been deciphered, it would be premature to draw definite conclusions.

IV. Molecular Aspects A. Neuronal Proteins Implicated in Synaptic Vesicle Trafficking: SNAPS, SNAREs, and Beyond 1. The SNARE Hypothesis The intracellular transport of vesicles and their fusion with other membrane-bounded compartments are universal processes of eukaryotic cells. More specifically, cells as varied as yeasts or neurons can both release vesicular cargo via different modes of secretion. Although the degree of regulation imposed on this vesicle-mediated secretion differs among cell types, research carried out during the past decade has disclosed striking similarities between the basic molecular mechanisms which operate in diverse constitutive or regulated forms of secretion. Notably, in this area of research neurotransmitter secretion has evolved as a paradigm for the study of ordered intracellular membrane flow. Numerous excellent reviews are available which discuss all aspects of the molecular protein machinery involved in vesicle trafficking, i.e., in vesicle biogenesis, transport, and membrane fusion (Ferro-Novik and Jahn, 1994; Siidhof, 1995; Linial and Parnas, 1996; Woodman, 1997). Although several details must still be worked out, it is quite clear that this molecular machinery relies on a complex sequence of multiple protein-protein interactions. Some facets of the respective current models will now be outlined since many synaptic vesicle trafficking proteins have proved to be important for the molecular and functional characterization of SLMVs in neuroendocrine cells. In the context of this review, we will concentrate on those proteins which have already been investigated in the pineal gland. According to the widely cited SNARE hypothesis for vesicle-membrane docking and fusion, a set of complementary proteins which are localized in distinct membrane compartments directs and specifies vesicle trafficking in both constitutive and regulated pathways (Sollner et al., 1993a,b). These cognate proteins are residents of the vesicle membrane (as so-called vSNAREs) and of the target membrane (as so-called t-SNARES) which constitute the specific transport destination for a given kind of vesicle. vSNAREs and t-SNARES are receptors for cytoplasmic proteins which are

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essential for all membrane fusion events. The latter are called NSF (Nethylmaleimide sensitive fusion protein) and SNAPs (soluble NSF attachment proteins). With regard to synaptic vesicles, the SNARE hypothesis predicts that the V-SNAREsynaptobrevin and the t-SNARES syntaxin and SNAP-25 (synaptosomal-associated protein 25) pair in order to form a stable ternary core complex (Hayashi et al., 1994) which mediates docking of the vesicle at the plasma membrane. Docked vesicles are those which are directly apposed to the presynaptic plasma membrane and thus experience the first close encounter with their target membrane. Following the docking step, the ATPase NSF can bind the SNARE complex through SNAPs, leading to the formation of a 20s complex. The original hypothesis holds that subsequent ATP hydrolysis then reorganizes this complex so as to render the vesicle competent for membrane fusion and exocytosis. The latter process is referred to as “priming” and allows synaptic vesicles to complete exocytosis rapidly in response to Ca2+influx into the nerve terminal. However, since the initial formulation of the SNARE hypothesis, several significant modifications of this concept have been put forth (Calakos and Scheller, 1996; Goda, 1997; Nichols et af., 1997; Jahn and Hanson, 1998; Weber et al., 1998). For example, although the central importance of the core complex for cellular membrane fusion remains undisputed, NSF and SNAPs are now thought to initiate disassembly of the core complex after membrane fusion has occurred (Rizo and Sudhof, 1998). Moreover, a reclassification of SNARE proteins based on structural features of the fusion complex has been proposed (Fasshauer et al., 1998). 2. SNARES a. Synaptobrevin Synaptobrevin (also denominated as VAMP) is a small integral protein of the synaptic vesicle membrane (Trimble et al., 1988; Baumert et al., 1989; Sudhof et al., 1989a). Several neuronal and nonneuronal isoforms and homologs of synaptobrevin are known (Elferink et af., 1989; Protopopov et al., 1993; Rossetto et af., 1996), in addition to a ubiquitous isoform called cellubrevin (McMahon et al., 1993). Since synaptobrevin is a V-SNARE,it has initially been proposed that this protein plays a pivotal role during membrane recognition and docking of synaptic vesicles (Sollner et al., 1993a,b). According to recent experimental studies synaptobrevin is probably also required for postdocking events during exocytosis (Hunt et af., 1994; Broadie et af., 1995). The importance of synaptobrevin for neurotransmitter release is highlighted by the selective cleavage of VAMP proteins by various serotypes of clostridial neurotoxins which impair neurotransmission (Niemann et af., 1994).

b. Syntaxin The isoform I A of the integral plasma membrane protein syntaxin, now recognized as a t-SNARE, was originally described as a

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surface protein (HPC-I) of hippocampal neurons and retinal amacrine cells (Barnstable et al., 1983, 1985; Inoue et al., 1992). Whereas syntaxins I A and IB are largely neuron specific, other syntaxin isoforms exhibit a broader tissue distribution (Bennett et al., 1993). The importance of syntaxin for the vesicle docking and fusion process is emphasized by many studies which have probed syntaxin function by means of genetic manipulations, introduction of syntaxin peptidedantibodies into secretory cells, or cleavage of syntaxin by clostridial neurotoxins (Bennett et al., 1993; Blasi et al., 1993b; Gutierrez et al., 1995). The biochemical interactions of syntaxin with a variety of proteins implicated in synaptic vesicle exocytosis (synaptobrevin, synaptotagmin, SNAP-25, munc-18, a-SNAP, and N-type CaZt channels) render syntaxin a key player in vesicle docking and fusion (Yoshida et al., 1992; Sollner et al., 1993a,b; Hodel et al., 1994; Pevsner et al., 1994a). The presence of substantial amounts of syntaxin in the synaptic vesicle membrane may be the result of missorting during vesicle recycling but has also given rise to speculations as to whether v-SNARE/t-SNARE interactions might occur at the level of synaptic vesicles (Walch-Solimena et al., 1995; Kretzschmar et al., 1996). c. SNAP-25 SNAP-25 was initially characterized as a neuron-specific protein (Oyler et al., 1989,1991) associated with the neuronal plasma membrane via palmitoylation (Hess et al., 1992). As discussed for syntaxin, an important fraction of SNAP-25 can also be detected on synaptic vesicles (Walch-Solimena e l af., 1995; Kretzschmar et al., 1996). Similar to synaptobrevin and syntaxin, the protein is a substrate for specific clostridial neurotoxins (Blasi et al., 1993a; Schiavo et al., 1993). As a t-SNARE, it is part of the synaptic core complex and strongly stabilizes the latter (Pevsner et al., 1994b) but probably also acts downstream of the vesicle docking process (Wilson et al., 1996). In addition, SNAP-25 has been implicated in the process of axon elongation during neuronal development (Osen-Sand et al., 1993).

3. Synaptophysin Synaptophysin, a major integral glycoprotein of the synaptic vesicle membrane (Wiedenmann and Franke, 1985; Jahn et al., 1985), belongs to a small gene family comprising neuronal (Buckley et al., 1987; Leube et al., 1987; Knaus et al., 1990) and nonneuronal (Zhong et al., 1992; Leube, 1994) isoforms. The cytoplasmic carboxyl terminus contains unique tyrosine-rich repeats (Siidhof et al., 1987) and is known to be phosphorylated by pp60'-"' kinase (Barnekow et al., 1990). Although various roles have been proposed for this long-known protein, its precise function is still equivocal.

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Earlier studies have suggested participation of synaptophysin in the formation of a fusion pore during vesicle exocytosis (Thomas et al., 1988; Betz, 1990) or have pointed to a role in the structural organization of synaptic vesicles (Johnston et al., 1989b). The results from recent experimental studies indicate that synaptophysin is actively involved in neurotransmitter release (Alder et al., 1992a,b, 1995). In this context, biochemical findings of a strong interaction between synaptophysin and the V-SNAREVAMP2 (Calakos and Scheller, 1994; Edelmann et al., 199.5; Washbourne et al., 1995) are of particular interest. Since this interaction seems to prevent the assembly of the core complex, a role for synaptophysin as a negative regulator of vesicle exocytosis has been considered. However, in view of the overall normal phenotype of synaptophysin knockout mice (Eshkind and Leube, 1995) the regulatory function of synaptophysin may be subtle, although it cannot be excluded that the observed phenotype is merely a consequence of isoform redundancy.

4. Synaptotagmin

Synaptotagmin is an abundant membrane protein of synaptic vesicles that belongs to a protein family consisting of at least 11members with differential tissue distributions (Mizuta et al., 1994; Ullrich and Sudhof, 1995; C. Li et al., 1995; von Poser et al., 1997). The protein possesses a cytoplasmic carboxyl terminus which is thought to be of major functional relevance since it contains two copies of a repeat that is highly homologous to the regulatory C2 domain of protein kinase C (PKC; Perin et al., 1990). Akin to PKC, the C2 domain is responsible for the Ca2t-dependent binding of synaptotagmin to negatively charged phospholipids (Brose et al., 1992; Davletov and Sudhof, 1993). The calcium-binding properties of synaptotagmin and the phenotypes resulting from genetic disruptions of synaptotagmin (Nonet et al., 1993; Geppert et al., 1994b; Littleton et al., 1993, 1994) have been taken as evidence that synaptotagmin serves as an important calcium sensor in neurotransmission, linking presynaptic calcium entry to exocytosis. Numerous interactions between synaptotagmin and other nerve terminal proteins such as the t-SNARE syntaxin (Yoshida et al., 1992), N-type Ca2t channels (Yoshida et al., 1992), the a-latrotoxin receptor (Petrenko et al., 1991), and the clathrin adaptor complex (Zhang et al., 1994) have been described. Recent observations of a high-affinity interaction of synaptotagmin with SNAP-25 have led to the suggestion that synaptotagmin also functions as a V-SNARE(Schiavo et al., 1997). Together, the available data show that synaptotagmin is a multifunctional protein which fulfills distinct roles during both exo- and endocytosis.

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5. Rab3

Rab proteins make up a large family of low-molecular-weight GTP-binding (G) proteins which participate in the directional flow of membranes between different cellular compartments (Simons and Zerial, 1993; Pfeffer, 1994). Members of the rab3 subfamily are specifically associated with secretory vesicles in various cell types (Fischer von Mollard et al., 1990, 1994; Lledo et al., 1993; Jena et al., 1994; Tang et al., 1996). The hydrophilic rab proteins are reversibly attached to membranes by isoprenylation and can shuttle between membranes and the cytosol (Johnston et al., 1991; Magee and Newman, 1992). This cycling, which is accompanied by cycles of GTP/ GDP exchange and hydrolysis, is thought to underlie vectorial intracellular membrane traffic (De Lisle, 1993; Novick and Brennwald, 1993). In particular, the isoforms rab3A and rab3C, which bind to synaptic vesicles, have been shown to undergo membrane dissociation-association cycles during stimulation of regulated transmitter release (Fischer von Mollard et al., 1991, 1994). Many experimental studies performed on various secretory cell types point to an important role of rab3 in the control of exocytotic steps (e.g., Lledo et al., 1993, 1994). Since the results of mouse rab3A knockouts and Caenorhabditis elegans rab3 mutants are hard to reconcile with an essential role of rab3 in transmitter release (Geppert et al., 1994a; Nonet et al., 1997), it has been proposed that rab3 may be required for the complete and efficient fusion of the synaptic vesicle membrane after the Ca2+-dependentstep of the docking/fusion process (Geppert et al., 1997) or regulates the recruitment of vesicles to the release zone (Nonet et al., 1997). Based on the latest experimental data, it is thought that rab3 primarily acts at a late step during exocytosis in a manner opposite to synaptotagmin so as to limit the number of vesicles that undergo fusion as a function of Ca2+(Geppert and Siidhof, 1998).

6. Cysteine String Protein Cysteine string proteins (csps) were discovered as synapse-specific proteins in Drosophila which contained a characteristic concatenation of 11 consecutive cysteine residues forming the cysteine-rich “string” region within the central domain (Zinsmaier et al., 1990). Subsequently, csps could also be identified as components of the synaptic vesicle membrane in the marine ray Torpedo (Gundersen and Umbach, 1992) and in rat brain (Mastrogiacomo and Gundersen, 1995). The presence of a J domain in the amino terminus of csp which may interact with members of the heat shock protein family Hsp70 has led to the suggestion that csps serve chaperone-like functions by affecting the folding and stability of protein complexes of the exocytotic machinery (Mastrogiacomo et al., 1994; Buchner and Gundersen,

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1997). These functions obviously include a modulation of presynaptic Ca2+ channels by controlling Ca2+entry into the nerve terminal (Gundersen and Umbach, 1992; Mastrogiacomo et al., 1994; Buchner and Gundersen, 1997) which might serve to restrict Ca2+ influx to those sites where synaptic vesicles await exocytosis (Umbach et al., 1998). According to mutational analyses, csps facilitate evoked synaptic transmission and stabilize the transmitter release machinery at elevated temperatures (Umbach et al., 1994; 1994). Zinsmaier et d., 7. Munc-18

Munc-18, also denominated n-secl (Pevsner et a/., 1994a) or rb-secl (Garcia et a/., 1994), is characterized by its specific and tight binding to syntaxin I (Hata et al., 1993; Garcia et al., 1994; Pevsner e f al., 1994a). This association with syntaxin as well as hydrophobic modifications may be responsible for the recruitment of a fraction of the cytosolic munc-18 to the plasmalemma (Garcia et a/., 1995; Riento et a/., 1996). Munc-18 is homologous to yeast seclp and C. elegans unc-18. The latter proteins are essential for vesicular transport from Golgi apparatus to plasma membrane and for neurotransmission, respectively (Aalto er al., 1992; Hosono eta/., 1992; Pelham, 1993; Hata et al., 1993; Gengyo-Ando et a/., 1996). The binding of munc-18 to syntaxin, which seems to be modulated by so-called DOC2 proteins (Verhage et a/., 1997), inhibits the association of synaptobrevin and SNAP-25 with syntaxin (Pevsner et a/., 1994b), compatible with a key role of munc18 in regulating the formation of the synaptic core complex. 8. Synapsin

The synapsin family of neuron-specific proteins encompasses three members (Siidhof et al., 1989b; Kao et al., 1998) which interact with the cytoplasmic surface of synaptic vesicles and the cytoskeleton of the nerve terminal in a phosphorylation-dependent manner. Based on these properties, it has been argued that the isoform synapsin I reversibly tethers synaptic vesicles to the cytoskeletal meshwork and thus determines the availability of synaptic vesicles for exocytotic neurotransmitter release (Bahler and Greengard, 1987; Llinas et al., 1991; Greengard et al., 1993). Moreover, synapsins are known to promote the functional maturation of axons and synapses in developing neurons (Han et al., 1991; Ferreira et a/., 1994). Experimental investigations including presynaptic injection of synapsin peptide fragments and mouse knockout mutants lacking synapsin I and/or I1 have revealed that synapsins determine the time course of transmitter release and probably fulfill more subtle roles in synaptic regulation which are related to certain forms of neuronal plasticity (Rosahl et a[., 1993, 1995;

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L. Li et al., 1995; Hilfiker et al., 1998). An interesting new discovery is the finding that synapsins are endowed with ATP-binding properties which are differentially regulated by Ca'+ (Hosaka and Siidhof, 1998).

6. Synaptic Vesicle Trafficking Proteins in Neuroendocrine Cells 1. General Findings Following the identification of synaptic vesicle trafficking proteins in neurons, it was determined that these molecules are also widespread constituents of neuroendocrine cells. Indeed, the proteins and their corresponding mRNAs have been localized to a variety of neuroendocrine cells and cell lines, e.g., endocrine cells of the gastroenteropancreatic system (Navone et al., 1986; Redecker et al., 1991; Jacobsson et al., 1994; Ahnert-Hilger et al., 1996; Regazzi et al., 1995,1996; Wheeler et al., 1996; Hohne-Zell et al., 1997), chromaffin cells of the adrenal medulla (Schmidle et al., 1991; Chilcote et ul., 1995; Marqukze et al., 1995), or endocrine cells of the anterior and intermediate lobe of the pituitary (Redecker et al., 1995; Jacobsson and Meister, 1996; Majo et al., 1997). Several studies of neuroendocrine cells succeeded in demonstrating the presence of synaptic vesicle-associated proteins in the membrane of electron-lucent pleiomorphic vesicles with a diameter in the same range as that of synaptic vesicles (Navone et al., 1986; Reetz et al., 1991; Thomas-Reetz et al., 1993; Chilcote et al., 1995). Such observations were instrumental for the classification of the respective microvesicles as SLMVs, i.e., as a novel secretory compartment (De Camilli, 1991; Thomas-Reetz and De Camilli, 1994). In addition, many synaptic vesicle-associated proteins could also be identified as components of the peptide hormone-storing granules of neuroendocrine cells, indicative of their involvement in the regulated exocytosis of both classical secretory granules (Schmidle et al., 1991; Chilcote et al., 1995; Regazzi et al., 1995, 1996) and SLMVs.

2. Current Status in Pinealocytes

Studies performed during the past decade have unequivocally shown that pinealocytes must be included in the list of neuroendocrine cells which express proteins implicated in synaptic vesicle trafficking (Table I). This has considerably fostered the view that pinealocytes are equipped with microvesicular organelles which correspond to SLMVs of other neuroendocrine cells.

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TABLE I Synopsis of Synaptic Vesicle-Associated and Presynaptic Plasmalemmal Proteins (or Their Respective mRNAs) Known to Be Present in Mammalian Pinealocytes

Membrane protein Synaptophysin

Species Rat

Gerbil Mouse Hamster Guinea pig Cattle

Synaptotagmin

Synaptobrevin

Rab3A CSP sv2 Munc-18 SNAP-25

Syntaxin

Human Rat Gerbil Cattle Rat Gerbil Cattle Gerbil Gerbil Rat Gerbil Gerbil Gerbil Rat Gerbil Rat

References Vollrath and Schroder, 1987; Schroder et al., 1990: Redecker and Bargsten, 1993; Yamada et al., 1996b; Fujieda et al., 1997 Redecker et al., 1990; Redecker and Bargsten, 1993: Redecker et al., 1997 P. Redecker, unpublished observation: see Fig. 5 Redecker et al., 1996a Vollrath and Schroder, 1987; Schroder et al., 1990; Redecker et al., 1996a Sat0 et ul., 1994; Moriyama and Yamamoto, 1995a Huang et al.. 1992 Redecker, 1996 Redecker, 1996; Redecker et al., 1997 Mariyama and Yamamoto, 1995a Redecker, 1996 Redecker, 1996; Redecker et al., 1997 Moriyama and Yamamoto, 1995a Redecker, 1995; Redecker et al., 1997 Redecker et al., 1998 Hayashi ef al., 1998 P. Redecker, unpublished observation; see Fig. 6 Redecker et aL, 1998 Redecker et al., 1996b. 1997 Boschert et ul., 1996; Redecker et al., 1996b; Yamada ef al., 1996b Redecker, 1996; Redecker et al., 1997 Redecker, 1996

Synaptophysin was the first synaptic vesicle-associated membrane protein to be detected in the mammalian pineal gland. The immunofluorescence pattern observed in sections of rat and guinea pig pineal glands after incubation with a monoclonal antibody against synaptophysin suggested that most pinealocytes were synaptophysin-positive (Vollrath and Schroder, 1987; Schroder et al., 1990). In the pineal gland of the Mongolian gerbil, synaptophysin-immunoreactive cells could then be readily identified as pinealocytes by the alternate immunostaining of serial semithin sections with antibodies directed against synaptophysin and neuron-specific enolase (NSE), an established molecular marker of pinealocytes (Redecker et al., 1990). Redecker et al. also showed that interstitial glial cells, visualized with anti-

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bodies against the intermediate filament proteins glial fibrillary acidic protein and vimentin, were devoid of overt synaptophysin immunoreactivity. Furthermore, immunoreactivity for synaptophysin was demonstrated among pinealocytes of hamster (Redecker et al., 1996a), bovine (Sato et al., 1994; Moriyama and Yamamoto, 1995a), and human (Huang et al., 1992) pineal glands as well as in pineal parenchymal tumor cells (Collins, 1987;Jouvet et al., 1994). Some examples of synaptophysin-immunoreactive pinealocytes in diverse species are shown in Fig. 4. According to a recent developmental study synaptophysin-immunopositive rat pinealocytes appear as early as Embryonic Day 17, which renders this synaptic protein one of the earliest markers of pinealocyte differentiation (Fujieda et al., 1997). That synaptophysin is indeed a membrane component of SLMVs has been proven by immunoelectron microscopy of rat (Redecker and Bargsten, 1993; Yamada et al., 1996b), gerbil (Redecker and Bargsten, 1993), and mouse (P. Redecker, unpublished observation; Fig. 5) pinealocytes in situ and of purified SLMV fractions of bovine pinealocytes (Moriyama and Yamamoto, 1995a). Notably, in rat pinealocytes antibodies against synaptophysin also bind to microvesicles of synaptic ribbons (Redecker and Bargsten, 1993). In contrast to other neuroendocrine cells in vivo, pinealocytes tend to enrich synaptic vesicle-associated proteins such as synaptophysin in dilated process endings (Figs. 4 and 6) which correspond to the vesicle-rich process terminals described at the ultrastructural level (Redecker et al., 1990; Redecker and Bargsten, 1993). In addition to synaptophysin, further essential trafficking proteins of both the synaptic vesicle membrane and the presynaptic plasmalemma have been detected in pinealocytes of several species in vivo and in vitro. Thus, the calcium-binding protein synaptotagmin (Moriyama and Yamamoto, 1995a; Redecker, 1996; Redecker et al., 1997), the small G-protein rab3 (Redecker, 1995; Redecker et al., 1997), and the V-SNARE synaptobrevin (Moriyama and Yamamoto, 1995a; Redecker, 1996; Redecker et al., 1997) could all be localized to pinealocytes and their compartment of SLMVs. Moreover, the t-SNARES syntaxin (Redecker, 1996; Redecker et al., 1997) and SNAP-25 protein and mRNA (Boschert et al., 1996; Redecker et al., 1996b, 1997; Yamada et al., 1996b), as well as the ATPase NSF (Moriyama et al., 1995), were shown to be present in these photoneuroendocrine cells. Recent updates of the list of synaptic proteins contained in pinealocytes consist of munc-18 (Redecker et al., 1998), csp (Redecker et al., 1998), and the synaptic vesicle protein 2 (SV2; Hayashi et al., 1998; P. Redecker, unpublished observations: for micrographs of the immunocytochemical detection of some of the aforementioned proteins in pinealocytes, including the detection of SV2, see Figs. 6 and 7). SV2 is not considered a vesicle trafficking protein sensu strict0 but is thought to function as a ubiquitous synaptic vesicle-specific transporter protein, although the nature of the transport substrate remains

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FIG. 4 A compilation of synaptophysin-immunoreactive pinealocytes in semithin sections of plastic-embedded pineal glands from gerbil (a), hamster (b), and guinea pig ( c ) . Densely immunostained dots (arrows) correspond to pinealocytic process endfeet. Synaptophysinpositive pinealocytes in a paraffin section of a human pineal gland are shown in d. a, interference contrast optics; b and c, phase contrast optics: d, brightfield illumination. Scale bars = 20 pm.

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FIG. 5 Immunogold labeling of a LR White-embedded section of a mouse pineal gland with antibodies directed against synaptophysin. In this electron micrograph gold particles indicating exposed synaptophysin decorate numerous SLMVs within a pinealocyte process ending. Note the presence of some dense-core vesicles (arrowheads). Scale bar = 200 nm.

elusive. It has also been suggested that SV2 may play a role on the nerve terminal surface (Carlson, 1996). No comprehensive studies have addressed the question of whether the pineal levels of synaptic membrane proteins and/or their respective mRNAs vary over the light-dark cycle. Only limited data are available for synaptophysin inasmuch as no marked diurnal changes in the immunostaining pattern for synaptophysin can be encountered in the gerbil pineal gland (P. Redecker, unpublished results). Because of the relatively long half-life of these proteins (Johnston et al., 1989a; Matteoli et al., 1992) and their presumably low rates of synthesis (Johnston et al., 1989a), diurnal fluctuations of their levels, if existent at all, are likely to be only subtle. Although the vast majority of pinealocytes express the synaptic vesicle trafficking proteins discussed above, pronounced intercellular differences of immunostaining densities can frequently be observed in immunocytochemical analyses of these proteins (Redecker et al., 1990; Sat0 et al., 1994; Redecker, 1996). Such heterogeneities may simply be brought about by different functional states of one pinealocyte cell type. On the other hand, it cannot be excluded that these cell-to-cell differences reflect the presence of fundamentally different types of pinealocytes (Vollrath, 1981; Schomerus et al., 1994; Korf, 1996). Indeed, new findings pertaining to the expression

FIG. 6 The presence of the synaptic vesicle-associated proteins synaptobrevin I1 (a), SV2 (b), and rab3A (c) in gerbil pinealocytes is revealed by immunostaining of semithin sections of plastic-embedded pineals with specific poly- and monoclonal antibodies. Arrows indicate strongly immunoreactive pinealocyte process terminals. In d, the neuronal plasma membrane protein SNAP-25 is visualized in rat pinealocytes by immunocytochemistry. Due to the obvious staining of their plasma membranes, rat pinealocytes are distinctly outlined by SNAP-25 antibodies. Phase contrast optics. Scale bars = 20 pm.

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FIG. 7 As demonstrated by this semithin section of a rat pineal organ, a very heterogeneous immunostaining pattern can sometimes be observed after labeling pineal sections with antibodies directed against synaptic membrane proteins. In this case, marked intercellular differences of immunostaining densities have been elicited by incubating the section with a monoclonal antibody against synaptotagmin I. Interference contrast optics. Scale bar = 20 pm,

of phototransduction components in the rat pineal organ reinforce the concept of functionally diverse pinealocyte populations (Blackshaw and Snyder, 1997). Regarding synaptic membrane proteins, it is possible that markedly pronounced heterogeneities of the immunostaining pattern (Fig. 7) are due to a differential distribution of protein isoforms among pinealocytes, taking into account that such a differential isoform distribution is a common phenomenon in the mammalian brain (Fykse el al., 1993; Ullrich and Sudhof, 1995) and probably also in endocrine organs (Jacobsson and Meister, 1996). As discussed for neurons (Fykse et al., 1993; Ullrich and Sudhof, 1995), individual endocrine cells might coexpress different combinations of nonisofunctional isoforms of vesicle trafficking proteins which could allow them to regulate secretory pathways in a cell-specific manner. To date, only few comparative data on the distribution of the many different isoforms of the respective membrane proteins are available for the pineal gland (Redecker, 1996; Hayashi et al., 1998). In conclusion, the demonstration that pinealocytes are endowed with the major proteins which are involved in the regulation of synaptic vesicle targeting and exocytosis has paved the way for the classification of pineal

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SLMVs as endocrine equivalents of synaptic vesicles. However, since the regulation of SLMV exocytosis cannot be expected to be completely identical to the highly specialized regulation of ultrafast synaptic vesicle exocytosis (Thomas-Reetz and De Camilli, 1994), discrete differences in the expression of synaptic vesicle trafficking proteins among neurons and neuroendocrine cells are feasible. To date, the synapsins are the only vesicle trafficking proteins investigated that seem to be absent from pinealocytes (Redecker and Bargsten, 1993; Moriyama and Yamamoto, 1995a). The absence of significant amounts of synapsins from pinealocytes, as well as from other neuroendocrine cells, signifies that these phosphoproteins are a unique molecular adaptation of the presynaptic nerve terminal. The neuronspecific expression of synapsins probably can be explained by their involvement in specialized synaptic functions such as the mechanisms underlying synaptic plasticity. It is interesting to note that the concomitant lack of synapsins in ribbon-containing terminals of retinal rod and cone cells (Mandell et al., 1990) further underlines the close relationship between pinealocytes and photoreceptor cells. This finding also shows that pinealocytes differ to a certain degree from “classical” neurons, although it is obvious that mammalian pinealocytes generally display more neuron-like traits than other neuroendocrine cells (Vollrath and Schroder, 1987; Oksche et al., 1987).

V. Emergence of Functional Concepts A. Microvesicular Storage and Secretion of Signal Molecules The final establishment of SLMVs as neuroendocrine counterparts of synaptic vesicles has been greatly advanced by the demonstration that SLMVs are able to store and secrete signal molecules which otherwise serve as neurotransmitters in the nervous system. For example, it has been reported that SLMVs of pancreatic p cells are surrounded by high concentrations of the y-aminobutyric acid (GABA)-synthesizing enzyme glutamic acid decarboxylase (Reetz et aL, 1991), and that SLMVs of these neuroendocrine cells take up GABA via a GABA transporter which is driven by a proton electrochemical gradient (Thomas-Reetz et al., 1993) akin to the vesicular GABA transporter of neurons. In a hybrid pancreatic cell line, GABA secretion could be elicited by elevated concentrations of potassium and was independent of the release of secretory granule contents (AhnertHilger and Wiedenmann, 1992). Moreover, several neuroendocrine pancreatic cell lines have been shown to release GABA in a Ca2+-dependent manner upon stimulation with either high potassium or the calcium iono-

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phore A23187 (Ahnert-Hilger et al., 1996). The physiological relevance of GABA secretion in the endocrine pancreas has been highlighted by Gaskins et al. (1995), who found that endogeneous GABA is released by a pancreatic @-cell line in a glucose-dependent manner. The differential sensitivity of GABA and insulin secretion to extracellular glucose concentrations in this @-cell line (Gaskins et al., 1995), together with the dissociation between GABA release and insulin release in normal rat @ cells (Smismans et al., 1997), indicates that the intracellular pathways controlling GABA secretion from SLMVs are distinct from those which regulate insulin release from secretory granules. Microvesicular storage and regulated release of another neurotransmitter, i.e., acetylcholine, has been documented in rat phaeochromocytoma PC12 cells (Bauerfeind et al., 1993, 1995; Ninomiya et al., 1997). Interestingly, SLMVs of chromaffin cells, the physiological ancestors of PC12 cells, may instead contain catecholamines (Annaert et al., 1993). In the pineal gland, the studies performed to date leave little doubt that SLMVs of pinealocytes are involved in the storage and secretion of neuroactive amino acids. Glutamate was the first amino acid which evolved as a candidate molecule for microvesicular storage in pinealocytes. In an immunocytochemical investigation of the gerbil pineal, glutamate immunoreactivity was found to be concentrated over pinealocytes at both light- and electron-microscopic levels (Redecker and Veh, 1994;Fig. S), reminiscent of similar observations in the rat pineal (McNulty et al., 1992). In the former study, the quantification of gold particle densities after postembedding immunogold staining provided some evidence for a microvesicular compartmentation of glutamate. Using a biochemical approach, Moriyama and Yamamoto (1995a) could unequivocally demonstrate the accumulation of L-glutamate in SLMVs that had been isolated from bovine pineal glands. The vesicular uptake of glutamate observed in the latter investigation was neuron-like because it was coupled to a proton electrochemical gradient established by a vacuolar type H+-ATPase.Since the kinetics of glutamate uptake into pinealocyte SLMVs strongly resemble the uptake of glutamate into synaptic vesicles, the (elusive) vesicular glutamate transporters of neurons and pinealocytes seem to be similar, if not identical (Moriyama and Yamamoto, 1995b). Experimental in vitro studies of isolated pinealocytes have also provided compelling evidence that pineal SLMVs release their very contents in a regulated fashion. According to Yamada et al. (1996b), cultured rat pinealocytes secrete glutamate after stimulation with depolarizing concentrations of potassium or a Ca2+-ionophorein a Ca2+-dependent manner. Furthermore, the treatment of cultured pinealocytes with botulinum neurotoxin type E (BoNT/E) inhibited potassium-evoked glutamate release through enzymatic cleavage of SNAP-25, comparable to the attenuating effects of BoNT/E on synaptic neurotransmission. The addition of various Ca2+-channelblockers to cultured rat pinealocytes revealed that

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FIG. 8 Immunogold staining for glutamate performed on a section of a gerbil pineal gland that was embedded in Epon. In this electron micrograph, a high density of gold particles is associated with two pinealocyte process terminals (asterisks) which contain abundant SLMVs. Scale bar = 500 nm.

Ca2+influx through plasmalemmal Ca2+channels was required for microvesicular glutamate secretion, and it was concluded that the channels involved were mainly L- and N-type Ca2+channels (Yamada et al., 1996a,b). In these studies, the rate of glutamate release was markedly slower than that of neuronal glutamate secretion brought about by synaptic vesicle exocytosis. This observation corroborates the notion that the mechanisms of microvesicular secretion in neurons and neuroendocrine cells, although basically similar, exhibit some discrete differences. In retrospect, the discovery of glutamate storage in SLMVs of mammalian pinealocytes was not completely unexpected, in view of the obvious role of this amino acid as a neurotransmitter in retinal photoreceptors (Barnstable, 1993) and probably also in pineal photoreceptors of submammalian vertebrates (Meissl and George, 1984; Vigh et al., 1995). Since this discovery, an

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additional signal molecule has been determined. Yatsushiro and coworkers (1997) reported the Ca2+-dependentcorelease of glutamate and the L-form of the amino acid aspartate from cultured rat pinealocytes. The characteristics of L-aspartate release from pinealocytes were similar to those of glutamate release, suggesting that pinealocytes also release L-aspartate through microvesicle-mediated exocytosis. In this context, it is worth remembering that the results from many studies support the hypothesis that aspartate serves as a neurotransmitter of retinal and pineal photoreceptors (Meissl and George, 1984). Although it is still a matter of dispute whether neuronal L-aspartate can be enriched at all in synaptic vesicles for subsequent exocytotic release (Fykse and Fonnum, 1996; Obrenovitch and Urenjak, 1998), recent data strengthen the evidence for a vesicular storage of L-aspartate as a neurotransmitter in certain neuronal circuits (Breukel et al., 1997; Gundersen etal., 1998). Remarkably, the D form of aspartate is also released by rat pinealocytes, although secretion probably occurs through a nonvesicular release mechanism (Ishio et al., 1998).

6. Pinealocyte Microvesicles: Components of an Extensive Paracrine System? 1. Molecular and Morphological Evidence The studies summarized thus far have lended ample support to the view that SLMVs constitute a novel secretory pathway of pinealocytes. Nevertheless, we are only just beginning to understand the physiological function of this particular secretory organelle. It has been argued that within the pineal gland SLMVs provide a means for intercellular communication which shares many properties with the process of synaptic transmission (Redecker and Bargsten, 1993; Yamada et al., 1996b; Redecker et al., 1997; Yatsushiro et al., 1997). This model predicts that pinealocytes release signal molecules from SLMVs which then diffuse in the extracellular space to act upon adjacent cells in a paracrine manner. Analogous to synaptic neurotransmission, signal transduction mediated by SLMVs relies on the presence of appropriate receptors on nearby target cells and the inactivation of messenger molecules by reuptake systems and/or degrading enzymes. With regard to glutamate, many data have accumulated that meet the requirements of this hypothesis. First, binding sites for glutamate have been described in the pineal gland (Ebadi et al., 1986; Govitrapong et al., 1986). Recent immunocytochemical and in situ hybridization studies have extended these initial observations by demonstrating that pineal cells can express different subunits of all major classes of glutamate receptors, i.e., the ionotropic receptors of NMDA (N-methyl-o-aspartate), AMPA (a-amino-3-hydroxy-5-methyl-4-isoxazole

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propionate), and kainate type (Wisden and Seeburg, 1993; Sat0 et al., 1993; Tolle et al., 1993; Petralia et al., 1994), as well as the metabotropic receptors (mGluRs; Petralia et al., 1996). It should be kept in mind, however, that the results of the respective investigations are not entirely consistent (cf. Sat0 et al., 1993; Kus et al., 1994), which may at least partly be due to interspecies differences. Furthermore, most of the aforementioned reports lack information concerning the precise cellular localization of the glutamate receptor subunits studied. Interestingly, a more detailed immunocytochemical analysis of the macaque pineal revealed the presence of AMPAtype receptors in glial cells, whereas pinealocytes seemed to lack any of the AMPA-type receptor subunits (Mick, 1995). At variance, according to Yamada et al. (1996b) the GluRl AMPA subtype of ionotropic glutamate receptors is expressed by pinealocytes in the rat. An important clue that glutamate affects melatonin secretion by binding to the class I1 metabotropic receptor mGluR3 has recently been provided by Yamada et al. (1998b). Likewise, our own preliminary observations show that certain members of the mGluR receptor family can be detected in the pineal gland by immunocytochemistry. Intriguingly, in the gerbil antibodies against receptors such as mGluR2/3 or mGluR.5 elicit a particularly strong immunostaining in interstitial glial cells (Fig. 9). Although several details remain

FIG. 9 Strongly immunofluorescent cells (arrows) stand out in this section of a gerbil pineal gland after incubation with a polyclonal antibody recognizing the metabotropic glutamate receptors mGluRZlmGluR3. The immunoreactive cells can be identified as interstitial cells by double-labeling experiments (not shown). Scale bar = 10 pm.

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to be elucidated, it can be inferred that pineal parenchymal cells are equipped with appropriate receptors which should allow SLMV-derived glutamate to modulate electrophysiological and metabolic properties not only of pinealocytes but also probably of interstitial glial cells. Second, the pineal gland obviously also contains reuptake systems for the sequestration of extracellular glutamate. In the central nervous system, the extracellular concentration of glutamate is maintained at low levels by a family of sodium-dependent plasma membrane transporters. One of these transporters, denominated GLT-1, has been localized in rat pinealocytes in which it operates with similar kinetics as in the nervous system (Yamada et af.,1997b). This finding has led to the suggestion that the efficient uptake of glutamate from the extracellular space is necessary for the precise control of melatonin synthesis (Yamada et al., 1997b). It should be pointed out that interstitial glial cells seem to contribute to the pineal expression of glutamate transporters such as GLT-1 (P. Redecker, unpublished observation). Taken together, the current knowledge, albeit still incomplete, suffices to conclude that the essential components for glutamatergic signal transduction and signal termination are all in place in the pineal gland (summarized in Table 11). Thus, it appears as if microvesicle-mediated intercellular communication is a new paradigm for the concept of paracrinia which was developed several

TABLE II Synopsis of Glutamate Receptors and TransportersThat Have Been Identified in the Pineal Gland at the

Protein or rnRNA Level Species Receptor subunit NMDARl

Cellular localization

Reference Sat0 et a/., 1993: Petralia et al., 1994 Tolle ef al., 1993 Yamada et al.. 1996b Mick, 1995 Mick,1995 Mick. 1995

Rat

Not specified

NMDAR2C GluRl GluRl GluR4 GluR213I4c

Rat Rat Macaque Macaque Macaque

GluRS KA2

Rat Rat

Not specified Pinealocytes Glial cells Glial cells Glial cells, neuronlike cells Not specified Not specified

rnGluR2/3 rnGluR3 Transporter type GLT-1

Rat Rat

Not specified Pinealocytes

Sato ef a/., 1993 To11e et a / . , 1993; Wisden and Seeburg, 1993 Petralia ef a/., 1996 Yamada ef al.. 1998b

Rat

Pinealocytes

Yarnada et al., 1997b

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FIG. 10 Distribution of immunofluorescence in dissociated primary cultures of rat (a and b) and gerbil (c) pinealocytes after 1 week in vitro and labeling of the cells with antibodies against synaptophysin. Within the only faintly visible pinealocyte processes shown in a, synaptophysin is concentrated in varicosities and terminal swellings (arrows) which often establish close contacts with other pinealocytes (arrowheads). In b, a pinealocyte soma is discernible (arrow) which gives rise to two processes (large arrows). One process can he seen to terminate upon an adjacent pinealocyte with a highly immunofluorescent swelling (arrowhead). A long

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decades ago (Feyrter, 1938). Currently, use of the term paracrinia mostly implies the local action of peptide messenger molecules in the close vicinity of their cellular site of synthesis (Grube, 1986). However, intercellular signaling that relies on nonpeptide molecules such as those stored in SLMVs may well constitute a primordial form of paracrinia. This view is supported by the probably universal function of the respective nonpeptide molecules in cell-to-cell communication during early cellular evolution (ThomasReetz and De Camilli, 1994). In principle, paracrine signals may reach their target cells via secretion into the extracellular space and/or after release into blood vessels via local circulatory systems (“short-loop’’ mechanism; Grube, 1986). With regard to pineal paracrine signal transmission based on SLMVs, it is conceivable that both modes of messenger molecule delivery come into play. This assumption is substantiated by the microtopographical arrangement of the dilated vesicle-rich process endfeet of pinealocytes which in every likelihood are major release sites for microvesicular signal molecules. Since these process endings frequently lie near or within the pericapillary space (Redecker and Bargsten, 1993), microvesicle-derived messengers such as glutamate or aspartate may well reach their target cells by capillary blood flow. This short-loop paracrine mechanism would allow simultaneous signaling to a number of adjacent or more distant cells which are endowed with the option to respond to the corresponding messenger molecules. Because the pineal organ is among the most highly vascularized tissues (Ohtani et al., 1983), a short-loop mechanism could efficiently operate throughout the whole gland. O n the other hand, the intraparenchymal bulbous process terminals which are located in close apposition to adjacent pinealocytes or interstitial cells (Redecker and Bargsten, 1993) could bring about a more spatially restricted form of paracrine communication. Interestingly, similar intimate contacts between pinealocyte process swellings enriched in SLMVs and other pinealocytes readily develop in dissociated cultures of gerbil pinealocytes (Redecker et al., 1997; Fig. 10) and are also established by rat pinealocytes in vitro (P. Redecker, unpublished observations; Fig. 10). The presumed point-to-point signaling mediated by these contacts clearly would constitute the closest approximation yet of neuroendocrine signaling to synaptic transmission. It must be stressed, however, that microvesicle-based paracrine communication in the pineal

branching process (arrow) of a synaptophysin-positive pinealocyte is depicted in c. Inside this process, synaptophysin immunofluorescence is pronounced in varicosities and dilated endings (arrowheads), some of which are closely apposed to a small group of adjacent pinealocytes (asterisk). The whole extent of the same process (arrow) isvisualized with anti-tubulin antibodies by double-labeling in d. Scale bars = 10 pm.

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gland may partially proceed in a less localized manner because an unknown percentage of synaptophysin-positive pinealocytes definitely lack vesiclerich process endings (Redecker and Bargsten, 1993), and interspecies differences concerning the frequency of these endings have to be taken into consideration. Finally, it cannot be excluded that messenger molecules of SLMVs additionally exert some autocrine effects, i.e., that they act back on the very pinealocytes which have liberated them. This kind of selfregulation has been viewed as a special type of paracrinia (Grube, 1986).

2. Clues to the Functional Significance and Regulation of Microvesicle-Mediated Paracrine Communication Results from several independent lines of research indicate that signal molecules of SLMVs interfere with the endocrine activity of pinealocytes. Currently, most of the available data refer to the impact of glutamate on melatonin production. Earlier studies have revealed that glutamate inhibits AA-NAT activity and melatonin synthesis (Govitrapong and Ebadi, 1988; Kus et a/., 1991). These observations have recently been confirmed by in vitro analyses showing that, at least in the rat, glutamate suppresses the adrenergic-stimulated secretion of melatonin (van Wyk and Daya, 1994; Kus et al., 1994; Yamada er al., 1996b). Thus, a dose-dependent inhibition of adrenergic-evoked melatonin release from cultured rat pinealocytes was observed when L-glutamate was added to the medium at concentrations between 100 p m and 1 mM (Yamada et al., 1996b). Glutamate concentrations of a comparable magnitude are probably also reached in close vicinity to microvesicular release sites in vivo (Yamada et a/., 1996b). Meanwhile, similar inhibitory effects on melatonin synthesis have been ascribed to Laspartate (Yamada et a/., 1997a). As mentioned previously, the intracellular signal transduction pathway mediating the inhibitory effects of glutamate on melatonin secretion seems to be connected to mGluRs since in rat pinealocytes activation of the mGluR3 receptor has been reported to inhibit melatonin synthesis by decreasing CAMPlevels and AA-NAT activity (Yamada et al., 1998b). Hence, it appears that SLMVs play an important role in the regulation of the synthesis of the major pineal hormone, melatonin. Paracrine regulation by microvesicle-derived signal molecules may be a mechanism for the fine-tuning of pineal hormone release and thereby contribute to the shaping of the profile of melatonin secretion. It follows from the aforementioned studies that a main function of these signal molecules is to attenuate the amplitude of melatonin production and release. Therefore, the question arises as to whether microvesicular messengers elicit either a tonic or a phasic suppression of melatonin synthesis in vivo. In the latter case, it is possible that the neuroactive amino acids participate in curtailing the

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nocturnal rise of melatonin production at the end of the dark phase, although it is well established that transcription factors such as ICER (inducible CAMP early repressor) are of prime importance for the endogenous downregulation of melatonin biosynthesis (Stehle, 1995; Korf et al., 1998). Definite answers to these important questions must await the determination of the release pattern of glutamate and aspartate in the pineal gland in vivo and the elucidation of the time-dependent relationship between SLMVmediated secretion and melatonin production. Sophisticated methods such as refined in vivo microdialysis techniques have the potential to provide new insights into the release kinetics of hormonal or paracrine pineal effectors in situ (Drijfhout et al., 1996a,b). Although exocytosis of pinealocyte SLMVs can be provoked by depolarization or Ca2' ionophores in vitro, the physiological stimuli which govern microvesicular release in the intact pineal gland are not yet fully understood. In vivo, depolarization-induced exocytosis of SLMVs may occur as a consequence of the spontaneous electrical activity of pinealocytes. Extracellular electrophysiological recordings performed in pineal organs of several mammalian species have disclosed the presence of spontaneously active pinealocytes which produce action potentials with spike amplitudes similar to those of nerve cells (Reuss and Vollrath, 1984) and exhibit diverse rates and patterns of firing (Semm and Vollrath, 1980; Stehle et al., 1987; Schenda and Vollrath, 1997). Since the innervation is mandatory for the regulation of pineal endocrine function, it is necessary to determine the effects of the various intrapineal neurotransmitters and neuromodulators (Korf, 1996) on SLMVs. To date, it has not been determined whether noradrenaline, the main driving force behind melatonin synthesis, or peptides such as vasoactive intestinal polypeptide, which mimicks the effects of /3-adrenergic agonists in pinealocytes (Chick et af., 1988), exert any influence on microvesicular secretion. Preliminary in vitro experiments performed by Yamada et al. (1996a) failed to induce microvesicular exocytosis upon increasing the intracellular Ca2+concentration in pinealocytes by addition of the above mentioned neurotransmittersheuromodulators. On the other hand, the observation that noradrenaline is able to modulate the electrophysiological properties of pinealocytes (Reyes-Vazques et al., 1986; Stehle et al., 1989) could indicate a potential adrenergic control of depolarization-evoked exocytosis of SLMVs in viva Because the electrophysiological responses of pinealocytes to noradrenaline in the latter studies were diverse, it is possible that the adrenergic impact on microvesicular exocytosis, if existent at all, may vary among individual pinealocytes. Interestingly, there is convincing evidence that cholinergic mechanisms could be critically involved in the control of microvesicular secretion. Indeed, the parasympathetic innervation may be of greater functional significance for the pineal gland than previously thought (Laitinen et al., 1995; Korf, 1996; Drijfhout et af.,1996a;

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FIG. 11 Demonstration of close neuronal-pinealocytic relationships. (a) Immunocytochemical localization of synaptophysin in a semithin section of a plastic-embedded gerbil pineal gland. Perivascular imrnunoreactive nerve terminals appear as punctate labels and are frequently juxtaposed (arrows) to dilated process endings (arrowheads) of pinealocytes. L, capillary lumen. Scale bar = 10 pm. At the electron microscopic level (b), varicosities with features

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Korf et al., 1998). In particular, observations of an increase in intracellular free Ca2+concentration induced by cholinergic stimulations of dissociated rat pinealocytes (Schomerus et al., 1995; Marin et al., 1996) can be interpreted in the context of a parasympathetic control of SLMV exocytosis. In support of this assumption, patch clamp recordings of isolated rat pinealocytes have shown that acetylcholine is able to depolarize pinealocytes about 16 mV on average by binding to nicotinic acetylcholine receptors (Letz et al., 1997). Moreover, acetylcholine-induced depolarization caused Ca2+influx mainly attributed to the opening of L-type Ca2+channels. These findings have led to the proposal that the stimulation of nicotinic acetylcholine receptors and the ensuing influx of Ca2+may induce exocytosis of SLMVs (Letz et al., 1997; Korf et al., 1998). Consistent with this conclusion, a recent in v i m study of cultured rat pinealocytes clearly documented the ability of acctylcholine to trigger microvesicular release of glutamate via activation of nicotinic acetylcholine receptors (Yamada et al., 1998a). Furthermore, the existence of an apparently extrinsic cholinergic innervation of the pineal gland has recently been corroborated by the immunohistochemical visualization of relatively scarce cholinergic nerve fibers in the rat pineal organ with antibodies against the vesicular acetylcholine transporter (Schafer er al., 1998). Since acetylcholine can attenuate melatonin synthesis both by presynaptic inhibition of noradrenaline release (Drijfhout et al., 1996a) and by provoking glutamate release, the sympathetic and the parasympathetic systems may function as antagonists in the pineal gland (Korf et al., 1998). The concept of a cholinergic regulation of pineal SLMVs gains additional complexity if one considers the possibility that pinealocytes themselves may release acetylcholine from a cytoplasmic pool during the dark period (Wessler et al., 1997). Finally, concerning the potential impact of neurotransmitters on microvesicular secretion, it is of interest that varicosities of sympathetic nerve fibers are frequently juxtaposed to vesicle-filled process swellings of gerbil pinealocytes (Redecker, 1993b; Fig. 11) and can establish multiple point-to-point contacts with pinealocyte process terminals. Although it is not known whether similar contacts are also established by nonsympathetic nerves, it is tempting to speculate that nerve fibers may regulate the compartment of SLMVs with highest efficiency at the level of these intimate neuronal-pinealocytic contacts. If future studies of the pineal gland confirm that SLMV-derived chemical messengers mainly act as inhibitory modulators of melatonin secretion, the

typical of sympathetic nerve fibers (arrows) can consistently be found that establish tight point-to-point contacts with vesicle-filled process endfeet (asterisk) of pinealocytes. Note the absence of membrane specializations at the axon-pinealocyte contact sites. Scale bar = 500 nm.

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hypothesis that pinealocytes can make up pineal circuits analogous to the inhibitory circuits established by local interneurons elsewhere in the brain (Rosenstein et af., 1990) would gain further support. As emphasized by Rosenstein et af., a tonic suppression of melatonin synthesis and release elicited by an intrapineal inhibitory pinealocyte network could enhance the information processing capacity of the pineal gland in response to stimulation by pinealopetal nervous input, thereby allowing individual pinealocytes to regulate melatonin secretion in a graded fashion.

VI. Biogenesis of Synaptic-like Microvesicles Due to their content of SLMVs, neuroendocrine cells and cell lines are well suited to serve as model systems for the exploration of the synaptic vesicle life cycle. A case in point is the de novo biogenesis of synaptic vesicles which has long been difficult to address experimentally. The identification of the major membrane protein components of SLMVs has been exploited by various in vitro studies of neuroendocrine cell lines as well as transfected fibroblastic cells in order to obtain new clues regarding the mechanisms that give rise to mature SLMVs (Cameron et al., 1993; Mundigl and D e Camilli, 1994;Thomas-Reetz and De Camilli, 1994). Prompted by the results of these studies, which made use of methods such as immunolocalization and immunoisolation techniques, it has been hypothesized that the biogenesis of microvesicles is related to early endosomal compartments. This welldocumented model holds that after constitutive transport to the plasmalemma, membrane proteins of SLMVs reach the endosome with the same vesicular carriers which internalize recycling plasmalemmal receptors, e.g., the transferrin or low-density lipoprotein receptor. Protein components of SLMVs are then sorted away from these receptors to become incorporated into newly formed SLMVs. A key finding in support of this model is the observation that membrane proteins of SLMVs and the transferrin receptor are largely codistributed in certain cell lines (Cameron et af.,1991). In contrast, Schmidt et al. (1997), in a study of PC12 cells, proposed the existence of a novel subplasmalemmal donor compartment for SLMVs which is distinct from classical early endosomes and therefore lacks significant amounts of the transferrin receptor. Likewise, synaptophysin and the transferrin receptor were mainly found in different compartments in transfected epithelial cell lines (Leube et af., 1994) and in PC12 cells (CliftO’Grady et al., 1990). These data favor a major role of a specialized endosome-like compartment, probably corresponding to that found in axons of nerve cells (Takei et a]., 1996), in the biogenesis of SLMVs. The axonal endosome also lacks the transferrin receptor and is thought to be strongly

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involved in the biogenesis of neuronal synaptic vesicles. In addition to their endosomal-related generation, some mature SLMVs may be assembled directly at the plasmalemma (Thomas-Reetz and De Camilli, 1994). In fact, there is good reason to believe that in neurons synaptic vesicles are partly derived from the presynaptic plasma membrane (Koenig and Ikeda, 1996). It has even been suggested that synaptic vesicles completely retain their identity during recycling without communicating with endosomal compartments (Murthy and Stevens, 1998). Also, for PC12 cells, evidence for at least two distinct pathways of SLMV biogenesis has been presented (Faundez et al., 1998; Shi et al., 1998). In pinealocytes, the de novo formation of SLMVs has not been investigated in depth. As a first approach to tackle this question, the intracellular localization of synaptophysin and the transferrin receptor was compared in cultured gerbil pinealocytes using confocal laser scanning microscopy (Redecker et nl., 1997). It is worth noting that some of these pinealocytes showed a neuron-like polarization inasmuch as a few pinealocyte processes excluded the transferrin receptor and the microtubule-associated protein MAP2 and therefore closely resembled axons. Importantly, in the remaining transferrin receptor-positive processes and somata, the distribution of synaptophysin largely differed from that of the receptor (Redecker et af.,1997). Thus, it is reasonable to assume that a substantial proportion of pinealocyte SLMVs is not generated at the level of classical early endosomes which is compatible with the suggested modes of vesicle biogenesis described previously, The small areas of overlapping immunoreactivities for synaptophysin and the transferrin receptor in cultured gerbil pinealocytes are consistent with the proposal that in neuroendocrine cells a fraction of synaptophysin cycles between the subplasmalemmal donor endosomes, the transferrin receptor-containing endosomes, and the plasma membrane (Schmidt et al., 1997). In future studies, additional aspects of the life cycle of pinealocyte SLMVs besides vesicle biogenesis deserve attention, such as the temporal and spatial characteristics of SLMV recycling including the mechanisms that govern vesicle endocytosis.

VII. Concluding Remarks and Future Perspective The structural and functional characterization of SLMVs has begun to provide novel insights into secretory pathways which operate in neuroendocrine cells. The mammalian pinealocyte is an interesting cellular model for investigations of this microvesicular compartment. Pinealocytes differ from other neuroendocrine cell types in situ in that they often contain intriguingly large amounts of SLMVs and tend to enrich these microvesicles in process

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P: pinealocytes I :interstitial gIiaI cells A : messenger molecules released from SLMVs such as glutamate

M :membrane receptors for microvesicular messengers (e.g., metabotropic/ionotropicglutamate receptors)

M :membrane transporters for reuptake of microvesicular messengers (e.g., GLT-1) FIG. 12 Schematic drawing of the proposed SLMV-mediated paracrine communication system in the mammalian pineal gland (the drawing takes no account of possible autocrine mechanisms). Pinealocytes may release neuroactive amino acids as intercellular messengers from SLMVs which are either concentrated within dilated process terminals (arrows) or are dispersed in the remainder of the cell (dashed arrows). Messenger molecules liberated from pinealocyte process terminals eventually reach their target cells either immediately at sites of close point-to-point intercellular contacts (straight arrows) or via capillary blood flow (curved arrow). The presence of appropriate receptors and transporters for the corresponding

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swellings which constitute likely sites of local exoendocytotic vesicle recycling. The findings discussed in this review have shed new light on the functional significance of the high concentrations of glutamate which have long been known to be present in the pineal gland (see references in Redecker and Veh, 1994). In addition to a variety of metabolic functions of pineal glutamate (Govitrapong and Ebadi, 1988), it appears that a sizable fraction of pineal glutamate is sequestered in SLMVs to serve as a signal molecule for paracrine intercellular communication (Fig. 12). The available data strongly suggest a role of SLMV-derived glutamate as a regulator of melatonin synthesis and release, although additional, unknown functions cannot be ruled out. For example, more work is required to unravel possible specific functions of the protein components of SLMVs and/or of SLMVderived chemical messengers in particular settings, e.g., during pineal ontogenesis. Of note, there is some indication that SLMVs perform functions other than secretion, such as roles in cell membrane repair (Ninomiya et af., 1997). The recent discovery that L-aspartate is coreleased together with glutamate from cultured rat pinealocytes by SLMV-mediated exocytosis (Yatsushiro et al., 1997) raises the interesting question of whether pinealocytes costore both glutamate and aspartate within the same or different SLMVs and whether different populations of pinealocytes exist which selectively store either of the two amino acids. Although L-aspartate is not taken up by the vesicular glutamate transporter, costorage of glutamate and aspartate in the same microvesicles is not remote, taking into account the possibility that L-aspartate may be synthesized from L-glutamate inside SLMVs (Yatsushiro et al., 1997). It also remains to be clarified to what extent the suppressive effects of glutamate and aspartate on adrenergic-driven melatonin release, as demonstrated in rat pinealocytes in vitro, are physiologically relevant in vivo. One important caveat concerning the proposed role of these amino acids as negative regulators of melatonin secretion refers to the observation that pinealocytic responses to a given neurotransmitter may vary considerably across different mammalian species (Olcese and Maronde, 1997). This underlines the need for comparative functional studies of SLMVs in a wider range of animal models than has been accomplished to date. It is possible that such studies will also reveal species-dependent variations of the type of signal molecule(s) stored within SLMVs. For exam-

microvesicular messengers on plasma membranes of pinealocytes and interstitial cells enables efficient paracrine signaling to both principal types of pineal parenchymal cells. Note that morphological and molecular details of this putative communication system are likely to vary among different species.

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ple, although GABA was not taken up by isolated SLMVs of bovine pinealocytes (Moriyama and Yamamoto, 1995a), there is increasing evidence that this neurotransmitter is synthesized and released by pineal parenchymal cells to act as an intrapineal paracrine signal (Rosenstein et al., 1990). Taking into account that the GABA-synthesizing enzyme glutamic acid decarboxylase (Rosenstein et al., 1990;P. Redecker, unpublished observations) and GABA plasma membrane transporters (P. Redecker, unpublished observations) are expressed by pineal parenchymal cells, it cannot be excluded that GABA will evolve as a further candidate molecule for microvesicular storage in pinealocytes. It should also be kept in mind that amino acids may be liberated as neurotransmitters from intrapineal nerve fibers in addition to SLMVs, although corresponding pathways innervating the pineal gland have not been identified (McNulty et al., 1992; Redecker and Veh, 1994). This review has made the point that the proposed SLMV-based communication system in the mammalian pineal gland strongly resembles neuronal intercellular signal transmission. Apart from pinealocytes, which have been the main object of the studies reviewed in this chapter, future investigations should give greater consideration to the second type of pineal parenchymal cells, i.e., interstitial cells. Interstitial cells comprise both macroglial and microglial cell phenotypes (Pedersen et al., 1993; Redecker et al., 1996a; Redecker, 1998). Remarkably, recent observations favor unknown Ca2'mediated functions of astrocyte-like interstitial cells (Redecker et al., 1996a; Redecker, 1998) which perhaps may come into play in the context of SLMVmediated intercellular cross talk. Analogous to neuronal-glial signaling, which is crucial for proper brain function (Kettenmann, 1996) and seems to be of particular importance at the level of the synapse (Smith, 1998), it is feasible that comparable interactions also take place between pinealocytes and interstitial cells. The observation of a compartmentalization of glutamine within pineal interstitial cells (Mc Nulty et al., 1992), akin to the glial partitioning of glutamine in the nervous system which is thought t o be essential for neuronal glutamatergic signaling (for recent in vivo evidence, see Hassel et al., 1997), strongly supports the existence of such a pinealocyte-interstitial cell interplay. Clearly, this promising aspect of SLMV-related research is more amenable to analysis in the pineal gland than in other endocrine organs. I hope this review has shown that the study of SLMVs is attractive for cell biologists with various scientific backgrounds. For example, since analyses of SLMVs obviously converge at the question of how cells shape the profile of hormone release of an endocrine organ, studies along this line of research are at the core of endocrinology. Moreover, the comparison between synaptic vesicles and SLMVs at the molecular level can help identify those mechanisms which are essential and unique for the highly specific regulation of synaptic vesicle trafficking in neurons, a topic which

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is of considerable importance for the neuroscientist. With our growing knowledge of structure and function of SLMVs, the continuing saga of endocrine-neuronal relationships has thus taken an interesting new twist, not the least in the pineal gland. Given the plethora of hormones and nonpeptide as well as peptide neurotransmitters which have the potential to modulate the secretory activity of pinealocytes (Cardinali and Vacas, 1987; Korf, 1996; Maller et al., 1996), a complex multifaceted picture of the fine control of hormone synthesis and release in this photoneuroendocrine organ emerges. As Alice says, “curiouser and curiouser!” (Lewis Carroll, “Alice’s Adventures in Wonderland”).

Acknowledgments I thank R. Jahn (Gottingen) and S. Steinlechner (Hannover) for helpful comments on the first draft of the manuscript and D. Grube (Hannover) for continuous support of my studies. Thanks are also due to the laboratory staff of the department, especially to Ms. H. Peesel and Ms. D. von Mayersbach for help with the micrographs and with the drawing, respectively.

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Invertebrate Integrins: Structure, Function, and Evolution Robert D. Burke Department of Biology, University of Victoria, Victoria, British Columbia, Canada V8W 3N5

lntegrins are a family of molecules that have fundamental roles in cell-cell and cell-matrix adhesion. It is thought that all metazoan cells have one or more integrin receptors on their surface and that these molecules may have been key in the evolution of multicellularity. Knowledge of the structure, function, and distribution of integrin subunits in invertebrate phyla remains incomplete. However, through the recent use of polymerase chain reaction, integrin subunits have been identified in at least five phyla; sponges, cnidarians, nemadodes, arthropods, and echinoderms. The structure of all of the invertebrate subunits is remarkably similar to that of vertebrate integrin subunits. Some experimental data and patterns of expression indicate that invertebrate integrins have a range of functions similar to those of vertebrate integrins. The ligands are not well characterized but at least two laminin-binding receptors have been identified and two other receptors appear to bind using Arg-Gly-Asp motifs. Invertebrate integrins are present during development, in adults, and on a range of cell types including cells with immunological functions such as hemocytes and coelomocytes. Analysis of the invertebrate p subunits indicates that the invertebrate integrins have diverged independently within each phylum. The two major clades of vertebrate integrins (p,, p2,p7 and p3,p5,p6,ps)appear to have radiated since the divergence of the deuterostomes and there are no distinct orthologous subunits in any of the invertebrate phyla. Since fundamental functions of integrins appear to be conserved, studies of invertebrate integrins have the potential of contributing to our understanding of this important group of receptors. KEY WORDS: Integrins, Cell adhesion, Extracellular matrix, Invertebrates, Evolution. 0 1999 Academic Press.

1. Introduction Integrins are a large family of cell surface receptors with diverse functions. They are probably the principal means by which cells attach to the extracelIntrmntro,ral Rrvirn of Cwology, Vol IYI 0074-7696199 $30.00

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M a r matrix and are crucial to many forms of cell-cell adhesion. Integrins are also involved in signaling processes in which attachment activates processes such as cell spreading, migration, proliferation, and differentiation. Integrin-mediated cell adhesion is essential to mammalian immune responses, wound healing, hemostasis, inflammation, metastasis, and many aspects of development. Thus, there has been considerable interest in integrins and many integrin receptors have been well characterized. We are fortunate to have numerous excellent reviews on many aspects of integrin functions (Hynes, 1987, 1992, 1996; Ruoslahti and Pierschbacher, 1987; Albelda and Buck, 1990; Hynes and Lander, 1992; Cheresh and Mecham, 1994; Schwartz et al., 1995; Sheppard, 1996). The secretion of a fibrous matrix of extracellular glycoproteins is a hallmark feature of metazoans and the ability of cells to attach to such a matrix was probably a key aspect of the evolution of multicellularity. Because integrins have been shown to be involved in secretion of the extracellular matrix (ECM) and are essential to cellular attachment to the ECM, the invention of integrins may well have been a key feature in the emergence of metazoans. Given their involvement in fundamental cellular processes associated with multicellularity, it is likely that all living metazoans produce integrins. Despite the rich literature on integrin structure and function in vertebrates, knowledge of integrins in invertebrates is fragmentary. An early indication that integrins were not restricted to vertebrates came from data that indicated the position-specific (PS) antigens of Drosophila to be integrin subunits (Leptin et al., 1987). Marcantonio and Hynes (1988) suggested that integrins were phylogenetically widespread. Using an antibody prepared against peptides representing the cytoplasmic domain of the chicken PI subunit, they demonstrated immunoreactive, heterodimeric complexes in extracts of cells from insects (Drosophifa),nematodes (Caenorhabditis elegans), and a fungus (Candida afbicans).There were also indications that P subunits were to be found in invertebrates in the low-stringency Southern blots of DeSimone and Hynes (1988), which showed hybridization between a chicken PI probe and DNA from sea urchins (Strongyfocentrotuspurpuratus) and flies (Drosophifa melanogaster). Largely due to the use of polymerase chain reaction (PCR) with degenerate primers based on conserved sequences (Pytela et al., 1994), integrin subunits have been identified in five invertebrate phyla. However, it is likely that all metazoan cells express one or more integrins and there is much to be learned about the function and the evolution of integrins from studies in invertebrates. In an effort to bring together what is know about the diversity of integrins throughout metazoa, I will briefly review invertebrate integrins, outlining the structure of the subunits, the putative ligands, patterns of expression, and proposed functions. Next, I will examine how the invertebrate integrins

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relate structurally and functionally with the much better known vertebrate integrins.

II. Invertebrate lntegrins A. Sponges

Brower et al. (1997) describe a p subunit cDNA isolated from the adult sponge, Ophlitospongia tenuis. The subunit, termed pPol(Porifera l), was identified using degenerate PCR primers and subsequent screening of a cDNA library. Northern analysis was used to demonstrate expression. The structure of the subunit is similar to that of all other p subunits, showing highest similarity in the cytoplasmic domain and the ligand-binding region of the extracellular domain. The principal distinguishing features appear to be changes in the position of cysteine residues in the third and fourth repeats of the cysteine-rich stalk. Brower et al. (1997) concluded that the subunit was a divergent member of a Dl class of subunits. In 3 of 10 clones isolated from the library an alternatively spliced form of ppolwas identified. These clones appeared to code for a protein lacking 400 carboxy terminal amino acids. Thus, these forms were missing the stalk, transmembrane, and cytoplasmic domains. A similar truncated form of p3 has been reported (&) from a human erythroleukemia cell library (Djaffar et al., 1994). This form of p3 terminates at a position almost identical to that of the ppoltruncated form and was shown by RT-PCR and immunoblotting to be expressed in several human and mouse cell types. How such a truncated form of human p3 or ppolfunctions has yet to be determined.

B. Cnidarians Brower et al. (1997) also report a novel p subunit isolated from presettlement planulae larvae of the coral Acropora millepora. Degenerate PCR primers were used to amplify a fragment from cDNA, which was subsequently used to screen a cDNA library. Expression of the subunit (pCnl) was confirmed by Northern analysis. As in ppol,the 4 cysteine repeats found in the cysteine-rich domain vary from the vertebrate pattern. All 56 cysteines of this region are present, but the spacing within the inner two repeats is altered. There are no data indicating that this subunit or ppol associate with (Y subunits, but the cytoplasmic sequence WKLLXXXHD, which is essential for (Y subunit association, is present in both subunits (Hughes et al., 1996).

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C. Nematodes

Gettner er al. (1995) characterized a /3 subunit, from the nematode C. elegans. Using degenerate PCR primers a fragment of a novel /3 subunit was amplified, and this fragment was used to screen a genomic and a cDNA library. The gene encoding this /3 subunit was mapped to a region of the left arm of chromosome 111. Mutants that mapped to this region included pat-3 (paralyzed, arrested elongation at two folds). A 9-kb fragment coding for the /3 subunit was able to rescue pat-3 mutants shown to contain nonsense or missense mutations in the /3 subunit gene. Thus, the phenotype resulting from the absence of a functional /3 subunit was determined. The coding region of is similar structurally to other /3 subunits, and the positions of four of its seven introns are identical to introns in human p2, p3,and p7.Also, two of the four introns in Drosophila ppsare identical to introns in Using antibodies to a peptide corresponding to the cytoplasmic domain Gettner et al. (1995) showed that the protein in 109-kDa of nonreduced and 120-kDa reduced. They also demonstrated that the protein is expressed in a temporal and spatially regulated manner throughout embryonic development and in adult worms. Muscle cells express where myofilaments anchor to membrane. There are some nonmuscle cells, such as in the uterus, spermatheca, and coelomocytes, that also express Muscle cells in the pharynx do not express the subunit in the adult, but in the embryo marginal cells of the pharynx express until the three-fold stage. There was some staining of what may be neuronal processes. This complex pattern of expression was consistent with the phenotype of the pat-3 mutant and may be in part explained by association with multiple a subunits. In immunoprecipitates, up to nine putative a subunits were identified, many of which appeared to be temporally regulated. Like other p subunits, appears to form a variety of distinct receptors based on the a subunit with which it associates. There are data on some of the a subunits that appear to associate with the subunit (Baum and Garriga, 1997). Initially, genome sequencing ~ 2 1 el al., 1994). revealed two a subunits termed aF54GX3 and ~ ~ 5 4 (Wilson One of these, ina-1 (formerly F54G8.3) has been shown to be expressed in a subset of the cells expressing Using immunolocalization and a green fluorescent protein fusion construct, Baum and Garriga (1997) showed that at gastrulation, the subunit is expressed by all cells but particularly strongly in the pharynx. In larval stages, overall expression is lower, but expression is high in migrating neuroblasts, distal tip cells of the gonad, uterus, vulva, and male tail. Some nonmigratory neurons of the head and ventral nerve cord express intermittently. Defects in mutants at the locus correspond with this pattern of expression. Defects in neurons

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affect neuron migration and axon fasciculation, but remarkably there are no defects in axon outgrowth. Mutant embryos have a notched head defect that apparently results from failure of attachment of hypodermal cells. Baum and Garriga (1997) also report defects in the pharynx, gonads, uterus, vulva, and male tail. Immunoprecipitates indicate that the a,na.,subunit subunit. forms a receptor with the The Ppat.3subunit, like vertebrate p subunits, has been shown to play a role in the assembly of focal adhesion-like structures. Moulder et al. (1996) found that talin, vinculin, and a-actinin colocalize to the focal adhesionlike structures found in adult body wall muscles. In embryos with mutations that eliminate the function of either Ppat.3or vinculin, they found that talin is abnormally organized in integrin mutants but not in vinculin mutants. This shows that the formation of Ppat.3is critical for the formation of talincontaining focal adhesion-like assemblies in muscle cells, which is consistent with similar roles played by some p subunits in vertebrates.

D. Arthropods: Crustaceans Holmblad et al. (1997) report the structure for a p subunit from the freshwater crayfish, Pacifasticus leniusculus. Using degenerate PCR primers and screening of a hemocyte cDNA library, a complete cDNA sequence was determined. The cDNA encodes an 801-amino acid preprotein that is processed to a 776-amino acid mature protein. Using antibodies prepared to peptides representing the cytoplasmic domain, Holmblad et al. demonstrated that a protein of 100 kDa (nonreduced) or 110 kDa (reduced) was expressed by hemocytes. They speculate that integrins may function as part of the immune response in cell-cell attachment or attachment of opsinized antigens to hemocytes.

E. Arthropods: Insects Of all the invertebrate integrins, those of Drosophila have been best characterized. There are several excellent reviews of Drosophila integrins and ligands (Fessler and Fessler, 1989; Wilcox, 1990; Bunch and Brower, 1993; Hortsch and Goodman, 1991; Brown et al., 1993; Brown, 1993; Gotwals et al., 1994b). There are two known P subunits and three a subunits, and because Drosophila has wonderfully tractable genetics, much is also known about how these subunits are produced and how they function. The PS integrins in Drosophila have been most fully characterized. The PS antigens were originally identified in a screen of monoclonal antibodies to cell surface antigens that had differential patterns of expression in imagi-

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nal discs (Wilcox et al., 1981). There are now three PS integrins known: three a subunits (aps1, aps2,and apsj),each of which combines with the same p subunit (ppsl).As in many of the vertebrate integrins, differential splicing of a and p subunits has been demosntrated, providing an additional level of structural and functional complexity (Brown et al., 1989; Zavortink et al., 1993; Zusman et al., 1993). The large number of putative a subunits that was found immunologically in the analysis of the Drosophila PS integrins (Wilcox et al., 1984; Brower et al., 1984; Wilcox and Leptin, 1985) appears to be due to proteolysis (Bogaert et al., 1987) and to differential splicing (Brown et al., 1989). Thus, there appear to be at least four PS genes, but at least eight heterodimeric receptors are derived from them (Gotwals et al., 1994b). The ppsl subunit was identified as the gene defective in the lethal nzyospheroid (mys) mutation by cloning of a cDNA identified with probes from chromosomal DNA adjacent to a transposable element insertion site (MacKrell et al., 1988). The ppslsubunit is expressed before the formation of the blastoderm and peaks in expression at the extended germ-band stage (Leptin et al., 1989). The subunit localizes to the basal cell surfaces of both ectodermal and mesodermal cells where they appose. Later, the ppslsubunit is expressed on the basal surface of the gut epithelium, the apical surface of the midgut epithelium, and between muscle cells at attachment sites (Leptin et al., 1989). In pupae the ppsl subunit is also expressed in wing discs (Brower et al., 1984; Fristrom et al., 1993) and in the eye disc (Brower et al., 1984). In lethal myospheroid mutants, there is no expression of ppsl (Leptin et al., 1989), and there are severe defects in muscle attachment, germ-band retraction, and failure of the dorsal epidermis to close (Wright, 1960). Using mosaics and heat-inducible rescue, Zusman et al. (1990, 1993) showed that ppslis required during metamorphosis of the wing and eye discs. In the wing, ppslappears to function in maintaining the dorsal and ventral epithelial attachment and blisters form when integrin function is interferred with (Brower and Jaffe, 1989). In the development of the eye disc, pPsl is required in the latter half of pupation, apparently for the attachment of pigment cells to an underlying basal lamina (Zusman et al., 1993). The proposed functions for ppslintegrins indicate that it is involved in mechanical adhesion of cells or epithelia to extracellular matrices. The cytoplasmic domain is thought to be critical to the function of ppsl subunits. Grinblat et al. (1994) analyzed the cytoplasmic domain of pPsl using mutant rescue techniques. Like the results of in vitro studies of mammalian cells (Reszka et al., 1992), the cytoplasmic tail appears to be essential to PS integrin function. Substitutions that would prevent phosphorylation of tyrosine residues have no effect on function, nor does elimination of an intron containing a sequence that is alternatively spliced in vertebrate /3 subunits. Alteration to a charged, a-helical domain adjacent

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to the membrane spanning domain is important but nonessential, and the extent of rescue suggests some modulation of the cytoplasmic interactions during development. The PS1 integrin, originally termed the PS1 antigen, consists of the apsl subunit associated with the pPslsubunit. Similarly, the aps21/3ps1 subunits in association are known as the PS2 integrin. These receptors are expressed after cellularization and have complementary patterns (Bogaert et al., 1987; Wehrli et al., 1993). The PS2 integrin is expressed in mesoderm and the PS1 integrin is expressed primarily in the ectoderm and the endoderm. As somatic muscles form and attach to the epidermis, the integrins concentrate at attachment sites-PS1 on the ectodermal cells and PS2 on the muscle cells (Leptin et al., 1989). Similarly, in the gut, the visceral muscles express PS2, whereas the endodermal cells express PS1. In larval tissues the PS integrins are broadly expressed. In wing discs, where PS integrins have been analyzed in detail, PS1 is expressed primarily in dorsal cells and PS2 in ventral cells (Wilcox et al., 1981; Brower et al., 1984). Mutants for both PS1 and PS2 integrins have been found and characterized (Brown, 1994; Brower et al., 1995). The multiple edematous wings (mew) gene encodes the apsIsubunit and the inflated (if) locus encodes the aPs2 subunit. The defects associated with null mutations are complementary and correspond with the patterns of expression (Brower et al., 1995; Brabant and Brower, 1993; Bloor and Brown, 1998; Brown, 1994) Mutant mew embryos have defects in the gut, eyes, and dorsal wing epidermis, whereas if mutants have defects in somatic muscles, dorsal closure, and ventral wing epidermis (Brower et al., 1995). All these defects appear to prevent strong mechanical attachment of cells and tissues to the ECM. However, the PS integrins are widely expressed in embryos and larvae and it is possible that additional functions will be revealed by analysis of the phenotypes of new mutants (Bloor and Brown, 1998). The ligands for these integrins are beginning to be identified. It is clear from in vitro experiments that PS1 and PS2 bind different ligands (Gotswal et al., 1994b). Bunch and Brower (1992) showed that cells expressing PS2 bind through a Arg-Gly- Asp (RGD) sequence; however, RGD-containing vertebrate substrates, such as vitronectin and fibronectin, are not known to be present in Drosophila. Tiggrin is a novel ECM component containing an R G D sequence that has been shown to support attachment of PS2producing cells (Gotwals et al., 1994a; Fogerty et al., 1994). Recent analysis of tiggrin mutants indicates that not all PS2 mutant defects are found in embryos lacking tiggrin or having tiggrin in which the RGD sequence has been mutated (Bunch etal., 1998). This suggests additional RGD-containing ligands remain to be found. Recently, a third PS a subunit, termed aps3,was characterized. Using peptide sequences from tryptic digests of a 90-kDa band that coprecipitates

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with anti-ppsl,Stark et al. (1997) were able to clone aPS3. From protein studies, the subunit appears to be posttranslationally cleaved in the extracellular domain and there are indications that it is a laminin receptor. However, in structural comparisons the sequence does not readily cluster with other cleaved laminin receptors such as a6,a7,and aj.The subunit was localized immunocytochemically in embryos to diverse tissues, all of which were undergoing morphogenesis. These included trachea, midgut, dorsal vessel, salivary glands, and ventral nerve cord. The previously described scab (scb) mutation appears to encode aPS3. Defects in scb mutant embryos correspond expression. These defects include incomplete dorsal closure, to sites of aPs3 abnormal salivary glands, mislocation of pericardial cells, and interrupted trachea. Several of the defects are similar to those found in mutants with defective laminin A chain. Laminin is associated with most of the sites of aPS3 expression, suggesting that it may be a ligand for the PS3 integrin. The same a! subunit was identified in volado (vol) mutants (Grotewiel et al., 1998). The locus was identified using enhancer detector lines in which expression was enhanced in mushroom bodies of adult flies. A long mRNA is expressed selectively in heads and a shorter mRNA is expressed throughout the body. Both encode a! subunits that differ only in the first 63 amino acids. Remarkably, vol mutations appear to affect olfactory memory. Using aversive olfactory conditioning, mutants lacking either form of the subunit exhibit memory deficits within 3 min of training. Using heat shock rescue, it was shown that when wild-type vol was expressed 3 h before testing, flies recovered wild-type memory levels. This indicates that the defect in vol mutants is associated with memory and not some aspect of neuronal develmay mediate opment. These studies suggest that integrins containing aPs3 synaptic plasticity and behavior associated with short-term memory. Using PCR with degenerate primer sequences, Yee and Hynes (1993) reported a p subunit they termed pv (Pneu).The sequence deduced from cDNA indicates that the subunit is lacking 4 cysteines (14,15,47, and 48) of the 56 cysteines conserved in the extracellular domains of most p subunits. Expression of pvappears to be restricted to the midgut throughout embryonic and larval development. Mutations in this subunit have yet to be identified and we lack details on the a! subunits with which it may associate. F. Echinoderms: Echinoids

The use of PCR with degenerate primers has permitted the cloning of several integrin subunits from sea urchins. There are three p subunits for which there are complete sequences, and there are partial sequences for two a! subunits (Marsden and Burke, 1997, 1998, unpublished data). The two p subunits that have been best characterized are pc and pL(Marsden

INVERTEBRATE INTEGRINS

265

and Burke, 1997,1998). These subunits have all the characteristic structural features of this family of proteins: however, the positioning of the cysteines within the cysteine-rich stem vary from the canonical pattern of the vertebrate integrins. Both pL and pc are expressed during early development. In blastulae, mRNA and protein localize to the blastodermal cells, but pLprotein appears to localize to basolateral domains and PG to apical domains. During formation of primary mesenchyme, PG is produced by ingressing cells, whereas pLappears to be downregulated. The distribution of pLchanges during the initial phase of gastrulation, resulting in accumulations in the narrow apices of bottle cells (Marsden and Burke, 1998). Bottle cells form a ring within the vegetal plate during the initial stage of gastrulation and have dense arrays of filamentous actin in their apices (Nakajima and Burke, 1996). During gastrulation PG and pL are expressed, but pLis restricted to the presumptive mesoderm cells at the tip and pG is expressed by endoderm. As the mesenchyme begins to form the skeleton, both /3 subunits appear to be expressed. In larvae, pLis expressed by mesenchyme and the aboral ectoderm and pG is expressed by mesenchyme and throughout the gut (Marsden and Burke, 1997.1998). Thus, there are complex and overlapping patterns of expression of these two p subunits during early development. Both PG and pL are expressed in adult coelomocytes, a heterogeneous population of motile phagocytic cells found within the coeloms of adult echinoderms (J. Henson and R. Burke, unpublished data). These cells attach to a range of ECM substrates and are most motile on collagen, fibronectin, and laminin. The PG subunit is expressed on the surface of the motile coelomocytes, suggesting it may function on the surface of cells with immunologic functions. The ligands for the sea urchin integrins are not known. However, Marsden and Burke (1998) report that dissociated cells from embryos will attach to pronectin F, an engineered protein that has multiple R G D sequences. Cell attachment and spreading can be inhibited with R G D peptides and antibodies against the pL subunit. This suggests that, like other integrins, pLforms receptors that recognize RGD. Antibodies against pL also interfere with developmental processes and have been used to examine the functions of PL-containing integrins in embryos. The antibodies appear to block the initial stage of gastrulation and interfere with the accumulation of actin in the bottle cells of the vegetal plate (Marsden and Burke, 1998). Nakajima and Burke (1996) proposed that the formation of the bottle cells was brought about by active contraction of the apices by filamentous actin and that this change in cellular shape resulted in the inward buckling of the vegetal plate. The antibodies against the pLsubunit had at least two other effects on embryos. Antibody treatment caused the cells of the blastoderm to elongate and lose their basolat-

266

ROBERT D. BURKE

era1 arrays of F-actin. Also, treating embryos with the antibodies resulted in abnormal shapes and positions of the larval skeleton. The pc subunit is expressed on the surface of unfertilized eggs of the sea urchin S. purpuratus (G. Murray, N. Lake, and R. Burke, unpublished data). Confocal immunofluorescence and immunogold preparations indicate that the subunit is associated with the tips of microvilli. Within a few seconds of fertilization, pc appears to be removed from the egg surface. These observations suggest that the subunit may form part of a receptor that functions in some aspect of the fertilization process. Because the 4 1 integrin has been implicated in binding sperm to the surface of the egg in mice (Almeida et al., 1995), there is precedence for integrins functioning during fertilization. Our knowledge of a subunits in sea urchins remains fragmentary. There have been at least three a subunits identified using PCR with degenerate primers (Susan and Lennarz, 1993; P. Hertzler and D. McClay, personal communication; C. Reed and R. Burke, unpublished data). Hertzler and McClay characterized asu2, which appears to be a subunit expressed throughout early development on the basolateral domains of epithelial cells. Structurally, asu2is most similar to the a5 subgroup of vertebrate a subunits. Expression in adhesion-deficient Chinese hamster ovary cells conferred adhesion to basal laminae and laminin but not to fibronectin or collagen I or IV, suggesting asu2 may function in attaching epithelia to basal laminae.

111. Evolution of lntegrins Hughes (1992) analyzed the vertebrate a and p genes to determine the patterns of evolution of the two subunits. The subunits appear to form two evolutionarily unrelated families, but their function as heterodimeric receptors imposes coevolutionary constraints making their evolutionary history worthy of examination. Using comparisons of the number of nonsynonymous nucleotide substitutions per site, the p subunits appear not to be monophyletic within the vertebrates. Hughes found that the PI and p2 subunits form one clade and the p3,ps,and 0 6 subunits another. The three regions of p subunits that are most highly conserved appear to be the ligand-binding region, an asparagine-rich region within the stem region, and the transmembrane region. Within the stem there appears to be a region in which there is a significant bias toward amino acid replacements that lead to a change in charge yet a tendency to conserve polarity. These changes in charged amino acids occurred at a rate significantly greater than expected for random substitution, leading Hughes (1992) to conclude that

INVERTEBRATE INTEGRINS

267

this represents evidence of directional positive selection leading to adaptive radiation. Brower et al. (1997) provided a phylogenetic analysis of some of the invertebrate p subunit sequences. They concluded that the integrin sequences do not permit the reconstruction of a rigorous phylogeny, and that the sequences do not support an early conserved divergence of p subunits. There are indications from their analysis that the diversification of p subunits within the vertebrates is a relatively late evolutionary event. However, they proposed a and class of subunits, including pl, ppSl,Ppat.3, ppol.They characterize the class as /3 subunits that associate with numerous a subunits and have an involvement in morphogenetic processes. The sponge (pcnl)and coral (ppol)subunits are the most divergent of this proposed class. Brower et al. (1997) suggested that because associates with a large number of a subunits, it is more constrained than the invertebrate p subunits. Thus, Brower et al. suggest that the stalk structure of the vertebrate represents the ancestral form and that sponge and coral p subunits have diverged independently. Using a larger data set I have undertaken a similar analysis in which the sequences were first aligned using the MAP alignment (Huang, 1994) (Fig. 1) and then analyzed using PAUP tree-making software. These analyses indicate that the vertebrate p sequences are polyphyletic and form at least two major groups. One group includes p8, p6, p3, and pS and the other includes p7,p2, and PI. The p4sequences are derived to the extent that they are not included with the other vertebrate sequences and may represent a third clade. These are consistent with the two major groups identified by Hughes (1992) using the smaller data set available at that time. Within the two major vertebrate groups there is strong support for conventional vertebrate phylogenies. For example, in the p6 groupings amphibian p6 is distinct from the amniote p6 and avian 0 6 from mammalian p6. There is also support for the diversification of the vertebrate p subunits after the origin of the deuterostomes since the three sea urchin p subunits form an independent cluster. The relationships of the other invertebrate p subunits are weaker and do not follow conventional phylogenetic analyses. For is more closely instance, as Brower et al. (1997) found, the C. elegans allied with Drosophila ppsl than is the other arthropod j3 subunit from crayfish. Similarly, the sponge ppol and Drosophila Pneuweakly cluster together independently of the arthropod and coral p subunits. These associations may be significant but are not strong because of the depth of the phylogenies. However, it is also possible that the subunits, although similar, are not truly orthologous proteins. Without knowing all the p subunits of each of these groups, it is impossible to determine if the comparisons made are reasonable. The relative similarities of all the invertebrate sequences are the same as those determined by Brower et al. (1997).

268

ROBERT D. BURKE

The known subunits are remarkably similar in several aspects of their structure. This is particularly true with respect to their putative ligandbinding domain (LBD) and the cytoplasmic domains. Using the LBD in tree-building programs results in trees with similar topology to those resulting from complete sequences (Fig. 2b). However, the separation of the two vertebrate clades is weaker and the coral LBD appears to be more similar to that of the vertebrate /3 subunits. Also, the association of the arthropod and nematode subunits is weaker in this comparison. In analyses using the cytoplasmic domain alone (Fig. 2d), the vertebrate subunits cluster in paralogous groups: p2clusters with p7 and p6,p3,and ps cluster together. The cytoplasmic domains forms a distinct cluster, but the Drosophilu ppsland C. eleguns appear more similar to p1forms than to any of the others. There appears to be a strong clustering of sponge and vertebrate p8 subunits. Most of the clusters formed between the vertebrate and invertebrate cytoplasmic domains are weak and do not follow established phylogenies, but they suggest there may be some functional groupings, such as the weak grouping of the sponge, coral, and crayfish with vertebrate px, p2, and p7. The shortness of the cytoplasmic domain and small number of informative characters causes trees built with it alone to lack resolution. Hughes (1992) identified a region of the extracellular domain that his analysis showed to be highly variable. In trees based on this region (Fig. 2c) the vertebrate subunits, other than p8, remain clustered, but the class differences are less clear. Although some of the invertebrate subunits cluster as they did in analysis of the full sequence (such as the urchin subunits), there is insufficient resolution to make reliable branchings of almost all the invertebrate sequences. Trees based on this region support the contention that there is greater variability in this region because most of the branches are weaker than those seen with the full-length sequences. A difference between the invertebrate p subunits and the vertebrate p subunits appears to be in the spacing of the 56 cysteine residues in the

FIG. 1 Alignment of p integrin subunits. Sequences in FASTA format were aligned using the MAP multiple alignment tool (Huang, 1994). The first 45 residues were adjusted by eye. The conserved 56 cysteines of the extracellular domain are double underlined and numbered consecutively. The following are the accession numbers for the sequences: mouse p2,3183523; mouse &, 400075; mouse PI, 124964; mouse PSA,309510; mouse p3, 2736194; chicken PI, 124965; chicken p2. 422686; Xenopus fit, 124965; Xenopiis P3, 467812; human 0,. ~26010; human Ps. ~ 1 8 0 8 4human ; pH.184521; human p2,825636; human p3,420082; human &, ~18564; U19744; Drosophila melanognster coral 0, AF005356; sponge p, AF005357; C. elegans pPs(myospheroid). 503251; D . rnelanogaster p,,, L1330S; crayfish p, X98852; urchin (Strongylocentrotus purpuratus), Pc;,U77584; urchin (S. purpuratus) pL,AF078802; urchin (Lyrechiruts variegatus) pc, 3089557.

1

2 3

4

5 6

7 8 9

10 11

12

13 14

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DrosophilaBneu Spon eB1 hum% MPSA coralBl - - - ....MRHRGILV crayfish celegansBpat-3 MPPSTSLLLLAALLP DrosophilaBrnyo RREQLRLLMIAM= urchinBc lv _..... MRLHRRQEL urchjnBGurchinBL hum86 XENOPUS chicka3 hum3 MOUSE MOUSE hum87 MOUSE87 hum82 MOUSEB2 chickB2 XENO hum1 MOUSEBl chickBl

16 30 GGRAFLWIYLVFLIA GLQWSILGSLLVGW RGPASFLWAAWVFSL ---MKRRLCLLVSIF WAWSVWJIN-- W G FALPASDWKTGEVTG IAAQTNAQILWXLTA LLQWIFSVFIISLHV WK LLVLTIAFSAGQ PG4LvVFFLT-v

46

2

3

4 5 613 6 1

75 76 KEYQ-_ _ -.-

6

90

QEDFISGG--APMF----DEK AW~QPDF------AWCAQPDFQDAEN-GWPPLLPSYTDDTG--

72 69 92 63 76 86

76 79 72 78

68

70

66

_ _ _ _ WATVLALGALA

63 72 73 89 89 71 72 73

PGQLWAALLALGAIA PATLYA LLGL SEmAPSTGE; QSELDKITSSGEAAE L-ALVGLLSLG L I L-GLFFLGgV TWVL---LLLTTAPPVFTVGLLT---LV P I NI G L I s s vVF LVSWIGLISLI ~;

74

73 73 77

LLTWAGIL---

105 106 120 DrosophilaBneu VLQDKPLKDYETSW Spon eB1 DTVDPPLQ------hum% IPTENEIN------coralBl IQQDSNVGAK----crayfish ITQDTPLTVAGhTNR celegangBpat-3 ITEDSKLSDQGQVEDrosophilaBrnyo ILVNNKLTNQYKAEL urchinBc lv ITQKTPLSKAG-A-urch+BGVLGWISLSNAGSA-urchinBL G--NITNPLPSAV PIEDKPLSEA-NAhum86 QLNFIENPVSQVE ILK"LSV-GRQ-XENOPUS VAENRPLSVKGSE-chickB3 VLEERPLSDKGS--huma3 PESIEFPVSEAR VLEDRPLSDKGS--MOUSE NCAPESIEFPVSEAQ ILEARPLSSKGS- MOUSE G&EGE - IESPASSTH VLRNLPLSSKGSS-humB7 PLEELEEPRWE VLQEQPLSwARG-VLQDKPLSWDRG-MOUSEB7 ETQEDHNGGQK---humB2 MOUSEBZ NPEFDQRGQRK---chickB2 RTQDSAFSNKI---XENO KLKDIPITSKGKG-hum81 1KKNK"RSKG-MOUSEBl IKKNK"RSKG-chicks1 VLEDREmKIG- -

180 ---VQVTPQRnYLKL -DLTQVSPSRVSINV ---TQVTPGNSIQL ---VQVQPKKIRLNL DKIVQLKPQRMKLKL EEAVQIKPQEMYVEI GEIVQIQPQSMRLAL DNLVQVYPQEIDLTL GQAVQVRPQQVDLKL DAIVQVKPQMMRIKV -DIVQIAPQSLILKL -EITQMSPQKIDLFL --TTQMSPQRIQLNL -QITQVSPQRIVLRL -QVTQVSPQRIALRL 166

I

-DVIQMTPQEIAVSL EGATQLAPQRIRVTL EGATQLAPQRVRVTL

_ _ _ _ QLSPQKVTLYL

__ __ __ __QLSPQKVTLYL QLTPQEVHLKL

ANITQLRPQQMVFEL EDIHQIQPQQLVLRL EDITQIQPQQLLLKL EAITQIQPQKLVLQL

FIG. 1

115 106

127 98 127 132 165

123

114 120

113 116 110

108 117 118 132 132 107 108 110 122 123 123 127

1

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

R

celegangBpat-3 DrosophilaBmyo urchinBc Iv urch+BGurchinBL hum86 XENOPUS chickB3 hum83 MOUSE MOUSE hum87 MOUSEB7 hum82 MOUSEB2 chickB2 XENO hum81 MOUSEBl chickBl

241 255 FGSFADKPTLPMI- FGSFVEKVVAPFTTL FGSYVDKTVSPYISI FGSFVDKAVSPFVRFGSFVDKVLMPYADFGSFIDKKLMPFIDFGSFVDKVLMPWSYGAFIDKTVMPYIDFGSFVDKTVMPWSFGSFVDKTVLPWSFGSFVEKPVSPFVKFGAFVDKPMSPYMFM FGAFVDKPISPYMYI FGAFVDKPVSPYMYI FGAFVDKPVSPYMYI FGSFVDWISPFSYFGSFVDKTVLPFVSFGSFVDKTVLPFVSFGSFVDKTVLPFVNFGSFVDKTVLPFVNFGSFVDKTVLPFVNFGSFVEKTVMPYISFGSFVEKTVMPYISFGSFVEKTVMPYISFGSFVEKTVMPYIS-

DrosophilaBneu Spon eB1 humB% corals1 crayfish celegansBpat-3 DrosophilaBmyo urchinBc lv urchlnBGurchinBL hum86 XENOPUS chickB3 hum83 MOUSE MOUSE hum87 MOUSEB7 hum82 MOUSEB2 chic-2 XENO hum81 MOUSEBl chickB1

MKLAGDGLLAGI~QR FHFAGDGKLGGLILP SHLALDSKLAGIWP FHIAGDGKLGGIVTP FHHAGDGRLAGIVAP FHFAGDGRLAGVVEP FHYAGDGKLGGVIAP FHIAGDGKLGGIVKP -SIMPETKLGGIITP FHFAGDGRLGGIVEP SHFGMDSKLAGIVIP THIALDGRLAGIVQP THIALDGRLAGIVQP THIALDGRLAGIVQP THIALDGRLAGIVLP PHIALDGKLGGLVQP FHTAGDGKLGGIFMP ---VSWVFTSDDTFHTAGDGKLGGIFMP ---VTRLLVFATDDGFHFAGDGKLGAILTP ---VTRLLVFATDDGFHFAGDGKLGAILTP ---VTRLLVYATDDGFHFAGDGKLGGILTP ---VTRLLVFSTDAGFHFAGDGKLGGIVLP ---VTRLLVFSTDAGFHFAGDGKLGGIVLP ---VTRLLVFSTDAGFHFAGDGKLGGIVLP ---VTRLLVFSTDAGFHFAGDGKLGGIVLP

DrosophilaBneu Spon eB1 hum% corals1

crayfish

N

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201 195 216 183 213 219 251 208 199 205 202 204 198 196 205 207 220 220 195 196 198 209 210 210 214 360

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FIG. 1 (continued)

~~~~~~

288 282 302 270 298 304 336 296 284 291 288 292 286 284 293 295 306 306 281 282 283 295 296 296 ~~

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DrosophilaBneu Spon eB1 hum% corals1 crayfish celegansBpat-3 DrosophilaBmyo urchinBc lv urchinBGurchinBL humB6 XENOPUS chickB3 humB3 MOUSE MOUSE humB7 MOUSEB7 humB2 MOUSEBZ chickB2 XENO humBl MOUSEB1 chickBl

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DrosophilaBneu Spon eB1 humB% coralBl crayfish celegansBpat-3 DrosophilaBmyo urchinBc lv urchjnBGurchinBL humB6 XENOPUS chickB3 humB3 MOUSE MOUSE humB7 MOUSEB7 humB2 MOUSEB2 chicks2 XENO humBl MOUSEBl chickBl

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406 420 VIFAV------TEEV PIFAA------QRDA VIFAV------QGKQ PIFAV------TKQF VIFAV------SSHE VIFAV------TKNN IIFAV------TASQ PIFAIGKAGVDKQDP PIFAV------1QKE PIFAV-- - ---TRDQ LIFAV------TQEQ LVFAV------TNEV LIFAV------TDTV LIFAV------TENV LIFAV------TENV LIFAV------TKNH PIFAV------TSAA PIFAV------TGAT PIFAV------TSRM PIFAV------TKKM PIFAV------TSKM TIFAV------TEDF TIFAV------TEEF TIFAV------TEEF TIFAV------TEEF

375 376 390 KAGE-YTGSLNYDYP

EANE-YTASNQMDYP SNGL-YSRSTEFDYP SNGV-YTNSAEFDYP DN-L-YKRSNEFDYP DN-M-YKRSNEFDYP DN-M-YKKSNEFDYP EN-M-YTMSHYYDYP NN-M-YTMSHYYDYP NN-v-YTMSHYYDYP NN-M-YTMSHYYDYP

363 365 375 346 371 380 410 377 359 366 362 366 360 358 367 369 380 380 354 355 356 368 369 369 373

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FIG. 1

(continued)

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DrosophilaBneu Spon eB1 humB% corals1 crayfish celegansBpat-3 DrosophilaBmyo urchinBc lv urch+nBGurchinBL humB6 XENOPUS chickB3 humB3 MOUSE MOUSE humB7 MOUSEB7 humB2 MOUSEB2 chickB2 XENO humBl MOUSEBl chickBl

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DrosophilaBneu Spon eB1 humB% coralBl crayfish celegansBpat-3 DrosophilaBmyo urchinBc lv urch+nBGurchinBL humB6 XENOPUS chickB3 hum3 MOUSE MOUSE humB7 MOUSEB7 humB2 MOUSEB2 chickB2 XENO humB1 MOUSEBl chickBl

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5R5

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IKGYLYCGMCEC~JYEG KTEQD------KCK~ NSESD------TLLC DV---PEPNSSKCSF- H G T F T C G A m G ‘SG GNGTLECGSCICNPG ‘Ss ‘HH

R E E

ND---Q-PNSTL~ QA---E-ANSSF~SK QA---E-PNSHRW FA---Q-PSSPRmGNGTFEEGVCRmG GL---E-PNSARCS-GNGTYTCGLCECDPG -T---Q-PQAF’HCSD GQGHLQCGVCSCAPG -A---Q-PHAPYCSD GNGMGICS EG---E-PNSPAcHDGNGTFEEGGRFG 7

38

691 705 DGDTDA GPTGEIE -Pvsi$PmGDLFS------GWEGDRC

603 594 594

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HP------GFEGSA KP------GYEGSA TP-- - - - -KYEGSA FP------NYSGSA NP-- - - - -NYTGSA YP------NYTGSA FP------NFTGSA

FIG. 1 (continued)

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KEPT---CS-GRCQE IEPD---m-GACEI DCPT---CS-GESR

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a

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766 780 IDPEKD---NKLCLF

DrosophilaBneu Spon eB1 DPTNRTSI NG--RG humB% Ns -K-GQ$sG- -RG coralB1 QDAK-SE -GGADRG crayfish celegansBpat-3 DrosophilaBmyo PPGG-GEICSG--HG urchinBc lv AP-N-GEImG--VG urchinBGurchinBL hum6 XENOPUS chickB3 h u m3 MOUSE MOUSE humB7 MOUSEB7 humB2 NP-R-RVECSG--RG MOUSEB2 NA-R-LVE SG--RG chickB2 NS-R-QNE SL--RG XENO AK-N-GQI G--RG humB1 AS-N-GQI G--RG MOUSEBl AS-N-GQI G--RG chickBl AG-N-GQImG--RG 811 825 826 DrosophilaBneu Spon eB1 NPNLPEGSMVSFDSE hum% --HPHNLSQAILD coralBl crayfish celegansBpat-3 DrosophilaBmyo urchinBc lv --AFHTGAFNKSQ-E urchinBG--AFGTG-LSKAD-E urchinBL --VFGTGRLTPEQhumB6 --LSAAGQAGEE-XENOPUS --KFERGPWFEDSS chickB3 - - KYERGTLVEQQS humB3 --KFDRGALHDENT MOUSE --KFNRGTLHEENT MOUSE --LLHQGK-PDNQ humB7 --AFRTGPL--Am MOUSEB7 --AFGTGPL--AANC humB2 --KFEKGPF--GIWC MOUSEB2 chickB2 XENO --AFQKGEK--QD humB1 MOUSEBl chickBl

I

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855

- - - - -. - - - QKQGMG

DAGAVIINGTERAD

55

871 885 E----TYPYNFVHEL DG-CJYRYFV----GI

DG TYYFTTE-----TEADSKNFVLWQKD EENVLEVQIVDDIKK --INSNKIDSLDGQV EGiiLmF-YIDSA SDTKPQQYQIFVQKD --FKVIPVE----EL -DEATDNATWWRKH --TQFVPVGVEKVEI -SEQGE-LHVYAQEN PIYVVTELRK----ILP-NGTNINQEE PVIMVDNL-E----KSA-NLALILWQKE NIINVTSIDE------YTEDNPKmFPLS-SE-NETVTVYVEGR AGATISEEEDFSKDG SV-----SSLQGET-DNEGKTIIHSINE EIES-VTELVDNGKN AV----- TYKDEE-DASGKSVLWINE EIET-VQELGDRGKD AV-----NTYKDEE-DSSGKSILWIEE AV-- -. TYKNE-EIES-VKELKDTGKD AV-----N E- DSSGKSILYVVEE DIEQ-VKELTDTGKN E-DTSGRAVLYVVEE AV-----LFYKTAE-LPNGRSNLTVLRE ILDDGW-- KERTL-DDARGTVJLRV--R VNVTLTLA-- - - - -P NLDDGW-- KERTI- DNQLFFFLVE------H?ASG-IVLRV--R LQLSNNPVKG-RT--- - - - - - - - CKERDS - EGCWVAYTLE-----QQDGMDRYLIYVDES MTLQTIPLKK-ICE-- - .- - - - - - CKERDS - EGmITYTLQ----QKDGRNIYNIHVEDS ENmISFYMA-----QDDGEEMYTCTTVDPK IQLADEPRAGSRQ-ED WNFTYS-----V-DSKNEVMVHWKE FNISLVDSREELPQP FNITKVESRDKLPQP DD WNFTYS-----V-NGNNEVMVHWEN DD WFYFTYS-----V-NGNNEAIVHVVET FNLTKVESREKLPQP GD WFYFTYS-----V-NSNGEASVHVVET FNMTRVESRGKLPQP

gm&IVXDDQ;

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FIG. 1 (conrinued)

692 757 664 711 722 729 768 728 706 717 699 708 702 700 709 711 715 714 692 6 94 694 720 720 720 725

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2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

901 915 916 930 931 945 946 960 DrosophilaBneu GI DSFFAYQVIDH SNFLTIQAVDCEPPD YVALVGYISAFTLLI GLLIIFIILWYIRAK Sponges1 d&DQQGGTAPW- - - - - - - - - - - - - - - --IIAISIIIpLIVL GLLLLLLLKGLLLLW VLPIVLGWGGILFL GLLILIVIKGLFTMV VLGIVFGLVAAIVAI GLLTLLIKLTTLH VLAIVLGVIAGIVIL GILLLLLWKLLTVLH MLGIVMGVIAAIVLV GLAILLLWKLLTTIH GLRLLLVWRLEVWa MLWVILGIIIGIILV GLKLLIWRLLTYIH IMHVIIGIWGIIIV IRWIVIGIILGIVLI GMIWWRLYTYVQ IPMIMLGVSLATLLI GVVLL IWKLLVSFH ILVVLMSVMGAILLI GLVALEIWKLLITIH ILVVLLSVMGAILLI GLRALLIWKLLITIH ILVVLLSVMGAILLI GLRALLIWKLLITIH ILVVLLSVMGAILLI GLATLLIWKLLITIH AMTILLAWGSILLI GMALLAIWKLLVTIH GLGLVLAYRLSVEIY GLGLVLAYFSSVEIY GILLLVIWKALIHLS VAAIVGGTVVGVVLI GVLLLVIWKALTHLT IALIVGSTIAGVALI GLLLLLTWRLLTEIF IIPIVAGVVAGIVLI GLALLLIWKLLMIIH IIPIVAGVVAGIVLI GLALLLIWKLLMIIH IIPIVAGVVAGIVLI GLALLLIWKLLMIIH IIPIVAGWAGIVLI GLALLLIWKLLMI IH

961 975 DAREYAKFEEDQKNS D-KFEREImA DRIEYQKFERERMHS DRREYAKFEKERKLP DRSEYATFNNERLMA DRREFARFEKERMNA DRREFQNFEKERANA DSREFASFEKERAGT DKREYAQWEND KKA DR-AKF-i%m DRREFAKFEEERAKR DRREFARFEEEKARA DRKEFAKFEEERARA DRKEFAKFEEERARA DRREFAKFQSERSRA DRREYSRFEKEQQQL DRREYRRFEKEQQQL DLREYRRFEKEKLKS DLREYRRFEKEKLKS DRREYRRFEKEKSKA DRREFAKFEKEKMNA DRREFAKFEKEKMNA DRREFAKFEKEKMNA DRREFAKFEKEKMNA

976 990 V--RQENPIYRDPVG KYTmNPLYRSATK KWTREKNPLYQRRKT SGKRAENPLYKSAKT KWDTNENPIYKQATT KWDTGENPIYKQATS TWEGGENPIYKPSTS HWGQNENPIYKPSTS QWDQSDNPIYKSSTT KWQTGTNPLmGSTS KWDTAHNPLYKGATS KWDTGNNPLYKEATS KWDTANNPLYKEATS KWDTANNPLYKEATS RYEMASNPLYRKPIS NWKQDSNPLYKSAIT NWKQDNNPLYKSAIT QWNN-DNPLFKSATT QWNN-DNPLFKSATT KWNEADNPLFKSATT KWDTGENPIYKSAVT KWDTGENPIYKSAVT KWDTGENPIYKSAVT KWDTGENPIYKSAVT

780

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664

780 790 797 836 796 774 793 767 776 770 768 777 779 783 782 759 761 762 788 788 788 793

838 737 792 801 809 846 806 783 802 788 788 781 778 787 799 798 806 769 770 772 798 798 798 803

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FIG. 1 (continued)

275

INVERTEBRATE INTEGRINS 1 2 3

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1111 799 DrosophilaBneu _ _ _ _ _ _ _ _ __ _ _ __ _ _ __ _ _ __ _ _ __ _ _ 838. _ Spon eB1 769 humB% ETRAVTYRREKPEEI KMDISKLNAHETFRE NF 792 coralBl 801 c rayf i 9h 809 celegansBpat-3 846 DrosophilaBmyo 806 urchinBc lv 783 urchlnBG802 urchinBL 788 hum6 788 XENOPUS 781 chickB3 778 hum3 787 MOUSE 799 MOUSE 798 hum7 806 MOUSEB7 769 hum2 770 MOUSEB2 772 chickB2 798 XENO 798 hum1 798 MOUSEBl 803 chickBl

__

FIG. 1 (continued)

extracellular domain. The cysteine residues have a stereotypic pattern in the vertebrates, with minor exceptions in P4,Ps, and p7.However, there is more variance in this spacing in the invertebrate sequences (Fig. 2), and exceptions to the canonical vertebrate pattern can be found in all the invertebrate subunits. Brower et al. (1997) and Marsden and Burke (1997) noted this previously for specific subunits. Brower et al. (1997) suggested that the spacing for the vertebrate integrins is closer to the ancestral structure and that association with multiple a subunits has acted to conserve this pattern. My analysis suggests an alternative interpretation. I believe it is possible that the invertebrate /3 subunits represent independent diversifications of ancestral forms. Although the /3 subunits are homologous members of a single family, the lack of evidence for a simple conserved divergence suggests there was considerable early divergence that cannot be resolved. This is supported by the range of variance in positioning of cysteines, which implies differences in the folding of the extracellular domain. However, the vertebrate P subunits appear to be derived from at least two radiations. These groupings of paralogs may be derived from gene duplications and represent diversification from a smaller set of ancestral P subunits. The retention of the spacing of the cysteines in the vertebrate form is a derived state, but one that is rigorously constrained by function. The question most often asked about invertebrate integrin subunits is which vertebrate type is it? Since the vertebrate subunits are associated with a set of functions, it would be useful if invertebrate subunits could be similarly classified. It appears that, at least for the /3 subunits, there have been independent diversifications of structure within most of the major taxa and there are no orthologs of the vertebrate subunits. By analogy,

276

ROBERT D. BURKE

one would be unable to determine which of the five classes of echinoderms is most like a mammal. Therefore, it is unlikely that there is a molecule that is structurally and functionally more like the PI or p2 subunit in any of the invertebrate phyla. Integrin subunits are remarkably diverse within the vertebrates. Not including differentially spliced or posttranslationally modified forms, there are 8 p subunits and 16 a subunits associating as heterodimers to form 22 different receptors in mammals. It is not known whether all vertebrates have such a wealth of integrin subunits, but in amphibians and birds, most of the paralogs are present. In fish, at least 7 of the 8 p subunits appear to be present (M. Marsden, personal communication). It is unknown if this diversity will be found in invertebrate phyla, but to date the data suggest that this is not the case. Using similar methods to those used with vertebrates, no more that 3 p subunits have been identified in any one species. In Drosophila, which has been extensively studied, the a subunits appear to be more abundant than the p subunits, as in the vertebrates, but it is unlikely that 16 a subunits will be found. Fortunately, some of the invertebrates are organisms for which the entire genome is to be mapped and sequenced and the full catalog of subunits will soon become known. Whereas the /3 subunits appear to have undergone diversification independently, there are indications that a subunits may be derived from molecules for which phylogenies may be more tractable. Gotwals et al. (1994b) noted that there appear to be two groups of a integrins that diverged prior to protostome and deuterostome separation. One group of a subunits includes vertebrate a3,ayg, a7,apsl,and aina-l. The other group includes as, ax, a", (YIIb, aps2, (YF54F2.1, and asu2(Gotwals et al., 1994b; P. Hertzler and D. McClay, personal communication). However, the number of invertebrate a subunits is relatively small and the strengths of these associations may prove no stronger that those found in the p subunits. Even less data exist regarding the positioning of intron boundaries, but both a and p subunits from Drosophila and C. elegans appear to have conserved at least some of these (Gettner er al., 1995; Wehrli et al., 1993).

FIG. 2 Trees generated using PAUP (version 4.0) and the aligned sequences. Trees were calculated using bootstrap analysis and 100 iterations of a neighbor-joining, distance algorithm, with no outgroups designated. The bootstrap values are shown for branch points; values over 50 are generally considered significant, but I have retained branchings with lower values that consistently arise. (a) Tree based on the complete sequences. (b) Tree based on the region containing the putative ligand-binding domain (195-365). ( c ) Tree based on a region proposed to have a high variability (677-816). (d) Tree based on cytoplasmic domains (950-1112).

INVERTEBRATE INTEGRINS

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FIG. 2

(continued)

280

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ROBERT D. BURKE

71

INVERTEBRATE INTEGRINS

28 1

IV. Concluding Remarks Invertebrate integrins appear to be generally similar to vertebrate integrins in structure and function. Features, such as being heterodimeric and the association of several different CY subunits with some /3 subunits, are well conserved. Although invertebrate integrin ligands have been less well characterized, ligands such as laminin and RGD-containing ECM components such as tiggrin appear to be involved. As with the vertebrate integrins, functions appear to be the mechanical adhesion of cells to ECM and the formation of focal adhesion-like complexes that associate with the actin cytoskeleton. Although data are largely circumstantial, the association of integrins with cells having immune functions, such as hemocytes and coelomocytes, suggests that at least some of the plethora of integrin-based adhesion phenomena seen in the vertebrate immune system may be found in invertebrates. Much remains to be learned about the invertebrate integrins. In particular, studies of function are needed, especially in adults. The impetus to study integrins in invertebrates stems largely from the involvement of integrins in morphogenesis and the current data are derived from organisms in which studies of development are tractable. With the complete sequencing of genomes, we will have access to the full complement of integrin subunits in some organisms. Also, the further development of molecular genetic methods employed in the context of questions of cell biology will permit a wider range of functional studies. Because integrin structure and function in invertebrates resembles that of vertebrates, these studies will remain relevant and potentially may contribute substantially to our understanding of this remarkable group of receptors.

Acknowledgments I am grateful to Ben Koop and Sheldon McKay for assistance with analysis. I thank Phil Hertzler and David McClay for summaries of their studies prior to publication. Matt Rise. Greg Murray, Chris Reed, and Norma Lake all provided comments on drafts of the manuscript. The Natural Sciences and Engineering Research Council of Canada support my research on invertebrate integrins.

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Almeida, E. A. C.. Huovila, A,-P. J., Sutherland, A. E., Stephens, L. E., Calarco, P. G., Shaw, L. M., Mercurio, A. M., Sonnenbrrg, A,, Primakoff, P., Myles, D. G., and White, J. M. (1995). Mouse egg integrin a6pl functions as a sperm receptor. Cell 81, 1095-1104. Baum, P. D., and Garriga, G. (1997). Neuronal migrations and axon fasciculation are disrupted in ina-1 integrin mutants. Neuron 19, 51-62. Bloor, J. W., and Brown, N. H. (1998). Genetic analysis of the Drosophila alphaPS2 integrin subunit reveals discrete adhesive, morphogenetic and sarcomeric functions. Genetics 148, 1127-1142. Bogaert, T., Brown, N.. and Wilcox, M. (1987). The Drosophila PS2 antigen is an invertebrate integrin that, like the fibronectin receptor, becomes localized to muscle attachments. Cell 51,929-940. Brabant, M. C . , and Brower, D. L. (1993). PS2 integrin requirements in Drosophila embryo and wing morphogenesis. Dev. Bid. 157, 49-59. Brower, D. L., and Jaffe, S. M. (19fi9). Requirement for integrins during Drosophila wing development. Nature 342, 285-287. Brower, D. L., Wilcox. M., Piovant, M.. Smith, R. J., and Reger, L. (1984). Related cellsurface antigens expressed with position specificity in Drosophila imaginal discs. Proc. Natl. Acad. Sci. USA 81,7485-7489. Brower, D. L., Bunch, T. A,, Mukai, L., Adamson, T. E., Wehrli, M., Lam, S., Friedlander, E., Roote, C. E., and Zusman, S. (1995). Nonequivalent requirements for PSI and PS2 integrin at cell attachments in Drosophila: Genetic analysis of the alpha PSI subunit. Developmenr U1, 1311-1320. Brower, D. L., Brower, S. M., Hayward, D. C., and Ball, E. E. (1997). Molecular evolution of integrins: Genes encoding integrin beta subunits from a coral and a sponge. Proc. Natl. Acad. Sci. USA 94,9182-9187. Brown, N. H. (1993). Integrins hold Drosophila together. BioEssays 15, 383-390. Brown, N. H. (1994). Null mutations in the alpha PS2 and beta PS2 integrin subunit genes have distinct phenotypes. Developrnenf 120, 1221-1231. Brown, N. H., King, D. L., Wilcox. M., and Kafatos, F. C. (1989). Developmentally regulated alternative splicing of Drosophila integrin PS2alpha transcripts. Cell 59, 185-195. Brown, N. H., Bloor, J. W., Duninborkowski, O., and Martinbermudo, M. D. (1993). Integrins and morphogenesis. Development SuppL, 177-183. Bunch, T. A., and Brower, D. L. (1992). Drosophila PS2 integrin mediates RGD-dependent cell-matrix interactions. Developmenf 116, 239-247. Bunch, T. A,, and Brower, D. L. (1993). Drosophila cell adhesion molecules. Curr. Topics Dev. B i d . 28, 81-123. Bunch, T. A., Graner, M. W., Fessler, L. I., Fessler, J. H., Schneider, K. D., Kerschen, A,, Choy, L. P., Burgess, B. W., and Brower, D. L. (1998). The PS2 integrin ligand tiggrin is required for proper muscle function in Drosophila. Development 125, 1679-1689. Cheresh, D. A., and Mecham, R. P. (Eds.) (1994). “Integrins Molecular and Biological Responses to the Extracellular Matrix.” Academic Press, San Diego. DeSimone, D. W., and Hynes, R. 0.(1988). Xenopus laevis integrins. J. Biol. Chem. 263,53335340. Djaffar, I., Chen, Y. P., Creminon, C . , Maclouf, J., Cieutat, A. M., Gayet, O., and Rosa, J. P. (1994). A new alternative transcript encodes a 60 kDa truncated form of integrin beta 3. Biochern. J . 300, 69-74. Fessler, J. H., and Fessler, L. I. (1989). Drosophila extracellular matrix. Annu. Rev. Cell Biol. 5,309-339. Fogerty, F. J., Fessler, L. I., Bunch, T. A., Yaron, Y., Parker, C. G., Nelson, R. E., Brower, D. L., Gullberg. D.. and Fessler, J. H. (1994). Tiggrin, a novel Drosophila extracellular matrix protein that functions as a ligand for Drosophila alpha(PS2)beta(PS) integrins. Developmenr UO, 1747-1758.

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Fristrom, D., Wilcox, M., and Fristrom, J. (1993). The distribution of PS integrins, laminin A and F-actin during key stages in Drosophila wing development. Development 117,509-523. Gettner, S. N., Kenyon, C., and Reichardt, L. F. (1995). Characterization of betapa,.i heterodimers, a family of essential integrin receptors in C. elegans. J. Cell Biol. 129, 112771141, Gotwals, P. J., Fessler, L. I., Wehrli, M., and Hynes, R. 0. (1994a). Drosophila PSI integrin is a laminin receptor and differs in ligand specificity from PS2. Proc. Narl. Acad. Sci. U S A 91, 11447-1 14.51, Gotwals, P. J., Painesaunders. S. E., Stark, K. A,, and Hynes, R. 0. (1994b). Drosophila integrins and their ligands. Curr. Opin. Cell. Biol. 6, 734-739. Grinblat, Y., Zusman, S., Yee, G., Hynes, R. O., and Kafatos, F. C . (1994). Functions of the cytoplasmic domain of the beta(PS) integrin subunit during Drosophila development. Development 120, 91-102. Grotewiel, M. S., Beck, C. D. O., Wu, K. H., Zhu, X.-R., and Davis, R. L. (1998). Integrinmediated short-term memory in Drosophila. Nature 391, 455-460. Holmblad. T., Thornqvist, P.-O., Soderhall, K., and Johansson, M. (1997). Identification and cloning of an integrin beta subunit from hemocytes of the freshwater crayfish Pacifastacus leniusculus. J. Exp. Zool. 277, 255-261. Hortsch, M., and Goodman, C. S. (1991). Cell and substrate adhesion molecules in Drosophila. Annu. Rev. Cell Biol. 7, 505-557. Huang, X. (1994). On global sequence alignment. Comp. Appl. Biosci. 10, 227-235. Hughes, A. L. (1992). Coevolution of the vertebrate integrin alpha- and beta-chain genes. Mol. Biol. Evol. 9, 216-234. Hughes, P. E., Diaz-Gonzalez, F., Leong, L., Wu. C.. McDonald, J. A,, Shattil, S. J., and Ginsberg, M. H. (1996). Breaking the integrin hinge. A defined structural constraint regulates integrin signalling. J. Biol. Chem. 271, 6571-6574. Hynes, R. 0. (1987). Integrins: A family of cell surface receptors. Cell 48, 549-554. Hynes, R. 0. (1992). Integrins: Versatility, modulation, and signalling in cell adhesion. Cell 69, 11-25. Hynes, R. 0.(1996). Targetted mutations in cell adhesion genes: What have we learned from them? Dev. Biol. 180,402-412. Hynes, R. 0..and Lander, A. L. (1992). Contact and adhesive specificities in the associations, migrations, and targeting of cells and axons. Cell 68,303-322. Leptin, M., Aebersold, R., and Wilcox, M. (1987). Drosophila position specific antigens resemble the vertebrate fibronectin-receptor family. EMBO J . 6, 1037-1043. Leptin, M., Bogaert. T., Lehmann, R., and Wilcox, M. (1989). The function of PS integrins during Drosophila embryogenesis. Cell 56, 401-408. MacKrell, A. J., Blumberg, B., Haynes, S. R., and Fessler, J. H. (1988). The lethal myospheroid gene of Drosophila encodes a membrane protein homologous to vertebrate integrin beta subunits. Proc. Natl. Acad. Sci. USA 85, 2633-2637. Marcantonio, E. E., and Hynes, R. 0.(1988). Antibodies to the conserved cytoplasmic domain of the integrin beta 1 subunit react with proteins in vertebrates, invertebrates and fungi. J. Cell Biol. 106, 1765-1772. Marsden, M., and Burke, R. D. (1997). Cloning and characterization of novel beta integrin subunits from a sea urchin. Dev. Biol. 181, 234-245. Marsden, M., and Burke, R. D. (1998). The PL integrin subunit is necessary for gastrulation in sea urchin embryos. Dev. Biol. 203, 134-148. Moulder, G. L., Huang, M. M., Waterson, R. H., and Barstead, R. J. (1996). Talin requires beta-integrin, but not vinculin, for its assembly into focal adhesion-like structures in the nematode Caenorhabditis elegans. Mol. Biol. Cell 7, 1181-1193. Nakajima, Y . , and Burke, R. D. (1996). The initial phase of gastrulation in sea urchins is accompanied by the formation of bottle cells. Dev. Biol. 179, 436-446.

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Pytela, R., Suzuki, S., Breuss, J., Erle, D. J., and Sheppard, D. (1994). Polymerase chain reaction cloning with degenerate primers: Homology-based identification of adhesion molecules. Methods Ezvmol. 245, 420-451, Reszka, A. A., Hayashi. Y.. and Horwitz. A. F. (1992). Identification of amino acid sequences in the integrin beta 1 cytoplasmic domain implicated in cytoskeletal associations. J. Cell Biol. 117, 1321-1330. Ruoslahti, E., and Pierschbacher, M. D. (1987). New perspectives in cell adhesion: RGD and integrins. Science 238, 491 -497. Schwartz, M . A., Schaller, M. D., and Ginsberg, M. H. (1995). Integrins: Emerging paradigms of signal transduction. Annu. Rev. Cell Dev. B i d . 11,549-599. Sheppard, D. (1996). Epithelial integrins. BioEssays 18, 655-660. Stark, K. A., Yee, G. H.. Roote, C. E., Williams, E. L., Zusrnan, S., and Hynes, R. 0. (1997). A novel alpha integrin subunit associates with betaPS and functions in tissue morphogenesis and movement during Drosophila development. Development 124, 4583-4594. Susan, J. M., and Lennarz, W. J. (1993). Identification of an integrin alpha subunit in gastrula stage sea urchin embryos. Mol. Biol. Cell 4, 378a. Wehrli, M., DiAntonio, A., Fearnley, I. M., Smith, R. J., and Wilcox, M. (1993). Cloning and characterization of alpha PS1. a novel Drosophila melanogaster integrin. Mech. Dev. 43,21-36. Wilcox. M. (1990). Genetic analysis of the Drosophila PS integrins. Cell Differ. Dev. 32, 391-400. Wilcox. M., and Leptin, M. (1985). Tissue-specific modulation of a set of related cell surface anligens in Drosophila. Nature 316, 351-354. Wilcox. M., Brower, D. L., and Smith, R. J. (1981). A position-specific cell-surface antigen in the Drosophila wing imaginal disc. Cell 25, 159-164. Wilcox, M., Brown. N.. Piovant, M., Smith, R. J.. and White, R. A. H. (1984). The Drosophila position-specific antigens are a family of cell surface glycoprotein complexes. E M B O J . 3,2307-2313. Wilson. R.. Ainscough, R., Anderson, K., Baynes, C., Berks. M.. Bonfield, J., Burton, J., Connell, M., Copsey, T.. Cooper, J., er al. (1994). 2.2 Mb of contiguous nucleotide sequence from chromosome 111 of C. elegans. Nuture 368, 32-38. Wright, T. R. F. (1960). The phenogenetics of the emhryonic mutant, lethal myospheroid in Drosophila melanogaster. J . Exp. Zoul. 143, 77-79. Yee, G. H., and Hynes, R. 0. (1993). A novel, tissue-specific integrin subunit, bv, expressed in the midgut of Drosophila melanogaster. Development 118, 845-858. Zavortink, M., Bunch, T. A., and Brower, D. L. (1993). Functional properties of alternatively spliced forms of the Drosophila PS2 integrin alpha-subunit. Cell. Adhes. Commun. 1, 251 -264. Zusman, S., Patel-King, R. S., french-Constant, C., and Hynes, R. 0. (1990). Requirements for integrins during Drosophila development. Development 108, 391-402. Zusman, S., Grinblat, Y., Yee. G., Kafatos, F. C.. and Hynes, R. 0. (1993). Analyses of PS integrin functions during Drosophila development. Development 118, 737-750.

INDEX

A Acetylcholine, effect on melatonin synthcsis, 235 Acetylcholine receptor, mdx muscle, 123 Actin accumulation in bottle cells, 265 purification from Physaricm, 62 role in Ca inhibition. 72-76 Actin-binding protein high-molecular-weight. plasmodium-specific, 88 fhysarum, role in Ca inhibition. 77-83 Actin-myosin interaction Ca inhibition CaLc effect, 79-80 Mg’+ effect, 78-79 P. polycephalitni Ca” inhibition, 58-66 maximal in absence of Ca”. 56 Activation Pi uptake in plasma membrane. light-induced, 171 zygote nucleus, 5 Activation pathway, MAP kinase, 35 Activin accumulation, as oocyte information, 32-33 as all-purpose signaling molecule, 41-42 concentrations, animal cap responses, 27-28 effect on animal cap explants, 9 induction dorsal marker gene expression. 24-25 endoderm. 39 mesoderm marker gene expression, 22-24

inhibition of neural induction, 36-37 mechanism of action, 28-30 MIF activity, 13-16 postgastrulation gene expression promoted by, 26 role in mesodermineural patterning. 34 regionalized pattern specification, 21-22 signal transduction pathways, models. 37-38 signaling mechanisms, embryonic. 31 -37 Activin receptors, and signal transduction, 33 Actomyosin crude observation of myosin-linked nature of Ca” inhibition. 61-66 study of Ca” effect on fliysaritni actin-myosin interaction. 58-61 hybrid. Ca” inhibition, 62-64 motile events related to. Ca” inhibition, 55.58 Adhesion proteins, N-CAM, mdx muscle. 124 Agrin, effect on utrophin expression. 129 Algae, Pi uptake, effects of light and temperature, 171, 174 Ameba, fhysur~inl,myosin. and amebalplasmodia1 transition, 88-90 Amino acid sequence alignment of /3 integrin subunits, 268-275 CaLc. 68 a-Amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors, see AMPA receptors

285

286

INDEX

AMPA receptors, pineal cells expressing, 227-228 Animal cap, responses to activin concentrations, 27-28 Animal cap assay activin effects, 22-26 in identification of MIFs, 8-9 Animal-vegetal axis, specification, 2-5 Antibodies, 40-kDa protein, immunostaining of vertebrate cells with, 88 Antigens, PS1, 263 Apoplasts canal, 191-192 Pi homeostasis, 188-189 Arsenate, inhibitory effect on Pi transport, 168 Arthropods, integrin a and 0 subunits, 261-264 Aspartate, and glutamate, corelease from pinealocytes, 227 ATP, role in Pi transport, 183 ATPase activity of actomyosin preparations, Ca2+ effect, 59-60, 62-64 Physarum myosin actin-activated, 64 Ca2+effect, 75 Axin, effect on Wnt action, 19

B Basal lamina, mdx mouse, role in muscle regeneration, 115-116, 130 Basic fibroblast growth factor mdx muscle, 120 as natural mesoderm inducer, 13 Becker muscular dystrophy, gene, 100 bFGF, see Basic fibroblast growth factor Bioassay, identification of MIFs, 7-11 Biogenesis, SLMVs, 236-237 Blastopore lip, from late gastrula-stage donor, 6 BMP, see Bone morphogenetic proteins Bone morphogenetic proteins, role in dorsal mesoderm patterning, 20-21 Bottle cells, actin accumulation in, 265 Brain bovine, calmodulin, CaLc homology, 68 dystrophic, 107

dystrophin expression, 105 mdx mouse, metabolism, 124-125

C CaLc, see Calcium-binding light chain Ca1ciu m effects actin-myosin interaction, 54 protein kinases and phosphatases acting on myosin, 76-77 homeostasis, dystrophin role, 112 ionic flux in mdx muscle, 121-122 in microvesicular glutamate secretion, 225-227 Calcium-binding light chain as actin-binding protein, 79-80 as Ca-receptive subunit of Physarum myosin, 67-70 Physarum, phylogenic considerations, 70-71 Calcium-binding proteins, Physarum recombinant 40-kDa protein, 84-88 supporting Ca inhibition, 83-84 Calcium-dependent inhibitory factor, supporting Ca inhibition, 83-84 Calcium inhibition actin-binding protein role, 77-83 Ca-binding proteins supporting, 83-84 motile events related to actomyosin, 55-58 Physarum actin-myosin interaction, 58-66 Calcium sensor, synaptotagmin as, 214 Caldesmon-like protein, Physarum actin-binding, 80-81 calmodulin-binding, 83 Calmitine, levels in mdx mouse, 121-122 Calmodulin bovine brain, CaLc homology, 68 Physarum, supporting Ca-inhibition, 83 Calpains, proteolytic role in mdx dystrophy, 114 Cells plant Pi distribution among, 153. 156-157 Pi homeostasis, 184-192 vertebrate, immunostaining with 40-kDa protein antibody, 88

287

INDEX

vertebrate and invertebrate, motile events, Ca inhibition, 57-58 Chimera, scallop and skeletal muscle myosin light chains, 70 Chloroplasts, Pi-deficient, 156-157 chordin, dorsalizing factor, 25 Circadian rhythm, synchronizing role of melatonin, 204 Cleavage furrow, Ca2+concentration effect, 57-58 Cnidarians, integrin subunits, 259 Coelomocytes, integrin subunit expression, 265 Communication intercellular, microvesicle-mediated, 229, 231 paracrine, microvesicle-mediated, 232-236 Competence, prepatterning, and signal timing, 30-31 Concentration gradient, apoplastic Pi, 191-192 Contact zone, neurohemal, 202 Contractile apparatus, dystrophin network in relation to, 106-107 Crustaceans, integrin a and fl subunits, 261 Cysteine residues, integrin subunit, spacing in extracellular domain. 268, 275 Cysteine string protein, chaperone-like functions, 215-216 Cytoplasm, plant cell Pi homeostasis, 185-188 Pi levels, 156 Cytoplasmic streaming, Ca2+inhibition P. polycephalum plasmodia, 56-57 plant cells, 55-56 Cytoskeleton, mdx muscle, 123

D Darkness, and pineal organ function, 203-204 Degeneration, and regeneration, mdx muscles, 113-119, 129 Dephosphorylation, Physarurn myosin, 7275, 91 Depolarization induction of SLMV exocytosis, 233, 235 plasma membrane, and Pi accumulation, 165

Diaphragm, mdx mouse, degeneration, 119-120, 129 Differentiation mesoderm, proteins inducing, 12-17 notochord, Brachyury gene role, 28-29 tissue, activin-generated, 38-40 Diurnal changes, synaptic membrane proteins, 221 Divergence, integrin fl subunits, 267, 275 Diversity, integrin subunits among vertebrates, 275-276 Dorsal axis, inducing activity of Wnt genes, 18 Dorsoventral signaling center, induction of mesoderm formation, 3, 5 Drosophila, and amphibia, gene structure conservation, 21 Duchenne muscular dystrophy gene, 100 therapeutic projects, 126-129 P-Dystroglycan, association with dystropbin, 108-109 Dystrophin associated glycoprotein complex, 108-109 expression, 104-105 gene delivery, 127-128 gene mutation in animal models, 101-102 localization, 105-107 as mechanical transducer, 120 role in cell motility, 112 structure, 102-104

E Echinoderms, integrin subunits, 264-266 Eggs, see also Oocytes amphibian, polarization phenomena, 2-3 Ca2+level changes, relation to cleavage cycle, 57-58 Embryo activin signaling mechanisms, life history, 31-37 amphibian, axis specification, 2-5 Xenopus, activin genes and proteins, 15-16 Endocrine cells, sharing common features with neurons, 201-202 Endoderm, induction by activin, 39 Epidermis, atypical, in animal cap assay, 8

200

INDEX

Erythroid differentiation factor. activins equivalent to, 14, 16 Essential light chains, Physurum and Dictyostelium, 70-71 Evolution, integrins. 266-280 Exocytosis, SLMVs, inducing factors, 233, 235 Extracellular matrix, cell attachment to, integrin role, 257-258, 262 Extraction, Pi, from tissues and cells, 151-152

Glucocorticoids, therapy of Duchenne muscular dystrophy, 126 GluR receptor family, detection in pineal gland, 228-229 Glutamate microvesicular compartmentation, 225-227 pineal, sequestered in SLMVs, 239 Glutamate receptors, subtypes, pinealocytes expressing, 227-229 Glycoprotein complex, associated with dystrophin, 108-109 goosecoin

F Fertilization breaking of egg radial symmetry by, 3 and removal of integrin from egg surface, 266 Fibers mdx diaphragm, 119-120 morphological defects in mdx mouse, 113-115 Fibroblast growth factor as competence factor for activin signaling system, 31 MIF activity, 12-13 Fibroblasts genetically modified primary, 128 mdx muscle, interaction with other cell types, 116-119 Follistatin, effect on mesoderm induction, 34 Fragmin, Physurum, actin-binding, 81 Furosemide, inhibition of Pi transport, 168-169 FXMD cat, dystrophin deficiency, 102

G GABA release by pineal parenchymal cells, 240 uptake by SLMVs, 224-225 Gastrulation, integrin subunit expression, 265 Gene delivery, dystrophin. 127-128 Genes activin-related, 15-16 dystrophin, mutation in animal models, 101-102

expression induction, 38 induction by activin, 24 GRMD dog, dystrophin gene mutation, 101 Growth, plant, relationship to Pi nutrition, 177 Growth inhibitory factors, secreted by mdx fibroblasts. 117-119

H Heart, dystrophin expression, 105 Heparan sulfate proteoglycan, expression in mdx muscle, 115-116, 130 Homeostasis, Pi in plant cells, 184-192 Hydrogen ion, Pi uptake in plasma membrane dependent on, 161, 165-166 Hypertrophy, muscular, in mdx mouse, 114

I Inhibins, subunits in common with activin, 14 Innervation, mdx muscle, 122-123 Inorganic phosphate distribution, 152-157 efflux measurement from vacuole, 184 and xylem loading, 178-180 forms in living organisms, 150-151 homeostasis apoplastic. 188-189 cytoplasmic and vacuolar, 185-188 whole plants, 189-192 influx measurement plasma membrane, 160-161 tonoplast, 180-182

289

INDEX

measurement techniques. 151-1.52 mechanisms of transport plasma membrane. 161-169 tonoplast. 182-184 regulation of transport plasma membrane, 170-178 tonoplast. 182-184 transporter molecular structure. 169-170 Insects, integrin LY and ,f3 subunits, 261-264 Insulin-like growth factor-binding proteins, mdx fibroblast. 117-119 In tegrins crustacean and insect, 261 -264 echinoid, 264-266 evolution, 266-280 nematodes. 260-261 role in cell attachment to extracellular matrix, 257-258, 262 sponges and cnidarians. 259 Interstitial cells, interaction with pinealocytes. 240 Intracellular compartments, plant cells. Pi levels. 156-157

L Laminin. Pl/rlchains. expression in mdx muscle, 116 Laminin receptors, PS integrin apslsubunit as, 264 Leaves, Pi retranslocation, 190-191 Light activation of Pi uptake in plasma membrane, 171 effect on cytoplasmic Pi level. 186-187 Lim-I, role in mesoderm formation, 25 Lipids. linolenic-like. mdx mouse, 124

Magnesium, role in Ca inhibition of actinmyosin interaction, 78-79 MAP kinase, activation pathway. 35 Marker genes dorsal activin-induced expression, 24-25 secondary gene expression responses. 25-26

mesoderm, activin-induced expression, 22-24 Mastocytes, mdx mouse, 124 Mdx mouse adhesion proteins. 124 bFGF effects, 120 cytoskeleton. 123 diaphragm, characterization, 119-120 dystrophin gene mutation, 100-101 innervation. 122-123 ionic flux, 121-122 mastocytes. 124 muscle degenerationhegeneration, 113-119 dystrophin-associated protein reduction in. 109 muscle cells, metabolism, 124-126 structural defects at myotendinous junction, 107 therapeutic projects, 126-129 utrophin expression, 111-1 12 Melatonin amino acid negative regulatory role, 239 rhythmic synthesis and secretion, 204 secretion, glutamate effect, 228 synthesis, SLMV regulatory role, 232-236 Mesoderm differentiation, proteins inducing, 12-17 formation. Lirn-l role, 25 induction activin role, 1 follistatin effect. 34 presumptive endoderm cell role, 5 patterning factors modifying action of MIFs on, 17-21 inductive signal networks, 34-36 Mesoderm-inducing factors bioassay procedures, 7-11 effects on body patterning, 40-41 natural inducer candidates, 11-12 Mesophyll cells Pi deficiency, 181-182 Pi levels, 1.53 Messenger RNA activin P subunits, 15-16 dystrophin, 107 Metabolism mdx mouse, 124-126 Pi, effect on cytoplasmic Pi level, 186- 187

290 Metazoans, production of integrins, 258 Microvesicles clear as common constituents of pinealocytes, 205-206 components of synaptic ribbons, 209-211 paracrine communication mediated by, 232-236 synaptic-like, see Synaptic-like rnicrovesicles MIF, see Mesoderm-inducing factors Mix. I , activin-induced expression, 22-24 Models activin role in signal transduction pathways, 37-38 animal, muscular dystrophies, 100-102 axial patterning generation, 26-27 Monomers, dystrophin, 104 Morphological defects, mdx muscles, 113-115 Motility cell, dystrophin role, 112 Physarum, Ca inhibition, 90-92 Motility assay actin-rich and -poor conditions, 75-76 detection of myosin-linked nature of Ca2+ inhibition, 64-66 Munc-18, tight binding to syntaxin I, 216 Muscle cells, mdx mouse degenerationhegeneration, 113-119 metabolism, 124-126 Muscle contraction, at high Ca2+,54 Mutants, PS1 and PS2 integrins, 263 Mutation, dystrophin gene, animal models, 101-102 Myoblast transplantation, mdx mouse, 126-127 Myosin ameba], and amebal-plasmodia1 transition, 88-90 Physarum calcium-binding properties, 67-71 kinase activity, Ca2+effect, 83 phosphorylated state and Ca inhibition, 72-77 phosphorylation and dephosphorylation, 91 Physarum actomyosin, site of action of CaZ+,61-66 Myotendinous junction, defects, in mdx mice, 107

INDEX

N Nematodes, integrin (Y and fl subunits, 260-261 Neural induction animal cap cell response, 9 inhibition by activin, 36-37 vertical signal, 39-40 Neuroendocrine cells, synaptic vesicle trafficking proteins in, 217-224 Neuromuscular junction dystrophin localization, 106 utrophin localization, 109, 111 Neurons dystrophin expression, 105 endocrine cells sharing common features with, 201-202 synaptic vesicle trafficking in, 240-241 Nieuwkoop organizing center formation, Siarnois role, 36 induction of mesoderm formation, 3, 5 Nitric oxide synthase, neuronal, complex with a-syntrophin, 108-109 Noggin gene products, rescue of dorsal axis, 19-20 transcribed in neurula stage, 25 Noradrenaline, effect on microvesicular secretion, 233 Nuclear magnetic resonance, measurement of Pi concentrations, 152 Nucleus, zygote, activation, 5 Nutrition, Pi, relationship to plant growth, 177

0 Oocytes, see also Eggs information, activin accumulation as, 32-33 Orthophosphoric acid, homeostasis, 150

P Paracrine system, pinealocyte microvesicles as components, 227-236 Patterning axial, by activin, 26-31 body, anterior-posterior, 6-7

INDEX

mesoderm factors modifying action of MIFs on, 17-21 inductive signal networks, 34-36 neural, guided by activin-induced organizer, 39-40 pH, Pi transport dependent on, 165-166 PH084, encoding Pi transporter, 169-170 Phloem, Pi retranslocation via, 191-192 Pho regulon, role of two-component system, 177-178 Phosphatase, acting on myosin, Ca” dependence, 76-77 Phosphate inorganic, see Inorganic phosphate intracellular, effect on Pi uptake, 174 Phosphorus accessible to plants, 192-193 characteristics, 150-151 Phosphorylatable light chain, myosin light chain, 69-70 Phosphorylation, Physarum myosin, 72, 91 Phylogenetic tree essential light chains, 70-71 integrins, 267-268, 277-280 Physarum polycephalum actin-binding proteins, role in Ca inhibitory effect, 77-83 actin-myosin interaction, Ca” inhibition, 58-66 amebal myosin, and amebal-plasmodia1 transition, 88-90 calcium-binding proteins, 83-88 muscle relaxation in low Ca”, 54 myosin Ca-binding properties, 67-71 phosphorylated state and Ca inhibition, 72-77 plasmodia cytoplasmic streaming, Ca” inhibition, 56-57 Pi, see Inorganic phosphate Pinealocytes clear microvesicles as constituents, 205-206 melatonin synthesis in, 204 neuron-like polarization, 237 secretory function, morphological correlates, 205 SLMVs arrangements, 209 components of extensive paracrine system, 227-236

291 enrichment in process swellings, 237, 239 storage and secretion of signal molecules, 225-227 synaptic ribbons, clear microvesicles as components, 209-211 synaptic vesicle trafficking proteins, 217-224 Pineal organ, mammalian, darkness mediator, 203-204 Pintallavis, coexpression with Xbra, 29 Plants phosphorus accessible to, 192-193 Pi distribution, 152-157 homeostasis, 184-192 membrane transport, 157-184 Plasma membrane measurements Pi efflux and xylem loading, 178-180 Pi influx, 160-161 Pi transport mechanisms, 161-169 regulation, 170-178 Pi transporter molecular structure, 169-170 Plasmodia, Physarum amebal-plasmodia1 transition, and amebal myosin, 88-90 cytoplasmic streaming, Ca inhibition, 56-57 organization, actin-binding protein role, 82-83 Polarization dorsal-ventral, molecular basis, 3 neuron-like, pinealocytes, 237 phenomena, amphibian eggs, 2-3 Polymerase chain reaction, identification of integrin subunits, 258 Position specificity, insect integrins, 261-264 Potassium, ionic flux in mdx mouse, 122 Prednisone, effect on mdx muscle culture, 126 Prepatterns, competence, and signal timing, 30-31 Process terminals intraparenchymal bulbous, 231 pinealocyte, SLMV concentrations, 206-209 Profilin, Physarum, actin-binding, 81

292 Protein kinase acting on myosin, Ca” dependence. 76-77 phosphorylation of myosin, 91-92 Proteins cell wall, Pi transport, 168 induction of mesoderm differentiation, 12-17 40-kDa differential expression, 85, 88 structure and Ca-binding properties. 85 neuronal, implicated in synaptic vesicle trafficking, 21 1-217 synaptic vesicle trafficking, in neuroendocrine cells, 21 7-224 Purification, myosin and actin from Plzysnnim,62

Rab3 detection in pinealocytes. 219 role in synaptic vesicle docking, 215 Regeneration. mdx muscles, 113-1 19. 129 Regulon, Plio, role of two-component system, 177-178 Retranslocation, Pi, between plant tissues, 189-1 92

Sarcolemma, dystrophin localization, 105- 107 Secretion melatonin glutamate effect, 228 and rhythmic synthesis, 204 microvesicular, noradrenaline effect, 233 signal molecules, from microvesicles. 224-227 Secretory function, pinealocytes, morphological correlates, 205 Signaling activin. embryonic, 31-37 bone morphogenetic protein, 20-21 Signaling pathway, Wnt, 19 Signaling systems combinations, in inductive event, 11

INDEX vertical and planar, 6-7 Signal molecules microvesicular, release sites, 231 microvesicular storage and secretion, 224-227 Signal transduction and activin receptors, 33 activin role, models, 37-38 Skeletal muscle, dystrophin expression and localization, 104-107 SLMV. see Synaptic-like microvesicles Smad2 link with activin, 38 transcription regulator action, 19 Smad7, inhibition of activin and BMP signaling, 26-27 SNAP-25. acting downstream of vesicle docking, 213 SNARE hypothesis, synaptic vesicle trafficking, 211-212 Sodium ion flux in mdx mouse. 122 Pi uptake in plasma membrane dependent on, 166-168 Split-root experiments Pi transporter gene expression, 190 Pi uptake regulation, 176-177 Sponges, integrin subunits, 259 Starvation, Pi, effect on Pi uptake, 174, 176-177 Stoichiometry, plasma membrane Pi transporter, 166 Storage, signal molecules, in microvesicles, 224-227 Synapsin, reversible tethering of synaptic vesicles, 21 6 Synaptic-like microvesicles biogenesis. 236-237 classification, 203, 223-224 GABA uptake, 224-225 glutamate sequestered in, 239 pinealocyte as components of extensive paracrine system, 227-236 concentrations, 206 regulatory role in melatonin synthesis, 232-236 synaptophysin as membrane component, 219 Synaptic ribbons, clear microvesicles as components, 209-21 1

INDEX

293

Synaptic vesicle protein 2, as transporter protein, 219, 221 Synaptic vesicles docking, 212-213, 215 reversible tethering by synapsin. 216 trafficking in neurons, 240-241 trafficking proteins, 21 1-224 Synaptobrevin, role in synaptic vesicle docking, 212 Synaptophysin pineal gland, 218-221 role in neurotransmitter release, 213-214 and transferrin receptor, overlapping immunoreactivities, 237 Synaptotagmin, interactions with other nerve terminal proteins, 214 Syntaxin presence in photoneuroendocrine cells, 21 9 role in synaptic vesicle docking, 212-213 a-Syntrophin, complex with neuronal nitric oxide synthase. 108-109

T Talin, dystrophin-binding, 106 Temperature, effect on Pi uptake in plasma membrane, 171, 174 Tenascin-C, in mdx muscle, 116 Therapeutic projects, Duchenne muscular dystrophy, 126-129 Tiggrin, extracellular matrix component, 263, 281 Timing, signal induction. and competence prepatterning, 30-31 Tissues differentiation, activin-generated, 38-40 plant Pi extraction, 151-152 Pi retranslocation among, 189-192 Tonoplast measurements Pi efHux from vacuole, 184 Pi influx. 180-182 Pi transport, mechanism and regulation. 182- I84 Transferrin receptor. codistribution with SLMV proteins, 236-237 Transforming growth factor p1, synergism with bFGF, 14

Two-component system detection of extracellular Pi, 187 Pho regulon and, 177-178

Ultrasctructure, pinealocytes, 20521 1 Utrophin and dystrophin, mice deficient in, 128-129 localization. 109-112. 129 organizational role, 113

v Vacuoles Pi efHux from, 184 Pi homeostasis, 185- 188 Pi levels, 157 sap, Pi inHux measurement, 181182 Vegetalizing factors, corresponding to activin, 11 Vertebrates, integrin subunit diversity among, 275-276 Vesicles captured by synaptic ribbons, 210 synaptic, see Synaptic vesicles "g1 as natural mesoderm-inducing factor, 16-17 signaling inhibition, 34

W Wnr genes, role in mesoderm patterning, 17-19

X Xbrn activin-induced expression, 22-24 specification of ventral mesoderm, 29

294 Xdsh protein, in formation of complete dorsal axis, 18 Xhh, immediate early response gene, 25-26 XTC cell line, MIF activity, 14-15 XTrR-I, embryonic expression, 35-36 Xylem loading, and Pi efflux, 178180 Pi levels, 157, 188-189

INDEX

Y Yeast Na+-dependent Pi uptake, 167-168 tonoplast Pi transport, 183

z Zygote, nucleus, activation, 5