International Review of Cytology: A Survey of Cell Biology, Volume 173

VOLUME 173

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

1949-1988 1949-1 984 19671984-1 992 1993-1 995

EDITORIAL ADVISORY BOARD Aimee Bakken Eve Ida Barak Howard A. Bern Robert A. Bloodgood Dean Bok Stanley Cohen Rene Couteaux Marie A. DiBerardino Charles J. Flickinger Nicholas Gillham Elizabeth D. Hay P. Mark Hogarth Anthony P. Mahowald M. Melkonian Keith E. Mostov Audrey L. Muggleton-Harris

Andreas Oksche Muriel J. Ord Vladimir R. Pantic Thomas D. Pollard L. Evans Roth Jozef St. Schell Manfred Schliwa Hiroh Shibaoka Wilfred D. Stein Ralph M. Steinman M. Tazawa Yoshio Watanabe Donald P. Weeks Robin Wright Alexander L. Yudin

Edited by

Kwang W. Jeon Department of Zoology University of Tennessee Knoxville, Tennessee

VOLUME 173

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Front cover photograph: Darkfield micrographs of collagen fibrils. (For more details, see Chapter 2. Figure IOa.)

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CONTENTS

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

vii

A Model for Flagellar Motility Charles 6.Lindemann and Kathleen S. Kanous I. II. 111. IV. V. VI. VII.

Introduction , . . , , , . , , , , . . . . , , , . . , , . . , , , , , , . . . . . . . . . . . Structural Components of the Eukaryotic Flagellum . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Motor.. ......................................

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

.................. Coordination of the Beat Cycle . . . . . , . . . . . . . . . . Modeling the Flagellum , , . . . . . , , , . , . . . , . . . . , . . . . . . . . . . .

1 2 13 21

29 34 55 56

Basement-Membrane Stromal Relationships: Interactions between Collagen Fibrils and the Lamina Densa Eijiro Adachi, Ian Hopkinson, and Toshihiko Hayashi I. II. 111. IV. V. VI. VII.

. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . Introduction . . , . . . . . . . . . Molecules Related to lnterac en Collagen Fibrils and Lamina Densa . . . . . . Regulation of Collagen Fibril Diameter by pNcollagen 111 and Collagen V . . . . . . . . . . . .................. Collagen IV and the Skeleton of Lamina Densa . . . . .................. Interactions between Collagen Fibrils and Lamina Densa Other Systems Involved in the Anchoring of Collagen Concluding Remarks . . . , . . , , . , , , , , . . . . . . . . . , . , . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . V

73 78 109 120 125 132 138 140

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CONTENTS

The Role of Endoxyloglucan Transferase in the Organization of Plant Cell Walls Kazuhiko Nishitani Introduction .... ... Overview of Cell Wall Architecture in Plants . . . . . . . . . . . . . . . . Endoxyloglucan Transferase ............................ XRPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation of XRP Gene Expression ........................................ Overview of Cell Wall Construction during Plant Growth and Development: A Hypothetical Scheme . . . . .. VII. Concluding Remarks .................................................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. II. 111. IV. V. VI .

182 186 192 196 197

Microtubule-Microfilament Synergy in the Cytoskeleton R. H. Gavin I. II. 111. IV. V. VI.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Basal Body-Associated Fibrillar Networks ..................................... Microtubule-Microfilament Interactions in Cell Organelle Transport on Microtubule and Microfilament Tracks . . . ....... Regulation of Microtubule-Microfilament Inter Concluding Remarks ..................................................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

207 208 222 235 236

Insulin Internalization and Other Signaling Pathways in the Pleiotropic Effects of Insulin Robert M. Smith, Shuko Harada, and Leonard Jarett Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mechanisms of Insulin Internalization ........................................ Translocation of Insulin to the Cytoplasm and Nucleus .......................... Insulin-Responsive Pathways Other Than IRS-1 Involved in Insulin’s Effects on Immediate-Early Gene Expression .......................................... V. Summary.. . . . . . . . . ............... ........ References ....

243 248 258

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I. II. 111. IV.

Index

267 272 273

CONTRIBUTORS

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

Eijiro Adachi (73), Department of Anatomy and Cell Biology, School of Medicine, Kitasato University, Sagamihara City, Kanagawa 228, Japan R. H. Gavin (207),Department of Biology, Brooklyn College-CUNY, Brooklyn, New York 1 1210

Shuko Harada (243), Department of Pathology and Laboratory Medicine, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104 Toshihiko Hayashi (73),Department of Life Sciences-Chemistry, Graduate School of Arts and Sciences, The University of Tokyo, Tokyo 153, Japan Ian Hopkinson (73),Wound Healing Research Unit, Department of Surgery, University of Wales College of Medicine, Cardiff CF4 4XN, Wales, United Kingdom Leonard Jarett (243), Department of Pathology and Laboratory Medicine, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104 Kathleen S. Kanous ( l), Department of Biological Sciences, Oakland University Rochester, Rochester, Michiganl 43809 Charles B. Lindemann (1), Department of Biological Sciences, Oakland University Rochester, Rochester, Michigan 43809 Kazuhiko Nishitani (157), Department of Biology, College of Liberal Arts, Kagoshima University, Kagoshima 892, Japan Robert M. Smith (243),Department of Pathology and Laboratory Medicine, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104

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A Model for Flagellar Motility Charles B. Lindemann and Kathleen S.Kanous Department of Biological Sciences, Oakland University, Rochester, Michigan 48309

Experimental investigation has provided a wealth of structural, biochemical, and physiological information regarding the motile mechanism of eukaryotic flagellalcilia. This chapter surveys the available literature, selectively focusing on three major objectives. First, it attempts to identify those conserved structural components essential to providing motile function in eukaryotic axonemes. Second, it examines the relationship between these structural elements to determine the interactions that are vital to the mechanism of flagellarlciliary beating. Third, the vital principles of these interactions are incorporated into a tractable theoretical model, referred to as the Geometric Clutch, and this hypothetical scheme is examined to assess its compatibility with experimental observations. KEY WORDS: Flagella, Cilia, Motility, Dynein, t-Force, Axoneme, Oscillator, Molecular motors, Motor proteins, Microtubules.

1. Introduction The eukaryotic flagellum, with its ‘‘9 + 2” internal arrangement of microtubules (MTs), is one of the most curious of all biological constructions. The axoneme, the core of all eukaryotic flagella and cilia, serves in innumerable capacities. It provides motility or other motive force for organisms that range from one-celled algae to human beings. However, throughout its vast array of naturally developed applications, the flagellar axoneme has maintained a remarkably consistent design. The basic arrangement of nine peripheral doublet MTs interlinked by connecting protein strands and surrounding a central pair of MTs forms the flagellar template. Certain flagellar adaptations may lack one or more components, whereas other modifications involve the addition of accessory elements, but by and large these variations

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CHARLES 8. LINDEMANN AND KATHLEEN S. KANOUS

appear to originate from the standard flagellar configuration. Perhaps most noteworthy is the preservation of the spatial relationship between the axonemal MT components. This strict conservation of geometrical form suggests that the spatial organization of the axonemal elements is integral to flagellar functioning. The first goal of this chapter is to examine some of the variations in form and function that may provide clues to flagellar operation. Because the basic axonemal structure is common to both eukaryotic flagella and cilia, the terms flagella and cilia will be used interchangeably, and information garnered from studies conducted on either form will be included when applicable. Where pertinent information is available, the nature of the dynein-tubulin motor mechanism will also be discussed. Additionally, experimental findings that may shed light on the regulation of this motor will be examined. Finally, we will attempt to consolidate what is currently known into a plausible scheme to elucidate how the flagellar axoneme functions.

II. Structural Components of the Eukaryotic Flagellum A. Basic Axoneme Figure 1 illustrates the component parts of the eukaryotic flagellar axoneme. Nine doublet MTs (each consisting of a semicircular B MT attached to a round A MT) encircle a pair of centrally located single MTs. A central sheath, consisting of two C-shaped projections along each central MT (Warner and Satir, 1974), and a central “bridge” of electron-dense material (Olson and Linck, 1977) hold the central pair MTs together into what is sometimes referred to as the “hub” of the axoneme. Spokes are connected to the A MT of the outer doublets and converge toward the central hub. The spokes of most flagella repeat in a triplet pattern along the axoneme, with a major repeat interval of ~ 9 0 - 1 0 0 nm (Warner and Satir, 1974; Summers, 1975; Witman et al., 1978; Goodenough and Heuser, 1985). Isolated spokes appear straight and unbending when viewed in either negatively stained or freeze-fractured preparations (Olson and Linck, 1977; Goodenough and Heuser, 1985). Additionally, when the axoneme is fractured, bent, or distorted, the spokes are not observed to elongate, but the connection of the spoke head to the hub detaches instead (Warner and Satir, 1974; Summers, 1975; Lindemann and Gibbons, 1975; Olson and Linck, 1977; Goodenough and Heuser, 1985; Lindemann et al., 1992). Protein linkages (nexin links) interconnect the nine outer doublets, stabilizing the outer circular arrangement (Stephens, 1970). The nexin links extend from a point on the A MT, near the inner dynein arm in register with the

FLAGELLAR MOTILITY

3

FIG. 1 The eukaryotic flagellar axoneme. Structures commonly found in the typical axoneme are labeled on a silhouette diagram traced from an electron micrograph. The outer doublets. arranged in a ring of nine, each possesses an inner and outer row of dynein arms. In many flagella, doublets 5 and 6 are permanently bridged. prohibiting interdoublet sliding between these doublets. Each of the outer doublets is also linked to its neighbors by nexin links. The outer doublets are connected to the central pair by a series of wagon wheel-like spokes that interact with the axonemal “hub.” Some flagella have demonstrated the existence of a stable connection between outer doublets 3 and 8 that includes the central pair and roughly partitions the axonerne into two unequal “halves.” The flagellar beat is in a plane perpendicular to this partition as indicated by the double-headed arrow.

first spoke of each triplet repeat, to the B MT of the adjacent doublet (Dallai et al., 1973: Warner, 1976; Olson and Linck, 1977; Witman et al., 1978). Unlike the spokes, which detach and reattach, nexin links have been observed to stretch many times their resting length (Dallai et a/., 1973; Warner, 1976: Olson and Linck, 1977; Goodenough and Heuser, 1989). Tryptic digestion of these connections allows sliding disintegration of the axoneme (Summers and Gibbons, 1971, 1973; Lindemann and Gibbons, 1975; Sale and Satir, 1977). When a flagellum bends, the bends are mainly planar in a plane perpendicular to the axis of the central pair (Afzelius, 1961; Gibbons, 1961; Tamm and Horridge, 1970; Gibbons et al., 1987), as indicated by the doubleheaded arrow in Fig. 1. A number of structural factors contribute to the planar beat orientation. If there is no central pair complex (as in the 9 + 0 configuration), a helical wave pattern is observed (Gibbons er al., 1985;

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CHARLES B. LINDEMANN AND KATHLEEN S. KANOUS

Ishijima et al., 1988). Another important feature is the ability of the spokes located in the plane of the flagellar bend to change position by detaching and reattaching (“jumping”) at the hub connection (Warner and Satir, 1974). Additionally, there is strong evidence that spokes connecting the central pair to doublets 3 and 8 may not be as free, and instead form a stable midline “partition” that bisects the axoneme (Afzelius, 1959; Fawcett and Phillips, 1970; Lindemann et al., 1992; Kanous et al., 1993). Although the presence of this partition is not yet confirmed as a universal characteristic of flagella and cilia, prior evidence derived from sliding disintegrations points t o the possibility that this is a general characteristic of the axoneme (Tamm and Tamm, 1984; Sale, 1986). Some flagella also exhibit a permanent bridge between doublets 5 and 6 (Afzelius, 1959; Gibbons, 1961; Olson and Linck, 1977), precluding interdoublet sliding at that location and thereby inhibiting the formation of bends in the axis parallel to the central pair. Dynein acts as the molecular motor to power the movement of eukaryotic flagella. First isolated and characterized by Gibbons (Gibbons and Rowe, 1965), the dynein motor molecules (dynein “arms”) are composed of either two or three heavy chains, each with a globular “head” attached to a stalklike projection. The head contains the ATPase site, and the stalk is fixed to the A subtubule by way of an intermediate chain that binds t o tubulin. The dynein head is capable of attaching to the B subtubule of the adjacent doublet (“bridging”). In the presence of Mg-ATP, these arms translocate one doublet relative to its neighbor. In an intact flagellum, interdoublet sliding is impeded at the flagellar base by a centriole or basal body. The nine axonemal elements are permanently linked in a circle at the centriole/ basal body, thwarting translocation of one relative to another. This restraining mechanism results in flagellar bending as the force produced by the dynein arms exerts torque against the basal anchor. Each A subtubule bears two types of dynein arms, inner and outer arms (Fig. 2). Both inner and outer arm dynein are capable of driving microtubule sliding (Gibbons and Gibbons, 1973; Hata et al., 1980; Kamiya and Okamoto, 1985; Mitchell and Rosenbaum, 1985; Okagaki and Kamiya, 1986; Paschal et al., 1987; Kagami et al., 1990; Kurimoto and Kamiya, 1991; Smith and Sale, 1991; Kagami and Kamiya, 1992). However, the presence of outer arms generally results in a faster rate of sliding (Gibbons and Gibbons, 1973; Hata et al., 1980; Mitchell and Rosenbaum, 1985; Brokaw and Kamiya, 1987;Kurimoto and Kamiya, 1991;Hard et al., 1992) while imparting greater driving force (Oko and Clermont, 1990; Minoura and Kamiya, 1995). Additionally, the lack of outer arm dynein does not prevent motility. Inner arm dynein, on the other hand, presents a more complex contribution to flagellar activity, probably due to the existence of three discrete subforms of inner arm dynein (Piperno er al., 1990) that alternately repeat along the A subtubule (Goodenough and Heuser, 1985). Unlike the optional presence of outer

FLAGELLAR MOTILITY

5

DRC

LC Base

FIG. 2 Structure of the outer doublets. This diagram attempts to incorporate information garnered from a number of sources into an overview of the structures associated with the axonemal outer doublets. It must be understood that not all details are (or can be) represented within one single diagram. Most of the available structural evidence has been obtained from studies on Clilnmvdornonos, and therefore the SI and S2 spoke pairs have been included. whereas the S3 spokes (found in many cilia and Hagclla) have been omitted. The nexin links attach adjacent doublets from a point just distal to the spoke pairs (Goodenough and Heuser. 1989). with the same 96-nm repeat distance. The outer arms (bottom row) are composed of three dynein heavy chains (DHC) that repeat at 24-nm intervals. are anchored to the doublets with two dynein intermediate chains (IC). and are associated with numerous dynein light chains (LC) (only three of which are shown) (Witnian. 1992). On the other hand, the inner arms (upper row) repeat in a dyad, dyad, triad pattern, with the dyads connected at roughly the same area as the spoke attachments. The dyad associated with spoke S2 is also in close proximity to the protein complex referred to as the dynein regulatory complex (DRC). found on the face of the doublet between the dynein rows (Piperno ef NI., 1992; Mastronarde et nl., 1992; LeDizet and Piperno, 199Sa). The dynein light chains associated with the inner arms are not shown. Both the inner and outer dynein at-ms generally angle baseward from their doublet attachment (Avolio er d.,1984) and evidence suggests their power stroke pulls the N + 1 neighbor tipward. Points of possible regulation have been identified in the DRC. the dynein light chains. the dynein heavy chains. and the nexin links (see text).

arm dynein, those axonemes missing two or more types of inner arm dynein appear immotile (Okagaki and Kamiya, 1986; Kamiya et al., 1991; Kato et 01.. 1993). These observations suggest that the inner and outer arms play differcnt roles in producing the flagellar beat. The axonemal dimensions are highly conserved along with the structure of the outer doublets and central pair. This maintenance of size and composition dictates that interdoublet spacing and microtubule sliding displacement must also be highly conserved. Under the premise that this strict preservation of certain features may be necessary for the operation of the

6

CHARLES B. LINDEMANN AND KATHLEEN S. KANOUS

flagellar beating mechanism, examination of the variations that nature has permitted on this basic theme are considered next because they provide some interesting insights.

6.Variations on a Theme

+

Naturally occurring variants of the 9 2 axonemal pattern are occasionally seen that have not experienced a complete loss of function. The 9 + 0 pattern found in the sperm flagella of the Asian horseshoe crab (Ishijima et al., 1988) is perhaps the most striking variation. The waveform is more helical (less restricted to one bending plane), but the flagella retain motility. Although it could be argued that such evolutionary departures may have developed a compensatory mechanism to replace the central pair function, ample evidence has been procured demonstrating motility in both normal flagella that have extruded the central pair microtubules (Hosokawa and Miki-Noumara, 1987) and central pair-deficient mutations (Phillips, 1974; SchrCvel and Besse, 1975; Prensier et al., 1980; Gibbons et al., 1985; Brokaw and Luck, 1983; Ishijima et al., 1988). Although it does not inhibit motility, the absence of the central pair does apparently impair the ability to maintain a planar flagellarkiliary beat. This waveform variance could be a result of the incomplete partition because the central pair hub complex is missing from the normally stabilizing 3-central pair-8 structure. This presents an additional argument for the role of the bisecting partition in the maintenance of a normal planar beat. Experiments utilizing both rat and bull sperm confirm that some mammalian sperm axonemes are divided into two halves by this partition, as illustrated in Fig. 3 (Lindemann et al., 1992; Kanous et al., 1993). This feature, initially recognized in simpler flagellakilia (Afzelius, 1961; Tamm and Tamm, 1984; Sale, 1986), has been conserved, even as flagella increased in size and stiffness. In fact, rat sperm yield the greatest percentage of intact partitions (of the flagella studied), suggesting that the partition was reinforced as the flagellum was evolutionarily modified to increase its size. The 9 + 0 axonemal configuration also implies that the spoke apparatus may not be essential to the fundamental mechanism generating the flagellar beat. This is also supported by the induction of motility in spoke-free flagellar mutants (SchrCvel and Besse, 1975; Luck et al., 1977; Huang ef al., 1982; Brokaw et al., 1982; Gibbons et al., 1985). However, these cells usually demonstrate both altered motility (Huang etal., 1982) and reportedly fragile axonemes (Goldstein and SchrCvel, 1982; Gibbons et al., 1985). Therefore, as in the case of the central pair, the spokes are implicated as contributing stability to the axonemal structure yet appear less than crucial to basic motile functioning.

FLAGELLAR MOTILITY

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9.1.2 FIG. 3 The central partition of a rat sperm. The structure of the central partition was reconstructed from electron micrographs of disintegrating rat sperm axonemes. Using two different methods to disrupt the rat sperm flagellum, the microtubule axoneme can be made to come apart by interdoublet sliding. When pH 9.0 extraction was utilized, elements 4-7 were expelled, as shown in the upper inset. Prolonged ATP reactivation of Triton X-100-extracted models at 37°C caused the pattern of sliding disintegration depicted in the lower inset. In those cells, elements 1. 2, and 9 were expelled, emerging as a loop from the head-tail junction. In either case. the complex formed by elements 3, 8, and the central pair remained behind as a stable feature of the axoneme. Reprinted from Journal of Cell Science (Lindemann el al., 1992) with permission.

Attention has focused on the possible role of the spokes in activating inner arm dynein through the cluster of proteins called the dynein regulatory complex (DRC) (Piperno et al., 1992, 1994). The DRC is located on the A subtubule near or at the inner arm dynein attachment (Mastronarde et al., 1992; Piperno et al., 1992, 1994; Gardner et al., 1994; LeDizet and Piperno, 1995a) as illustrated in Fig. 2, and appears to be a factor in repressing dynein activity. The presence of radial spokes can counteract the inhibitory effect of the DRC on inner arm dynein (Smith and Sale,

8

CHARLES 6.LINDEMANN AND KATHLEEN

S. KANOUS

1992). Inner dynein arms exposed to spokes undergo a modification that persists even following the removal of the spokes (Smith and Sale, 1992). This points to the ability of the spokes to convey an “activating” signal to the inner dynein arms that coordinates the beat cycle. Observations that the central pair MTs appear to rotate during the beat cycle in certain axonemes (Omoto and Kung, 1979,1980: Omoto and Witman, 1981;Kamiya et al., 1982) have led to hypotheses describing a “distributor” scheme that uses the central pair to selectively activate particular doublets as a form of motility control (Omoto and Kung, 1979,1980; Huang et al., 1982; Huang, 1986).This selective activation of dynein-driven MT sliding may be involved in the flagellar motility of some species. However, those spokeless mutants that also lack an intact DRC are “derepressed” and capable of exhibiting coordinated beating (Porter et al., 1992; Piperno et al., 1994). From experimental evidence that protein kinase inhibitor improves motility in spokeless mutants, others surmise that the spokes regulate dynein activity by suppressing a CAMP-dependent protein kinase mechanism (Howard et al., 1994). In either case, a fairly complex interaction between the spokes and inner dynein arms seems adequately established. Evidence for derepression and kinase A regulation supports the premise that the primary system generating the flagellar beat can function without a spoke-based activation scheme. However, it also appears very likely that the spoke-dynein interaction plays a pivotal role in regulating or modulating the basic beat. This ability to modify the flagellar beat is essential for adaptations such as chemotaxis, phototaxis, and the capacitation/hyperactivation of mammalian sperm. Nature’s evolutionary diversions, resulting in larger flagellar structures that still maintain the basic axonemal apparatus, contribute additional information from a structural/functionaI perspective. One solution for creating a larger cilium that can basically perform the same task as a small cilium (only on a larger scale), without sacrificing velocity, was to unite a number of basic axonemes side by side (Sleigh, 1962). Such “compound” or “macro” cilia are widely observed in nature (Sleigh, 1968, 1974). To harness the power of multiple axonemes most beneficially in a compound cilium, each individual axoneme is oriented such that they all beat in the same plane, as shown in Fig. 4. In the compound cilia of the Ctenophore Beroe, each axoneme comprising the macrocilium is linked t o its neighbor by way of protein connections between doublet 8 of one axoneme and doublet 3 of the adjacent one, cementing them together in lateral rows (Afzelius, 1961; Tamm and Tamm, 1981, 1984). This arrangement, which is depicted in Fig. 4, substantiates that the permanent bonding of these elements between neighboring axonemes does not impair the basic beating mechanism. It also introduces the possibility that the 3-central pair-8 elements may be permanently interconnected because they do not need to slide relative to one another during the course of a normal beat. If this feature is actually

FLAGELLAR MOTILITY

9

FIG. 4 The construction of compound cilia. Thc stability of the partition formed by doublets 3 and 8. with the central pair, appears to form the basis for compound cilia assembly. A s illustrated in the diagram, many adjacent axonemes can be functionally linked by 340-8 connections while still permitting individual axonemes to retain their basic function. This arrangement was first described by Afzelius (lY61). Work by Tamm and Tamm (1984) established the stability of the 3-central p a i r 4 linkages in these structures by examining the pattern of microtubule sliding disintegration in the compound cilia of Beroe.

incorporated into the flagellar beat mechanism, maximal sliding must consequently occur between the doublets that interact with this 3-central pair-8 partition. In other words, doublets 2-3-4 and 7-8-9 must account for 60% of the dynein bridge turnover. The results of Warner’s (1979) experiments looking at ATP turnover as a function of axonemal position also point to these as the most active bridge sites.

C. Special Adaptations in Mammalian Sperm The development of compound cilia was only one of nature’s methods to scale up to a bigger flagellum. The sperm of mammals, insects, and birds incorporate a single axoneme to propel substantially larger flagella. The fundamental axoneme is similar lo that of smaller, simpler flagella in both size and interelement spacing (Fawcett and Phillips, 1970; Pedersen, 1970; Linck, 1979). Although the central axoneme displays spatial similarities to simple flagellakilia, there have been discreet evolutionary alterations that may have functional significance. The A subtubule of each outer doublet stains darkly in mammalian sperm (Pedersen, 1970), a phenomenon not observed in simpler axonemes. Additionally, the outer arm dynein of mammalian sperm is not easily removable using simple high-salt extraction (Marchese-

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CHARLES 6.LINDEMANN AND KATHLEEN S. KANOUS

Ragona et al., 1987), an effective method in more rudimentary axonemes (Gibbons and Fronk, 1972; Gibbons and Gibbons, 1973; Piperno and Luck, 1979). These incongruities caution that it would be an oversimplification to assume that the central axoneme itself has not been modified to accommodate an increase in flagellar size. Nevertheless, the basic proportions and structural composition of the basic axoneme appear to have been conserved. In addition to the aforementioned potential differences within the axoneme, an interesting pattern of indisputable modifications evolves allowing the increase in size. The predominant and most conspicuous flagellar modification is the presence of accessory MTs andlor non-MT auxiliary fibers contiguous with the basic nine outer doublets (Fawcett and Phillips, 1970; Fawcett, 1975; Baccetti, 1982; Dallai and Afzelius, 1993). The auxiliary fibers in some of the largest mammalian sperm (referred to as outer dense fibers; ODFs) can be as large as 260 nm in diameter, literally dwarfing the central axoneme (Phillips, 1972) . A cross section of a bull sperm axoneme illustrating the accessory structures is shown in Fig. 5. What was nature’s intended purpose for including these ODFs in the scaled-up version of the motile organelle? Although initially presumed to be contractile motor elements, originating as outgrowths of the outer doublets (Fawcett and Phillips, 1970), later biochemical studies have not successfully identified contractile proteins from isolated ODFs (Price, 1973; Baccetti et af., 1976; Olson and Sammons, 1980). Several investigators propose that the ODFs in mammalian sperm act to reinforce the structure, making the longer flagellum both stronger and stiffer (Phillips, 1972; Fawcett, 1975; Baccetti et af., 1976; Baltz et al., 1990). This view gains support from micromanipulatory techniques that yield a direct flagellar stiffness measurement at the bull sperm flagellar base 20 times greater than that of sea urchin sperm (Lindemann et af., 1973). The measured stiffness was also found to diminish along the flagellar length (Lindemann et af.,1973), corresponding to the fact that the ODFs taper toward the flagellar tip and fail to reach the endpiece (Telkka et af.,1961; Pedersen, 1970; Serres et af.,1983a). Motile human and bull sperm flagella demonstrate an increase in bend curvature as the bend propagates down the tail (Gray, 1958; Rikmenspoel, 1965; Serres et af., 1983b), where the ODFs progressively disappear. However, in sea urchin sperm, which are devoid of ODFs, the maximal curvature is achieved not far from the flagellar base (Gray, 1955; Gibbons, 1982). These motility characteristics implicate O D F stiffness as impacting the waveform of larger flagella. O D F size has been correlated to the length of mammalian sperm flagella, with the longest sperm generally containing the largest ODFs (Phillips, 1972; Baltz et af., 1990). Comparison of spermatozoa1 motility from a variety of mammalian species demonstrates that the achievable bend amplitude is basically inversely proportional to the magnitude of the dense fibers (Phillips, 1972;

FLAGELLAR MOTILITY

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FIG. 5 Special features of the mammalian sperm axoneme. A TEM cross section of a bull sperm axoneme is displayed, with the outer doublets numbered in the same convention applied to other cilia and flagella. The presence of a fibrous (dense staining) outer sheath around the axoneme identifies the section as one from the flagellar principal piece. The outer dense fibers (ODFs) are attached to their respective doublet over much of their length, particularly in the principal piece region. These ODFs are largest at the flagellar base and taper away to a termination point part way down the principal piece. Reproduced from Kanous ei al. (1993) with permission.

Phillips and Olson, 1973). Regarding the strengthening ability of the ODFs, tensile strength measurements of large sperm flagella (which are generally more vulnerable to the killing effect of shear forces than shorter flagella) demonstrate that the ODFs account for an increasing proportion of overall tensile strength relative to the length of the sperm (Baltz et al., 1990). ODFs appear to have other functions in addition to their contribution as structural strengtheners and stiffeners. Studies show that the ODFs in

CHARLES B. LINDEMANN AND KATHLEEN S. KANOUS

12

mammalian sperm are physically attached to the connecting piece at the flagellar base (Pedersen, 1970; Fawcett, 1975) as diagramed in Fig. 6. They are also attached to the outer doublets along much of their length (Lindemann and Gibbons, 1975; Olson and Linck, 1977), particularly in the distal end (Fawcett and Phillips, 1970). When the mammalian sperm axoneme is disrupted by proteolytic digestion, the doublets can be induced to slide apart with Mg-ATP. Upon inspection, it is generally found that the ODFs remain attached to the connecting piece and the extruded doublets (Olson and Linck, 1977; Lindemann and Gibbons, 1975; Lindemann ef al., 1992; Kanous et al., 1993). In simple flagella, the outer doublets must be anchored in a basal body or centriole to produce motility. In mammalian sperm, the distal centriole (which nucleates the development of axonemal MTs) disintegrates during spermatogenesis (Fawcett and Phillips, 1970; Woolley and Fawcett, 1973). This leaves the ODF-connecting piece complex as the sole basal anchor for the entire axonemal-periaxonemal structure. Figure 6 illustrates this concept. Therefore, in the mammalian sperm, force produced by interdoublet sliding is transferred to the ODFs and thereby transmitted to the basal anchor-connecting piece. The peripheral location of the ODFs amplifies the amount of torque that can be developed between

Basal anchor Outer doublet

I

T 1 I Striated columns

Central pair

FIG. 6 Force transfer in the mammalian sperm axoneme. A schematic, longitudinal view of a mammalian sperm is depicted. Unlike simple cilia and flagella. the basal body (distal centriole) of mammalian sperm flagella disassembles during development (Fawcett. 1975). leaving the doublets without a direct anchor to the flagellar base. The proximal centriole (PC) remains but is perpendicular to the flagellar shaft. However, the ODFs are securely anchored into the striated columns of the connecting piece, which forms a cap-like structnre at the flagellar base. In mammalian sperm the doublet attachment to the ODFs acts to supply the necessary basal anchoring that allows bend production. The doubletlODF connections also allow the dynein-tubulin interdoublet sliding force to be transferred to the ODFs. Because the distance between the ODFs can be considerably larger than the interdoublet distances, the flagellar force development acting over a larger working diameter produces substantially greater bending torque than would be possible with a simple axoneme. The ODFs and fibrous sheath also serve to stiffen and stabilize the axoneme. a necessary function in accommodating the greater torque development. Adapted from Lindemann (1996). Reproduced with permission.

13 ODFs (over that possible between outer doublets) due to the increased separation distance (which determines the lever arm length for torque production). In some mammalian sperm with extremely large ODFs, such as the ground squirrel, the separation distance between the ODFs becomes many times that of the isolated axoneme (Fawcett and Phillips, 1970). The curvature of the bending waves in large mammalian sperm is less than in smaller flagella. This requires each single bend to include a much longer section of the axoneme, thereby involving more dynein arms. The force contributing to bend formation is proportional to the number of dynein bridges pulling together. Consequently, the force developed to bend the flagellum in mammalian sperm must be greater. If this force is exerted across the longer lever arm provided by the ODFs, the torque (force X lever arm) is substantially magnified. Ultimately, the secret of the megaflagellum probably resides in this relationship (Lindemann, 1996). The ODFs allow a greater accumulation of dynein force plus an increased lever arm, which results in greater bending torque production. Simultaneously, ODFs provide additional stiffening to balance this enhanced force generation. This modified mammalian axoneme is additionally surrounded by a substantial sheath of mitochondria at the midpiece and a fibrous protein sheath at the principal piece (Fawcett and Phillips, 1970; Fawcett, 1975). Although simple invertebrate sperm possess a minimal mitochondria1 sheath, the fibrous sheath of the principal piece is unique to mammalian sperm (Fawcett, 1975). The presence of these supplemental exterior coverings may counteract the increased internal forces in large mammalian sperm, maintaining the integrity of an axoneme that might otherwise rupture. The feasibility of using one power source, provided by the central axoneme, to drive substantially larger mammalian sperm has been tested using a computer model (which will be discussed in more detail later in this chapter). When scaled to incorporate both the measured stiffness of bull sperm and the greater bending torque produced by ODF involvement, the model does in fact beat much like a bull sperm flagellum (Lindemann, 1996). FLAGELLAR MOTILITY

111. The Motor A. The Dynein ATPase The molecular motor dynein was first identified as an adenosine triphosphalase protein from KCl (0.1-0.6 M ) extracts of Tetruhymenupyriformis and isolated as 30 and 14s fractions using sucrose density gradient fractionation (Gibbons and Rowe, 1965). The ATPase activity of these fractions could be activated by either Ca2+or Mg”, with the 14s fraction more specific

14

CHARLES B. LINDEMANN AND KATHLEEN S. KANOUS

for Mg ion activation. Both dyneins were quite specific for ATP, hydrolyzing other nucleoside triphosphates at less than 10% of the ATP rate, and even ADP was only hydrolyzed 30% as rapidly (Gibbons and Rowe, 1965). Gibbons and Rowe (1965) speculated that dynein formed the “arms” of the axoneme, and experiments demonstrated that KCl extraction of 30s dynein coincided with the disappearance of the outer arm projections on the axonemal doublets (Gibbons, 1965; Gibbons and Fronk, 1972; Gibbons and Gibbons, 1973). KC1-extracted outer arm dynein, from Tetrahymena (Gibbons, 1965; Shimizu, 1975) or Triton X-100 stripped sea urchin sperm (Gibbons and Gibbons, 1973), was found to be capable of microtubular reattachment when the salt concentration was reduced. Using this method, it was demonstrated that removal of outer arm dynein reduced the beat frequency of Mg-ATP-reactivated sperm flagella (Gibbons and Gibbons, 1973), whereas reattachment could reinstate an increased beat frequency (Gibbons and Gibbons, 1976). Experiments such as these definitively established the axonemal arms as the location of the dynein ATPase, also confirming a role for dynein in the mechanism of flagellar motility. Furthermore, there exist outer arm-deficient Chlamydomonas mutants (oda) that beat at half the frequency of wild type (Brokaw and Kamiya, 1987). However, adding outer arm dynein (extracted from wild-type Chlamydomonas) to demembranated flagella of oda mutants increases their beat frequency to nearly that of the wild type (Sakakibara and Kamiya, 1989;Takada et al., 1992). Correspondingly, comparisons of ATPase activity between wild-type and oda mutants demonstrate that the activity of axonemes with outer arms was 5-12 times that of the arm-depleted mutant (Kagami and Kamiya, 1990). The ATPase activity of dynein can be facilitated (up to 30 times) by the presence of microtubules or outer doublets (Warner et af., 1985; Omoto and Johnson, 1986; Warner and McIlvain, 1986; Shimizu et al., 1989, 1992), much as the ATPase activity of myosin is facilitated by actin. Holzbaur and Johnson (1989) postulate that this ATPase activation is due to the microtubule effect of accelerating the rate of ADP release. Brokaw and Benedict (1968) established early on that there exists both a motility-dependent and a motility-independent rate of ATPase activity in intact axonemes. A number of studies have since confirmed a sliding-dependent enhancement of dynein ATPase activity (Gibbons and Gibbons, 1972; Penningroth and Peterson, 1986). A straightforward interpretation of motility-dependent ATPase activity could be based on the possibility that coordinated beating increases MT enhancement of dynein ATPase compared to nonmotile axonemes. Utilizing this simple viewpoint, the coordination mechanism of the beat cycle augments the opportunity for dynein-tubulin cross-bridge formation.

FLAGELLAR MOTILITY

15

Dynein ATPase has been extensively investigated, and it is now known that axonemal dyneins constitute a variety of unique proteins. These proteins are all members of a larger group of related molecular motors found in association with the MT cytoskeleton and involved in myriad applications. In their functional, nondenatured state, dyneins are huge protein complexes composed of a number of individual polypeptides (Piperno and Luck, 1979), designated as heavy chains (DHC, 400-500 kDa), intermediate chains (IC, 55-125 kDa), and light chains (LC, =20 kDa) (see Fig. 2). Each dynein is composed of from one to three DHCs and a variable number of ICs and LCs (Porter and Johnson, 1989). The basic form of each dynein arm consists of two or three globular “heads” connected by a “stalk” or “stem” to a common base attachment on the A tubule of the outer doublet. The DHCs make up the heads and a portion of the stems and contain the ATP binding sites (Johnson and Wall, 1983; Shimizu and Johnson, 1983; Pfister et al., 1984; Pfister and Witman, 1984). Each globular head is attached to a stalk composed of an a-helical portion of the DHC (Mitchell and Brown, 1994; Wilkerson et al., 1994).The DHC portion of the stalk does not bind directly to the A tubule but is anchored by an attached IC protein of the dynein complex (King and Witman, 1990; King et al., 1991, 1995; Witman, 1992; Mitchell and Kang, 1993; Wilkerson et al., 1995). The reader may refer to Fig. 2 for an illustration of the structure. The function of ICs in the ATP-insensitive, structural coupling of dynein to the A tubule is still not clear, although there is evidence for a role in attachment and localization (King and Witman, 1990; King et al., 1991; Gagnon et al., 1994). Immunoelectron microscopy was used to localize 78and 69 (70?)-kDa ICs to the base of Chlamydomonas outer arm dynein (King and Witman, 1990). A direct association of IC78 and IC69 has also been established (King et al., 1991,1995).The tendency for IC78 to interact with a-tubulin in an ATP-insensitive manner (King et al., 1991) and its recent identification as a microtubule-binding protein (King et al., 1995) suggest that IC78 plays a role in outer dynein arm attachment to the A tubule of the outer doublets. Certain outer arm-deficient Chlamydomonas mutants ( o d d and o d d ) require the addition of a 70-kDa polypeptide (IC69?) to facilitate attachment of isolated 12s and 18s outer arm dyneins to the outer doublets (Takada and Kamiya, 1994). This 70-kDa fraction is present in wild-type and other oda mutants and forms a pointed structure on the A tubule. Experiments (Takada et al., 1992) had shown that the complete three-headed Chlamydomonas outer arm dynein could combine with o h axonemes, although the separate 12s and 18s dyneins could not, Takada et al. speculated that a functional component necessary for reassociation was missing. In an even earlier study (Mitchell and Rosenbaum, 1986),monoclonal antibody examination found that anti-70 kDa did

16

CHARLES

B. LINDEMANN AND KATHLEEN S. KANOUS

not comigrate with 18s dynein following sucrose gradient extraction. The 70-kDa intermediate chain had dissociated from the 18sdynein and instead was part of a smaller protein aggregate. It could be deduced from these results that the 70-kDa IC is necessary for proper attachment and localization of the outer dynein arms, and this protein can either be part of the extracted outer arm dynein or removed by certain extraction methods. Dynein LCs have been identified as sites of CAMP-dependent phosphorylation (Hamasaki et af., 1989, 1991; Tash, 1989; Stephens and Prior, 1992; Salathe et af., 1993; Barkalow et al., 1994; Satir et al., 1995). Some LCs are associated with the DHCs (Pfister et al., 1984; Mitchell and Rosenbaum, 1986; Witman, 1992; King and Patel-King, 1995) and are believed to play a part in modulating motor function. A recently investigated LC that binds Ca2+ and is associated with the y-DHC of Chfamydomonas outer arm dynein demonstrated significant homology with calmodulin (King and Patel-King, 1995). Based on its in vitro affinity for 3 X lo-’ M Ca’+, it was speculated that this LC may modulate Ca”-mediated dynein activity (such as waveform symmetry and the flagellar reversal of photophobic responses). LCs have also been localized with the ICs at the A tubule connection (Mitchell and Rosenbaum, 1986; Stephens and Prior, 1992; Witman, 1992) and are thought to regulate dynein arm flexibility or interaction (Stephens and Prior, 1992). A 28,000 MW light chain (p28) has been detected that associates with a subset of Chfamydomonasinner arm D H C (LeDizet and Piperno, 1995a,b). ida4 mutants are missing the p28 protein (encoded by the IDA4 gene) (LeDizet and Piperno, 1995b). The specific DHC subset that complexes with p28 is also missing, suggesting that p28 participates in either the binding of these arms to the axoneme or their assembly (LeDizet and Piperno, 1995a,b). Although morphologically similar, comparison of inner and outer dynein arms demonstrates both structural and functional diversity. Dynein outer arms possess two heads (dyads) in many species (Le., pig tracheal cilia and sea urchin, bull, and trout sperm), whereas three-headed (triad) versions are common in protists (i.e., Chfamydomonas,Tetrahymena,and Paramecium) (Johnson and Wall, 1983; Shimizu and Johnson, 1983; Holzbaur and Vallee, 1994). In either case, the outer arms maintain one form throughout the axoneme, repeating at 24-nm intervals (Warner et al., 1985; Warner, 1989). Dynein inner arms also display dyad and triad formations but combine both varieties on each axoneme in a pattern of two dyads to each triad (Warner et af., 1985; Goodenough and Heuser, 1985, 1989). These arms maintain a periodicity of 24, 32, and 40 nm (a total 96-nm repeat), in agreement with the radial spoke repeat pattern (Warner et af., 1985; Goodenough and Heuser, 1985). The pictorial representation of the relationship of the dynein arms on a doublet as presented in Fig. 2 is modeled on accumulated data obtained from studies on Chlamydomonas.

FLAGELLAR MOTILITY

17

B. The Dynamics of Dynein-Tubulin Sliding Dyncin powers the flagellar beat by translocating each outer doublet relative to its neighbor in a process known as “microtubule sliding.” Satir (1965, 1968) was the first to experimentally confirm that outer doublets of intact axonemes slide. He examined TEM fixed, serially sectioned beating cilia of Elliptio cornplanatus (freshwater mussel) to locate doublet termination points and found the MTs to be uniform in length, verifying that they do not stretch or contract. The development of a method to create detergentextracted “models” of cilia/flagella, which could be “reactivated” with MgATP to simulate the motility of their intact counterparts, permitted induced biochemical modifications within the axoneme and examination of the effects on flagellar motility (Gibbons and Gibbons, 1972). Summers and Gibbons (1971, 1973) demonstrated that brief tryptic digestion of detergentextracted (modeled) sea urchin sperm axonemes, followed by application of Mg-ATP, resulted in axonemal disintegration by longitudinal sliding of outer doublets. In the absence of the basal body, the doublets were observed to telescope up to eight times the original length of the flagellar fragment. MT sliding in this manner implied that the dynein arms are in a unipolar arrangement around the axoneme. The extent to which the MTs were observed to telescope demonstrated that each doublet pair could participate in sliding (except the 5-6 pair. which is permanently bridged, as described earlier). Sale and Satir (1977) conclusively identified the direction and order of MT sliding. The outer doublets were recognized to be sliding baseward on their higher numbered neighbor (based on the numbering system of Afzelius, 1959). This axonemal organization has proven t o be a uniform attribute thus far. As a consequence of this arrangement, the dynein arms on doublets 1-4 would generate sliding to bend the flagellum in one direction, whereas those on doublets 6-9 work to bend it in the opposite. Although efforts to “see” how the dynein arms produce sliding have resulted in interesting findings, the evidence has not been conclusive. Micrographs from the work of Goodenough and Heuser (1982,1985, 1989) with freeze-etch replicas (flagella that have been fast frozen during activity) suggest that, rather than bridging by way of the globular heads, dynein arms are linked to adjacent doublets through thin connections called “B links.” However, it is difficult to conceive of a mechanism capable of conveying lateral force through what appears to be such a slender thread of material. The axonemal dynein arms must exert force between adjacent doublets separated by a considerable distance (19-21 nm) (Warner, 1978; Goodenough and Heuser, 1982). It seems likely that some means of mechanical triangulation would be required to achieve this goal. A structural scheme with this conceptual advantage was proposed by Avolio et al. (1984) based on their own electron micrographs, whereby some dynein arms link

18

CHARLES B. LINDEMANN AND KATHLEEN

S. KANOUS

MTs by uniting the multiple globular heads into a triangular-shaped structure that angles baseward toward the higher numbered neighboring doublet. Although these two descriptions of dynein arm structure seem to be mutually exclusive, other studies demonstrate that conformational changes occur in the presence or absence of ATP (Witman and Minervini, 1982), and varied interpretations of dynein structural composition can be obtained depending on the angle of viewing (Witman and Minervini, 1982.) A substantial amount of information on dynein-tubulin interaction has been obtained utilizing either partially disrupted axonemes or isolated dynein in combination with MTs. The MT sliding kinetics of disintegrating axonemes reveals maximal free-sliding rates in the 12-18 pmls range (Yano and Miki-Noumura, 1980; Okagaki and Kamiya, 1986; Sale, 1986; Kurimoto and Kamiya, 1991). Estimates of sliding velocities in working flagella (proportional to shear amplitude X frequency) yield rates in the range of 10-19 pmls (Brokaw and Luck, 1983; Brokaw and Kamiya, 1987; Eshel and Gibbons, 1989). The sliding rate is sensitive to load, and a force-velocity relationship has been measured for dynein-tubulin sliding in sea urchin sperm (Kamimura and Takahashi, 1981; Oiwa and Takahashi, 1988). Functional dynein motors can be isolated by low ionic strength buffer dissociation or high salt extraction of flagella or cilia. These isolated dyneins, when adsorbed to a glass slide or coverslip, can translocate MTs or doublets applied to the glass surface in the presence of ATP (Paschal et a/., 1987; Sale and Fox, 1988; Vale and Toyoshima, 1988, 1989a; Vale et a/., 1989; Kagami et al., 1990; Hamasaki et al., 1991; Kagami and Kamiya, 1992; Yokota and Mabuchi, 1994). Reported MT translocation rates induced by dynein are quite high (3.5-5.6 pmls) (Paschal et a/., 1987;Sale and Fox, 1988) considering the inability to control the number of actively participating arms or their orientation (because MT translocation by dynein is unipolar). These velocities are higher than those reported for other motor proteins such as kinesin at 0.3-0.6 pmls (Vale et al., 1985; Vale and Toyoshima, 1989b; Sheetz, 1989; von Massow et al., 1989; Shirakawa et al., 1995). The rapid MT sliding produced by axonemal dynein is necessary to maintain the rapid beating of cilia and flagella, whereas other molecular motors, such as those driving chromosomal motility, can operate at ik the velocity (Vale, 1992). Additionally, the translocation rate does not appear to be directly dependent on the number of participating dynein arms. Using adsorbed dynein concentrations of 44 pg/ml, MTs would not even attach to the glass, whereas 50 pglml of dynein translocated numerous MTs at near maximal velocity (Vale ef al., 1989). Borderline critical concentrations of adsorbed dynein produced slower sliding velocities, most probably due to the tendency for MTs to pause more frequently during translocation (Vale and Toyoshima, 1989a) rather than a decrease in the actual speed of MT movement. When experiments were conducted in which dynein molecules were densely

19 adsorbed to glass and aligned with the same polarity (Mimori and Miki-Noumura, 1994), MT translocation rates were consistently higher (12 pm/s). When utilizing low concentrations ( 4 0 pg/ml) of randomly adsorbed dynein, longer MTs were translocated faster and over longer distances, most likely due to their increased potential for maintaining contact with randomly distributed dyneins (Vale and Toyoshima, 1989a). An earlier study (apparently using higher concentrations of adsorbed dynein) revealed that translocation rates were independent of MT length (Paschal et al., 1987), whereas a later study (Hamasaki et al., 1995a) demonstrated an initial velocity increase with increased MT length, which then reached a plateau. The experimental data imply that velocity is independent of the number of dyneins producing the force, beyond a certain critical limit. This concurs with observations that MT sliding rates in disintegrating axonemes do not change as sliding progresses, even though the number of dyneins involved in force production must change as the area of doublet overlap decreases (Takahashi et al., 1982). These results point to a threshold-type mechanism in which the minimal number of properly aligned dyneins necessary to bind the MT are capable of propelling it at near maximal velocity. A similar finding was observed involving kinesin molecules (Vale et al., 1989), in which MT translocation speed was independent of kinesin density as long as ATP was available at a saturating concentration of 1 mM. FLAGELLAR MOTILITY

C. Axonemal Dynein Diversity-Variations in Function Both outer arm dynein (Paschal et al., 1987; Vale and Toyoshima, 1988; Sale and Fox, 1988) and inner arm dynein (Kagami et al., 1990; Smith and Sale, 1991; Kagami and Kamiya, 1992; Yokota and Mabuchi, 1994) will translocate MTs in vitro. There is a reported difference (Vale and Toyoshima, 1989a) between the maximal translocation rates of 8-12 pm/s for Tetrahymena 22s outer arm dynein and 4 or 5 pmls for 14s inner arm dynein. 22s dynein was positively identified as outer arm by Ludmann et al. (1993); 14s is not conclusively inner arm dynein, although supportive evidence has been presented (Warner et al., 1985; Warner and McIlvain, 1986; Vale and Toyoshima, 1988). This concurs with higher sliding velocities observed in disintegrating axonemes possessing outer arms (Hata et a/., 1980: Okagaki and Kamiya, 1986; Kurimoto and Kamiya, 1991). As described earlier, three different inner arm dynein complexes alternate within each 96-nm section along the flagellar axoneme. This substantiates the existence of three forms; however, seven distinct subspecies of inner arm dynein have been identified in Chlumydomonas (Kagami and Kamiya, 1992). MT rotation during translocation has been attributed t o certain inner arm dyneins (Vale and Toyoshima, 1988; Kagami and Kamiya,

20

CHARLES B. LINDEMANN AND KATHLEEN S. KANOUS

1992), a function that may prove integral in ultimately determining the specific role inner arm dynein plays in flagellar/ciliary motility. In addition, differences in the inner dynein arms located in the proximal and distal areas of Chfamydomonas flagella have been reported (Piperno and Ramanis, 1991). Support can be found for the idea that inner arm dynein force production is negligibly affected by viscous resistance (Minoura and Kamiya, 1995), whereas outer arm functions change under increased viscosity (Brokaw, 1996). This inner arm immunity to viscous load exists even though internal and external resistances vary along the axoneme, such that the force produced by proximally located inner arm dyneins varies from the force produced in the distal portion of the axoneme (Asai. 1995). Although distributed over the length of the cilia (Moss and Tamm, 1987), variability in the localized sensitivity to calcium in Beroe has also been identified (Tamm, 1988), with the highest response residing in the basal region. This lengthwise axonemal inner arm dynein differentiation could also be related to the functions of bend initiation (in the proximal region) and bend propagation (distally) (Witman, 1992). There is also evidence that dyneins that bend the flagellum in each of the two planar bend directions may be different from each other or at least have different activation controls. The asymmetric beat stroke of many flagellakilia consists of two distinct bending waves, a more tightly curved principal (P) bend in one direction and a reverse (R) bend of lesser curvature in the opposite direction. This suggests that the axoneme is structurally asymmetric, with opposite halves of the axoneme active at different times during the flagellar/ciliary beat ( Wais-Steider and Satir, 1979; Satir, 1985). A significant difference between the forces producing opposite flagellar bends in sea urchin sperm has been observed (Eshel ef al., 1991). although the source of this disparity has not been established. Morphological asymmetries of Chlamydomonas axonemes have been identified but not explained (Hoops and Witman, 1983). The sliding of MTs on the 2,3,4 side of the rat sperm axoneme can be selectively suppressed using a pH 9.0 extraction, whereas the same procedure does not disable the 7,8,9 dynein bridges (Lindemann et af., 1992). pH sensitivity differences between the mechanisms generating R and P bends in sea urchin sperm have been recognized (Goldstein, 1979). A more recent study has discovered a Chfumydomonas mutation (bop2-1)that demonstrates (i) flagellar motility patterns similar to those of inner arm mutants (ii) a missing 152-kDa phosphoprotein, and (iii) ultrastructural, doublet-specific, radial asymmetry in the dynein inner arm region of the axoneme (King et af., 1994). When exposed to threshold levels of Ca2+,many flagellakilia exhibit either an arrest response (Walter and Satir, 1978; Gibbons and Gibbons, 1980; Sale, 1986; Stommel, 1986; Satir et af.,1991; Shingyoji and Takahashi, 1995) or a change in beating waveformlsymmetry (Miller and Brokaw,

FLAGELLAR MOTILITY

21

1970; Brokaw ef al., 1974; Brokaw, 1979; Brokaw and Goldstein, 1979; Bessen et al., 1980;Brokaw and Nagayama, 1985; Izumi and Miki-Noumura, 1985: Lindemann and Goltz, 1988). The Ca” arrest response usually leaves the flagellum at one extreme of the beat cycle rather than a straight, relaxed (equilibrium) position. Gibbons and Gibbons (1 980) proposed that calcium-induced quiescence (in reactivated sea urchin sperm) is not a totally passive state but rather an asymmetric activation of dynein arms on only one side of the axoneme. Sale (1985) credits calcium with the ability to override or bypass the normal activation mechanism, thus eliciting quiescence in dcmembranated sperm. The arrest position, in the principal bend direction, may result because the flagella are trapped at the end of the principal bend. Nickel ion addition arrests motility in cilia and flagella (Naitoh and Kaneko, 1973; Lindemann et ul., 1980; Larsen and Satir, 1991) and has been shown to block the flagellar Ca2+response (Lindemann and Goltz, 1988; Satir etal., 1991). Ni” also demonstrated the ability to block sliding between MT doublets 2 and 3 during bull sperm flagellar disintegration while allowing sliding between doublets 7 and 8 (Kanous et al., 1993). Mechanically manipulating Ni?+-arrestedbull sperm revealed that force production in one bending direction is selectively inhibited as shown in Fig. 7A (Lindemann et a/., 1995). The normal beat of bull sperm was altered when Ni” was added to ATP-reactivated cells. Flagellar bending became more and more asymmetrical because bending in one direction decreased progressively until the motility arrested with the flagellum curved in a sustained bend in the opposite direction (Lindemann et al., 1995). This not only implies that nickel selectively inhibits only the dynein bridges on one side of the axoneme but also that switching during normal beating depends on reciprocal action between the two halves of the axoneme.

IV. Regulation of Flagellar Motility A. Signal Pathways Two types of regulatory control are widespread (if not universal) attributes of eukaryotic flagella and cilia. First, both flagella and cilia can be turned on (activated) or turned off (deactivated) by phosphorylation/dephosphorylation of axonemal proteins. Second, the shape of the flagellar/ciliary wave can be altered to produce a more symmetrical (equal in both bend directions) or asymmetrical (lopsided, more pronounced in one direction than the other) beat. The activation/deactivation control has been linked to the cAMP/kinase A signaling pathway (Garbers et a/., 1971, 1973a,b; Morton

CHARLES B. LINDEMANN AND KATHLEEN S. KANOUS

22

A

Equilibrium

B

FIG. 7 Restoring a beat cycle in Ni2+-treated sperm. Spontaneous beating can be inhibited in ATP-reactivated, Triton-extracted bull sperm models by the addition of 0.4-0.6 mM nickel ion. The flagella cease moving, arresting in a curved endpoint of the beating pattern. (A) If these inhibited sperm are manipulated with a microprobe so as to bend the flagellum in the direction opposite to the prevailing curvature, the flagellum exhibits an active response to the probe. This active response is not elicited if the same flagellum is pushed in the same direction as the prevailing curve. (B) If the flagellum is pinioned and held in the position that triggers an active response, a pattern of repetitive beating can be reestablished in flagella following Ni2+treatment. Reproduced from Lindemann et al. (1995) with permission.

FLAGELLAR MOTILITY

23

eta/., 1974; Lindemann, 1978; Morisawa and Okuno, 1982; Morisawa et al., 1983, 1984; Opresko and Brokaw, 1983; Brokaw, 1984; Lindemann et a/., 1987; Brokaw, 1987; Ishida et al., 1987; Tash and Means, 1988; Tamaoki et al., 1989; Chaudhry et al., 1995). The modification of the beat waveform has been traced to a cytosolic Ca2+-mediatedcontrol (Miller and Brokaw, 1970; Brokaw et al., 1974; Brokaw, 1979; Brokaw and Goldstein, 1979; Bessen ef al., 1980; Brokaw and Nagayama, 1985; Lindemann and Goltz, 1988). These two control pathways make a variety of responses possible in the living cells.

6 . Responses in the Living Cell It may be beneficial to take note of the role that flagellarcontrol mechanisms play in nature. Gametes lack RNA to synthesize replacement proteins (parts of the living machinery), consequently the high metabolic rate required t o support flagellar motility ultimately leads to senescence (Norman et al., 1962). Therefore, it seems likely that the ability to turn off the motor mechanism during sperm storage could delay the period of effective motility to coincide with the opportunity to achieve successful fertilization. The flagella of preejaculatory sperm are quiescent in many species, becoming activated to motility only upon release/dilution just prior t o fertilization. This is true of sperm from invertebrates, such as sea urchin and tunicates (Lee ef al., 1983; Brokaw, 1984), as well as vertebrates, including fish (Morisawa et al., 1983; Morisawa and Ishida, 1987; Morisawa and Morisawa, 1988) and mammals (Morton et a/., 1974, 1979; Cascieri et al., 1976; Turner and Howards, 1978; Mohri and Yanagimachi, 1980; Wong et al., 1981; Chulavatnatol, 1982; Carr and Acott, 1984; Usselman et al., 1984; Turner and Reich, 1985). A sperm motility activation mechanism is triggered by various stimuli in different species. Sperm dilution into hypotonic (freshwater) or hypertonic (seawater) environments at spawning (which lowers K + and increases CAMP)activates flagellar motility in some fish and amphibians (Morisawa and Suzuki, 1980; Morisawa el af., 1983; Morisawa and Ishida, 1987: Christen et a/., 1987; M. Morisawa and Morisawa, 1988; S. Morisawa and Morisawa, 1990). Intracellular alkalinization after exposure to seawater stimulates sea urchin sperm motility (Nishioka and Cross, 1978; Christen et al., 1982; Lee et al., 1983; Shapiro e f al., 1985). In most mammals, mixing with seminal fluid (to lower Ca”. dilute an inhibiting factor, or contribute H C 0 3 - ? ) induces sperm flagellar activity (Morisawa and Morisawa, 1990). Once sperm have been activated, the duration of flagellar motility is usually fairly brief, ranging from seconddminutes in some freshwater fish (Ginsburg, 1963; Nelson, 1967; Okuno and Morisawa, 1982; Christen et al., 1987; Billard and Cosson, 1990) to hourddays in most mammals (Soderwall and

24

CHARLES B. LINDEMANN AND KATHLEEN S. KANOUS

Blandau, 1941; Green, 1947; Vandeplassche and Paredis, 1948; Tyler and Tanabe, 1952; Perloff and Steinberger, 1964; Doak et al., 1967; Thibault, 1973; Parker, 1984; Critchlow etal., 1989). It should be noted that the actual in vivo motility period is difficult to determine in mammals (and other animals in which internal fertilization takes place) because of the tendency for sperm to be stored in a virtually dormant state for various periods within the female reproductive tract (Thibault, 1973; Katz, 1983; Smith and Y anagimachi, 1990). The ability of a flagellated cell to change its beating waveform in response to external stimuli makes the observed behaviors of chemotaxis and phototaxis possible. A chemotactic waveform response of sperm flagella was first characterized in studies of Nereis, Arbacia (Lillie, 1913), and Tubuluria sperm (Brokaw et al., 1970; Miller and Brokaw, 1970). Exposure to compounds released from egg jelly induced a change in swimming direction by an asymmetry in flagellar beating. The flagellar bends in one beat direction became increasingly curved, biasing the overall beating curvature to one side and causing the sperm path to curve into a circular arc. This pattern of motility was later duplicated in reactivated sea urchin sperm by elevating intracellular Ca2+within the range of lo-’ to lo-‘ M (Brokaw e? al., 1974; Brokaw and Goldstein, 1979; Brokaw, 1987). The phototactic response (beat reversal) in wall-less Chlamydomonas mutants (Schmidt and Eckert, 1976), and the “mechanoshock” avoidance response of the green alga Spermatozopsis similis (Kreimer and Witman, 1994), were found to be M calcium. The “photostop” dependent on the presence of at least response of intact Chlamydomonas was found to require a minimum of 300 nM external calcium, with increased calcium inducing prolonged stop durations (Hegemann and Bruck, 1989). Ciliary beat reversal in Parameciitm was also shown to be triggered by increasing internal calcium levels (Naitoh, 1968, 1969; Naitoh and Kaneko, 1972, 1973), and the mutant Paramecium “pawn” (which does not exhibit beat reversal while intact) will demonstrate backward swimming in response to calcium when the membrane has been extracted with Triton X-100 (Kung and Naitoh, 1973). A calcium-induced change in waveform has been identified in Crithidia (Holwill and McGregor, 1976) and reactivated Chlamydomonas (Hyams and Borisy, 1978; Bessen et al., 1980) and Tetrahymena (Izumi and MikiNoumura, 1985) as well, thus demonstrating this calcium modification of waveform to be a fairly prevalent phenomenon in flagellakilia. The calcium response requires the presence of calmodulin (Rauh etal., 1980; Brokaw and Nagayama, 1985), which has been detected in cilia and flagella ( Jamieson et al., 1979; Gitelman and Witman, 1980; Jones et al., 1980; Feinberg et al., 1981; Ohnishi et al., 1982; Stommel et al., 1982; Gordon et al., 1983). Mammalian sperm also demonstrate modifications in both swimming pattern and flagellar waveform in response to physiological conditions en-

FLAGELLAR MOTILITY

25 countered in the female reproductive tract (Katz and Yanagimachi, 1980; Katz, 1983; Suarez et af.,1983; Suarez and Osman, 1987; Shalgi and Phillips, 1988). Sperm undergo a transition from linear swimming to a nonprogressive tumbling form of motility that Yanagimachi (1981) dubbed “hyperactivation.” External calcium is necessary for the transition to hyperactivated motility (Yanagimachi and Usui, 1974; Cooper and Woolley, 1982; Fraser, 1987; White and Aitken, 1989). Suarez et af. (1993) demonstrated that Ca2+ enters the cell during the transition to hyperactivated motility and that the addition of calcium ionophores can trigger the same transition in intact sperm (Suarez et al., 1987). It is also possible to induce hyperactivationlike beating in demembranated sperm models by adjusting the free Ca2+ levels in the reactivation mixture (Lindemann and Goltz, 1988; Mohri et af.,1989; Lindemann et al., 1991b). Like the chemotaxic responses of invertebrate sperm, hyperactivation may serve to localize sperm in the vicinity of the cumulus oophorus by terminating progressive swimming, thereby causing sperm to accumulate near the egg. At its most severe, the flagelladciliary response to high levels of calcium ion is a conformational “arrest” in one extreme curvature of the beat cycle. In sea urchin sperm, flagella take on the form of a “candy cane,” with a sharp bend at the proximal end of the flagellum (Gibbons, 1980; Gibbons and Gibbons, 1980). In Mytifus gill cilia, the arrest is also a curved, hooklike configuration that is similar to the end of the recovery stroke (Tsuchiya, 1976, 1977; Waiter and Satir, 1978; Wais-Steider and Satir, 1979; Satir et al., 1991). In rat and mouse sperm, the arrest condition resembles a fishhook, with the most severe bending occurring in the middle piece (Lindemann et a/., 1987, 1990, 1992; Lindemann and Goltz, 1988). This calcium arrest configuration does not appear to involve permanent “rigor-like’’ crossbridge formation because arrested Mytifus gill cilia are capable of resuming a beat if mechanically stimulated by using a microprobe to bend the cilia in the direction opposite that of the arrest position (Stommel, 1986).

C. Underlying Mechanisms of Control The two flagellar regulatory responses, mediated through CAMPand Ca”, have been elusive to define at the structural/functional level. Many false starts have diverted the quest to identify the regulatory sites. The endeavor has been complicated by the axonemal localization of multiple Ca2+-binding proteins (Salisbury et al., 1986; Otter, 1989; Salisbury, 1989; King and PatelKing, 1995) and a plethora of intracelluiar phosphoproteins (Hamasaki et al., 1989; Stephens and Stommel, 1989; King and Witman, 1994; Chaudhry et a!., 1995).

26

CHARLES 6. LINDEMANN AND KATHLEEN S. KANOUS

Some significant progress has been made, in the past several years, toward identifying phosphorylation sites that appear to impact dynein activity (Pipern0 et af.,1981; Tash, 1989; Hamasaki et af., 1989,1991; King and Witman, 1994). Possibly the best candidate site of kinase A control currently identified is located on one of the dynein light chains associated with outer arm dynein (Barkalow et al., 1994). Phosphorylation at this site appears to modulate in vitro outer arm dynein-dependent MT translocation (Hamasaki et af., 1995b; Satir et af.,1995). As mentioned previously, the outer arms are not essential for beat coordination but do add power to the beat and increase its frequency (Hata et af., 1980; Mitchell and Rosenbaum, 1985; Okagaki and Kamiya, 1986; Kurimoto and Kamiya, 1991). In most flagella and cilia, CAMP-dependent phosphorylation results in an increase in beat frequency (Lindemann, 1978; Nakaoka and Ooi, 1985; Bonini and Nelson, 1988, Hamasaki et af., 1991), indicating that at least one of its actions is to augment both the speed and the power output of the beat. In what may be a related phenomenon, Hard’s laboratory demonstrated that newt respiratory cilia can be induced to exhibit two distinct states of motility (Weaver and Hard, 1985; Hard and Cypher, 1992; Hard et af., 1992). In both states the beat pattern is maintained, but the power output and beat frequency are biphasic. These ciliary axonemes function in two distinctly different modes of operation, one low output and one high output. This transitional behavior was initially induced by adjusting Mg-ATP concentrations and experimental temperature, although it was suggested that the transition to the higher beat frequency was also CAMP dependent (Hard and Cypher, 1992). Most significantly, Hard’s group demonstrated that conversion to the energetic mode was eliminated if the outer dynein arms were extracted, clearly establishing the role of outer arms in mediating the transition to the higher frequency condition (Hard et al., 1992). This transition between the two modes of motility has since been established to be under the control of CAMP-dependent kinase (R. Hard, personal communication). This links both the phosphorylation site and the motile response within the same experimental system. Similarly to the previous work (Gibbons and Gibbons, 1973; Brokaw and Kamiya, 1987), these findings suggest that outer arms contribute power to the beat without markedly affecting the beat cycle coordination. This corroborates the findings that the regulation site for flagellar power output resides with the outer arms (Hamasaki et af., 1991, 1995b). On the other hand, where outer arms are unnecessary for beating (serving as power amplifiers or auxiliary power sources), genetic dissection of the axoneme has demonstrated that the ability to beat is lost when all inner arm dyneins are dispensed with. Therefore, inner arms must be capable of carrying out all the phenomena necessary to initiate and perpetuate the

FLAGELLAR MOTILITY

27

beat cycle. If the initiation of MT sliding is a function of the inner arms, this would make them the most logical site for control of axonemal Ca2+bias. In both sea urchin (Gibbons and Gibbons, 1980; Okuno and Brokaw, 1981) and rat sperm (Lindemann and Goltz, 1988), the calcium response can be induced even when active beating has been blocked with vanadate. This finding presents an enigmatic situation. On the one hand, as noted earlier, ciliary/flagellar beat arrest seems to represent a switching failure at one extreme of the beat cycle. This view is supported by the demonstration of progressive lopsidedness in the flagellar beat at increasing Ca2+ concentrations. One would reason that the uneven activation of the bridge set on one side of the axoneme ultimately stalls the beat cycle when the dominant bridge set fails to disengage (or the opposing set fails to engage), causing the beat to arrest at one extreme of the beat cycle. The fact that the same flagellar configuration can slowly develop under vanadate-induced inhibition of beating is problematic to the basic view that flagellar arrest is the result of a switching failure. These events were reconciled by Brokaw’s “biased baseline” hypothesis (Brokaw, 1979; Eshel and Brokaw, 1987) contending that calcium ion controls the equilibrium position (or baseline) of nonbeating flagella. In other words, the beat, but not the baseline curvature, of the flagellum is selectively inhibited by vanadate. Therefore, the biased baseline concept suggests that one process is at work in controlling the beat, whereas an independent one controls the arrest formation of the candy cane or fishhook. In this view, the normal beating action is superimposed on the baseline curvature. In experimental support of Brokaw’s view, examination of Ciona, sea urchin, and ram sperm flagellar motility established that the observed asymmetric beating patterns were developed by the propagation of basically symmetric bending waves on a flagellum with a sharp static basal curvature (Brokaw, 1979; Eshel and Brokaw, 1987; Chevrier and Dacheux, 1991). This baseline curvature is probably maintained by a separate system within the axoneme that modifies the structural equilibrium. Because this function is relatively insensitive to vanadate, it could be presumed that the underlying mechanism is independent of the dynein bridges, which are the target of vanadate’s inhibitory action (Kobayashi et al., 1978; Gibbons et al., 1978). Examination of the literature documenting the action of vanadate as a dynein inhibitor suggests a possible resolution of the seemingly contradictory experimental observations mentioned previously. Low concentrations of vanadate (0.5-5.0 puM) are highly effective in blocking coordinated beating in demembranated cilia/flagella (Gibbons et al., 1978; Sale and Gibbons, 1979; Okuno and Brokaw, 1981; Penningroth, 1989). However, vanadate is considerably less effectual in completely blocking MT sliding or dynein ATPase activity, generally requiring 5-10 times greater concentration than that needed to suppress motility (Gibbons and Gibbons, 1980;

28

CHARLES B. LINDEMANN AND KATHLEEN S. KANOUS

Bird et af., 1996). Therefore, in cilia or flagella inhibited with less than 10 pM vanadate, it should be possible to have internal MT translocation and bend formation based on dynein-tubulin sliding. If the triggering mechanism for bridge switching is suppressed by vanadate inhibition, the residual sliding in the vanadate-treated axoneme would move the cilium/ flagellum to one beat end point, at which point the beating process would stall. There is a possible explanation for the aforementioned vanadate effect. If vanadate-complexed dynein heads are rendered dysfunctional at low vanadate concentrations, it would be reasonable to expect that most dynein would be complexed and thereby inhibited. However, some dynein should remain uncomplexed and therefore functional. In vitro studies of dynein-MT translocation demonstrate that high translocation velocities can be obtained with remarkably few dynein arms (Vale et al., 1989). Hence, in motility inhibition utilizing low vanadate concentrations, there may be adequate functional dyneins to translocate doublets and bend the flagellum most of the way to the end of a normal beat cycle. If the switching mechanism requires not only bending but a critical summated force production between the outer doublets, then decreasing the number of contributing dyneins reduces the summated force necessary to activate the switching mechanism. The Geometric Clutch mechanism (Lindemann, 1994a,b) states that the product of force X curvature is a key component of the switching mechanism. In this context, the inability of the internal force to reach the critical level (due to subminimal numbers of functional dyneins) results in the expected switching failure observed in vanadate-treated cilia and flagella. To validate the calcium regulation concept, it is necessary to demonstrate that Ca2+exerts a selective influence over which dyneins will dominate when the flagellum is passive. Studies have implicated Ca2+ in this role, either through direct application of calcium to induce asymmetric dynein sliding (Sale, 1986) or localized asymmetric bending (Okuno, 1986) or by using Ni2' as a probe (Lindemann and Goltz, 1988; Kanous et al., 1993) to block the calcium response. Once again, a key feature of dynein is its propensity to form bridges spontaneously and initiate sliding episodes. As discussed earlier, this capability is necessary to explain observed flagellar behavior, and it must be addressed in any analysis of axonemal functioning. The Geometric Clutch model includes a formulation for giving a bridgeformation advantage to the dynein arms on one side of the axoneme, with Ca2+modifying the bias on that side. Work by Sale (1986) can be viewed as supporting this hypothesis because he demonstrated a calcium-induced selectivity of MT sliding in disintegrating sea urchin sperm axonemes. Additionally, good candidates for Ca2'-sensitive sites have been discovered. Centrin (also known as caltractin), a Ca2'-responsive contractile protein,

FLAGELLAR MOTILITY

29

has been localized to the axoneme (Salisbury et al., 1986; Salisbury, 1989). It has been identified specifically to the D R C in association with the inner arms (Piperno et al., 1992) and affiliated with certain subsets of inner arm dynein heavy chains (LeDizet and Piperno, 1995a). If the distribution of calcium regulatory sites were bilaterally differentiated (with the set of inner arms that bend in one direction being more sensitive to calcium than the opposing set), the very type of bridge biasing predicted by the Geometric Clutch mechanism could exist under calcium concentration control. To date, there are several studies suggesting differences in the dyneins on opposite sides of the axoneme (Kanous et al., 1993; King et al., 1994; Lindemann et al., 1995). However, conclusive evidence to support this view has not yet been obtained. A compilation of studies provides overwhelming experimental testimony that the Ca2+response is subject to modulation by the CAMP-kinase A pathway (Nakaoka and Ooi, 1985; Izumi and Nakaoka, 1987; Bonini and Nelson, 1988; Lindemann et al., 1991a,b). Additionally, exposing Mytilus gill laterofrontal cirri to greater than physiological CAMP levels results in ciliary arrest at the end of the effective stroke, the opposite direction of the calcium-induced arrest (Sanderson et af., 1985). Consequently, it is likely that control via phosphorylation and/or calcium binding is involved in at least two, if not more, functional sites within the axoneme. Possible candidates for regulatory sites include the outer arm dynein light chains (Barkalow et al., 1994; Hamasaki et al., 1995b; Satir et al., 1995; King and Patel-King, 1995), outer arm heavy chains (King and Witman, 1994), the nexin links (Ohnishi et al., 1982), the DRC (Piperno et al., 1992), and the inner arms (LeDizet and Piperno, 1995a).

V. Coordination of the Beat Cycle A. Minimal Requirements for Beating The isolated flagellar axoneme is a self-contained mechanical oscillator. This fact was established by microdissection (Lindemann and Rikmenspoel, 1972a,b), glycerin extraction (Hoffman-Berling, 1955; Brokaw, 1968), and detergent demembranation of intact cells (Gibbons and Gibbons, 1972; Morton, 1973; Lindemann and Gibbons, 1975). Using detergent-extracted flagella, it was possible to define the minimal requirements for axonemal functioning without the interference of a plasma membrane. It was demonstrated that under the proper conditions, using a suitable pH and sufficient Mg-ATP concentration, the isolated flagellar axoneme was still a fully functional motile organelle (Gibbons and Gibbons, 1972). In light of this

30

CHARLES 8. LINDEMANN AND KATHLEEN S. KANOUS

evidence, the search to uncover the underlying mechanisms that generate flagellar beating was directed away from membrane potentialslionic signaling, and toward intrinsic structurallchemical components of the axoneme itself. The simplicity of the basic chemical requirements for beating must be qualified due to a number of observations indicating that the basic oscillator is sensitive to other contributing factors in addition to the Mg-ATP concentration. Most ciliary and flagellar beating will arrest in the presence of a sufficient (10-6-10-5 M ) Ca2+concentration (Satir, 1975; Satir and Reed, 1976; Tsuchiya, 1976, 1977; Walter and Satir, 1978; Wais-Steider and Satir, 1979; Gibbons and Gibbons, 1980; Sale, 1986; Lindemann and Goltz, 1988; Satir et al., 1991). Other reports also implicate increasing the free Mg2' concentration in generating an arrest-like response (Lindemann and Gibbons, 1975; Sale, 1985; Yeung, 1987). However, because it is difficult to control and monitor the effect increased Mg2+has on the free Ca2+concentration, it cannot be ruled out that the magnesium effect is actually due to a cross-interaction of Mg2' on the free Ca2' level. The calcium response itself is well documented and clearly modifies the performance of the flagellar oscillator. In addition to provoking outright arrest, the free Ca2+level can also bias the P and R bend contributions to the beat cycle, thereby altering the flagellarlciliary waveform (Miller and Brokaw, 1970; Naitoh and Kaneko, 1973; Brokaw et al., 1974; Holwill and McGregor, 1976; Brokaw, 1979; Brokaw and Goldstein, 1979; Okuno, 1986; Lindemann and Goltz, 1988). Another interesting avenue of investigation highlights the flagellar oscillator's response to alternate nucleotides, primarily ADP and ATP analogs. Although ATP is undisputively the natural fuel for the axonemal motor, ADP (another physiologically present nucleotide) has a powerful impact on the oscillation mechanism. Lindemann and Rikmenspoel (1973) first noted that the beat cycle of impaled, dissected sperm was facilitated by the inclusion of ADP in the external medium. ADP was observed to reduce the beat frequency of Triton X-100-extracted sperm models while improving the maintenance of a coordinated beat (Lindemann and Gibbons, 1975) and increasing amplitude/bend angle (Okuno and Brokaw, 1979). In recent studies of the dynein-tubulin cross-bridge cycle, accumulated evidence suggests that ADP lengthens the duty cycle of dynein by slowing down the bridge release step (Johnson, 1985; Omoto, 1989,1991). This action results in a slowing of the interdoublet sliding rate (Bird et al., 1996) but also serves to convert the hyperoscillation observed in paralyzed mutant Chlamydomonas to a form of undulation (Yagi and Kamiya, 1995). Additionally, although the presence of either ADP or ATP analogs hampered MT sliding velocity, their addition increased the extent of sliding disintegration possible in demembranated Tetrahymena (Kinoshita et al., 1995). A number of ATP

FLAGELLAR MOTILITY

31

analogs have been investigated to determine their effect on dynein activity (Shimizu, 1987; Inaba ef al., 1989; Omoto and Brokaw, 1989; Omoto and Nakamaye, 1989; Shimizu et af., 1989, 1991; Omoto, 1992). Some analogs found to be capable of inducing in v i m dynein-driven MT translocation (ATP,S and formycin 5’-triphosphate) could not elicit ciliary reactivation (Shimizu et a/., 1991). This suggests a number of possible differences between the dynein-mediated processes of MT translocation and axonemal motility, including a stricter substrate specificity for initiation of beating, a requirement of multiple motor participation (each with its own substrate specificity), or a greater sliding velocity/force production necessary than the analogs are capable of supplying (Shimizu ef al., 1991). Omoto et al. (1996) demonstrated that ribose-modified ATP analogs, as well as ADP, were capable of restoring motility to paralyzed Chlamydomonas mutants in the presence of millimolar ATP concentrations (motility could also be induced if the ATP levels were reduced below 50 p M ) . All the above results implicate cross-bridge cycle dynamics as having an impact on the oscillation mechanism. If the interpretation of ADP’s action is correct, a prolongation of the force-producing step (bridge attachment) facilitates the events that coordinate the beat cycle.

B. Mechanics of the Beat Cycle One of the most compelling features of the isolated flagellar axoneme is its sensitivity to mechanical stimulation. Isolated distal sections of bull or starfish sperm flagellum will not beat spontaneously, even if supplied with Mg-ATP. Nonetheless, if the isolated fragment is bent, using a microprobe, a pattern of repetitive beating can be triggered, which persists as long as the imposed bend is mechanically maintained (Lindemann and Rikmenspoel, 1972a; Okuno and Hiramoto, 1976). This behavior in isolated flagellar pieces can be explained if the imposed bend activates the dynein bridges that act to bend the flagellum in the direction opposite t o the imposed bend. Naturally, when the dynein-tubulin sliding episode terminates, the flagellum will snap back to its original position. If the equilibrium (original) position is controlled by the microprobe, then this original bend (which provoked the initial flagellar response) will again develop, and the events will repeat. This scheme can only explain the observed phenomenon if the episode of dynein-tubulin sliding can self-terminate. Kamiya and Okagaki (1986) demonstrated just such a self-terminating mechanism, a result of two adjacent MTs undergoing sliding in an axoneme bent beyond a critical limit. In an intact beating flagellum, it is likely that the action of one set of dynein bridges is sufficient to bend the flagellum beyond the activation trigger point of the opposing set. Therefore, action termination of the

32

CHARLES B. LINDEMANN AND KATHLEEN S. KANOUS

first set would relinquish control to the newly activated second set, and reciprocation could proceed without the need for external triggering. Ni2+ inhibits sliding of the doublets on one side of the axoneme (Kanous et af., 1993) and also blocks spontaneous flagellar/ciliary beating. The nickelinhibited cells retain the ability to reinstate a beat in response to micromanipulatory bending (Lindemann et af., 1980, 1995), but only if a microprobe is used to impose a bend in the direction normally produced by the inactivated bridges (see Fig. 7B). Based on experimental observations, Satir and co-workers proposed that each phase of the beat cycle is initiated or terminated by activation of a switch point to activate the opposing set of bridges (Wais-Steider and Satir, 1979; Satir, 1985; Satir and Matsuoka, 1989). The beat cycle of most cilia can be subdivided into two opposing phases, referred to as the effective and recovery strokes, each possessing fairly well-defined characteristics. The effective stroke is rapid and powerful (Rikmenspoel and Rudd, 1973) and often described as stiff or “oar-like.’’ The recovery stroke, on the other hand, is more of a rolling wave of bending that propagates along the cilium. Satir’s laboratory demonstrated that the ciliary beat could be arrested at opposite extremes of the beat cycle by Ca2+ and vanadate ( Wais-Steider and Satir, 1979; Satir, 1985; Satir and Matsuoka, 1989; Satir et aL, 1991). Nickel ion has also been used to produce ciliary/flagellar arrest (Naitoh and Kaneko, 1973; Lindemann et af., 1980, 1995; Larsen and Satir, 1991). Apparently, any of these inhibitory agents prevent the switching to the next phase of the beating cycle. The parallel of the Ca2+ and Ni2+ arrest phenomena between MytiZus cilia and rat sperm has been noted, along with the similarities in arrest positions (Satir etaZ., 1991). These arrest patterns, in both cilia and sperm flagella, involve the maintenance of a unidirectionally curved configuration, suggesting the continued dominance of a one-sided episode of dynein-tubulin engagement (implying that the capability of switching dominance to the other side is somehow impaired). Although open to more than one interpretation, it is important to note that the ciliary switch point hypothesis can be reconciled with the results of bull sperm micromanipulation studies presented here. If the basic beat mechanism requires a degree of reciprocation between the dynein bridges on the two opposing sides of the axoneme, and if the action of each set normally triggers the activation of the opposite set, this proposal is still lacking several important details. First and foremost, no mechanism is provided to explain how a nonbeating flagellum/cilium could assume a coordinated beat without an external push. A crucial factor in the initiation of spontaneous flagellarkiliary beating is the presence of a basal anchor. An early study (Douglas and Holwill, 1972) of isolated, reactivated Crithidia flagella, and flagellar fragments, revealed that freely suspended flagella did not demonstrate wave propaga-

33 tion. However, fragments that became attached by one end t o aglass surface were observed to generate waves originating from the attached end. A later study examining the effect of mechanically reanchoring clipped sperm flagella using a microprobe discovered that creating an anchor could restore beating (Woolley and Bozkurt, 1995). This information specifically points to anchoring as a requirement for normal beat production. Although the bending wave of most flagella travels from base to tip, it is interesting that some flagella are capable of a reversed wave propagation direction. The sperm of certain rotifers maintain an axonemal basal body at the distal tip of the flagellum; undulations originate there to pull the cell along (Melone and Ferraguti, 1994). In addition, certain eukaryotic protozoa can reverse wave propagation direction during flagellar beating (Walker, 1961; Holwill, 1965). A protein was immunologically localized to both the basal body and the flagellar tip of Trypanosoma brucei (Woodward et af., 1995), pointing to the possibility of a common structure at both ends of the flagella. Micromanipulation studies of Crithidia oncopelti revealed that microprobe dissection of the flagellum allowed continuation of tip to base wave propagation in severed portions of the flagellum (Holwill and McGregor, 1974), whereas laser irradiation usually resulted in a base t o tip direction reversal (Goldstein et af., 1970). It was speculated that laser irradiation welded the components of the newly formed base together, whereas microprobe cutting probably left the axonemal elements free from each other (eliminating an anchoring device at that end). Fractionated distal fragments of flagella given Mg-ATP do not beat (Brokaw and Benedict, 1968; Lindemann and Rikmenspoel, 1972a; Summers, 1975; Woolley and Bozkurt, 1995). Close examination of some “immotile” fragments reveals that they are no1 completely inactive but actually exhibit small-amplitude “jittering” all along the flagellar shaft (Lindemann and Rikmenspoel, 1972a). Lindemann and Gibbons (1975) also demonstrated the same type of small-amplitude, uncoordinated “twitching” in reactivated bull sperm exposed to Mg-ATP concentrations outside the range established to support spontaneous beating. Close examination of isolated sea urchin sperm (Kamimura and Kamiya, 1989, 1992) or Chfamydomonas (Yagi ef af., 1994: Yagi and Kamiya, 1995) axonemes in later studies revealed “nanometer-scale’’ high-frequency oscillations in nonbeating flagella. Mutant Chlamydomonas specimens lacking various axonemal components were also observed to vibrate, although in a slightly different manner (Yagi et af., 1994). Vanadate was shown to decrease the amplitude of oscillation with no effect on frequency (Yagi et af., 1994), whereas the addition of high concentrations of ADP ( 3 mM) increased the amplitude of these oscillations and (at high enough ATP concentrations) could result in a kind of beating (Yagi and Kamiya, 1995). This concurs with the earlier results of Lindemann and Rikmenspoel(1972b) who converted the jittering FLAGELLAR MOTILITY

34

CHARLES B. LINDEMANN AND KATHLEEN S. KANOUS

of bull sperm flagellar fragments to a regular form of beating at high ADP concentrations. The significance of these observations resides in the concept that, when the flagellum fails to coordinate the activity of the dyneins into regular beating under suboptimal conditions, there is still spontaneous force production. This disorganized development of force is insufficient to supply the initial bending necessary to trigger reciprocal activation. The meager level of stochastic dynein bridge formation is below the threshold level necessary to initiate substantial multiple dynein activation. However, the presence of a basal anchor helps convert this sporadic bridging force into an effective bend. This was demonstrated when reanchoring the cut end of a flagellar fragment reestablished beating (Douglas and Holwill, 1972; Woolley and Bozkurt, 1995). The addition of A D P also has a facilitating effect by increasing the amplitude of the nanometer-scale vibrations (Lindemann and Rikmenspoel, 1972b; Yagi and Kamiya, 1995). This may be due to the previously mentioned supposition that high concentrations of A D P alter the duty cycle of dynein by prolonging the force-producing stroke of the bridge cycle (Johnson, 1985; Omoto, 1989, 1991). The concepts that emerge from the results of these studies can be summarized as follows: 1. Bending the flagellum in one direction activates the dynein bridges acting to bend the axoneme in the opposite direction. 2. When a flagellum is beating, each episode of dynein-driven sliding acts as the stimulus to activate the opposing bridge set. 3. The dynein bridges exhibit spontaneous, random (stochastic) subthreshold bridging, even when the flagellum is not beating. 4. The random bridge activity can self-organize into coordinated beating if a basal anchor is present to assemble the summated forces from random bridging into a bend sufficient to activate a whole group of dyneins.

VI. Modeling the Flagellum

A. Physical Parameters of Flagellar Movement The flagellum is nature’s answer to the need for a biological propeller. It is an organelle that, in most of its applications, is providing motive force in a fluid environment. Not surprisingly, it has drawn the attention of biophysicists interested in an intriguing problem of interfacing hydrodynamics and biology. Some of the earliest studies attempting to understand the propulsive mechanism of flagellar motility were conducted by biophysicists

35

FLAGELLAR MOTILITY

who had to find methods of dealing with the complex problem of a flexible, thin beam (the flagellum) interacting with a viscous fluid. The first hurdle was to address the effect of fluid (viscous) drag on the generation of propulsive force. As a flagellum moves through its fluid surroundings, the movement is countered by viscous drag. Because the flagellum is a long, thin, flexible structure, all the viscous drag encountered by the flagellum as it moves must be opposed by active forces produced within the structure and conveyed to the areas of viscous resistance. This force transmission must occur through the long, thin shaft of the flagellum itself. Perhaps the first significant headway into a practical evaluation of the drag on a flexible beam moving in sinusoidal waves comes from Taylor (1952). Gray and Hancock (1955) used a somewhat different approach by segregating the drag into two categories, components transverse to or parallel to the long flagellar axis. They concluded that the flagellum creates twice as much drag moving transversely (like an oar) as when it moves longitudinally (like a dragging rope). This difference in drag is the basic source of flagellarkiliary propulsion, making possible both the swimming of sperm and the fluid transport of cilia (Fig. 8). This relationship between drag and propulsion, derived by hydrodynamic theory, was experimentally confirmed (Brokaw, 1965; Rikmenspoel, 1965). The physics of viscous drag sufficiently explains why a flagellum can do its job as a propeller of fluid. Biophysics can also tell us something about the internal forces needed to push the flagellum. By Newton’s laws, viscous drag must be countered by equal and opposite force contributed by the flagellum. Machin (1958,1963) further elaborated on the description of the Newtonian balance of forces, expressed as moments (force times lever arm). He divided the internal forces that balance the viscous drag (Mviscous or M y )into those derived from the contractile machinery (Mactiveor Ma) and those forces created by bending the elastic structures in the flagellum (Melastic or M e ) . Consequently, the basic equation of flagellar motion actually has three parts; an active term (Ma), a viscous drag term (My), and an elastic bending term ( M e ) , the sum of which must equal the Newtonian balance at any point along the flagellum:

M, + M ,

+ Ma = 0.

(1)

This equation, and its derivative forms, has become the basis of flagellar motility analysis. It is reasonable to expect that solving for both the viscous drag and the elastic term at each point along the flagellum would reveal the internal forces. Using this approach, Machin made a major conceptual contribution by showing that force applied only to one end of a passive elastic rod could not reproduce the beating patterns observed in live flagella. This led to the conclusion that the active forces must somehow be locally produced along the flagellar length (Machin, 1958, 1963).

36

CHARLES B. LINDEMANN AND KATHLEEN

S. KANOUS

A \

net

FIG. 8 Fluid propulsion in cilia and flagella. Stylized renderings of both ciliary (A) and flagellar (B) beating are displayed. The trajectory of a specific point on each of the cilium and flagellum is followed and indicated by the dotted lines. Note that the ciliary axis along the path traversed by a point on a distal segment is mainly perpendicular to the direction of movement during the effective stroke while being predominantly parallel to the direction of motion during the recovery stroke. Because the drag on a cylindrical structure is approximately twice as great when the structure passes through the fluid perpendicular to its long axis, there is a net propulsive drag that moves the external fluid in the direction indicated by the arrows. In the case of the flagellar beat, points on the flagellum follow a figure eight-like trajectory. Movement of each point is proximally directed at the upper and lower extremes of the “8”shaped pattern. During this proximally directed movement, the flagellar shaft is roughly parallel to the direction of motion. Distally directed motion is generated at the center portion of the 8-pattern, and the perpendicular component of motion is greatest there. Because the perpendicular component of the fluid drag is mainly limited to the distally directed part of the beat path, the net fluid drag is distally directed. If the flagellum is on a free (unanchored) cell, this net drag provides the propulsion necessary for swimming.

Rikmenspoel also adopted Machin’s approach. If a rigorous specification of the two physical terms of the equation of motion could be reached, the active term should then become apparent and open to examination. Deducing the Ma term by analyzing the motion of a number of different cilia and flagella might be conducive to understanding the underlying function. In a series of progressively more rigorous approximations, Rikmenspoel(l965, 1966a, 1971; Rikmenspoel and Rudd, 1973) produced computed simulations in an effort to duplicate the motion of cilia and flagella, as observed in nature. In the process of developing his computer model, he did some of

FLAGELLAR MOTILITY

37

the most thorough analyses of the physical parameters of flagellar and ciliary systems (Rikmenspoel, 1965, 1966a, 1971, 1978). Rikmenspoel and Rudd (1973) produced a rigorous, large-amplitude solution to the equation of motion that allowed virtually any ciliary or flagellar waveform to be analyzed. A number of intriguing findings that pertain to the nature of the active forces emerged. Rikmenspoel(1965, 1966a) demonstrated that the traveling waves normally observed in bull sperm flagella could be produced using a standing waveform of the active moment, without complex timing functions or wave phase dependency. The ciliary effective and recovery strokes could be modeled only if forces producing the effective stroke were activated very abruptly (simultaneously) along most of the axonemal length. The active forces producing the recovery stroke were much more localized, propagating along with the physical bend (Rikmenspoel and Rudd, 1973). The flagellar beat in both bull and sea urchin sperm appears more evenly distributed in the two bending directions. Nevertheless, there is a dominant bending direction (referred to as the principle bend) and a subdominant (reverse) bend. Rikmenspoel (1971) found a component of M ain flagella that had a more broadly distributed onset. Naturally, any successful mechanistic explanation of the beating cycle will, of necessity, have to be capable of reproducing these characteristics of M,derived from live cilia and flagella. Based on his deduction that active forces must be produced all along the length of the flagellum, Machin (1958,1963) hypothesized that flagellar bending participates in the activation of the localized force production mechanism (which at the time was considered to be a contractile event). This concept, that the local curvature in some way activates a local forceproducing mechanism, led to later curvature control theories. In order to “push” a bending wave effectively down the flagellum, the control function must turn on the flagellar motor apparatus somewhat out of phase with the mechanical wave. Consequently, in the curvature control models, the control function requires the incorporation of a phase delay (time delay) from the current curvature. This line of reasoning precipitated a number of models, and the basic underlying assumptions still play a dominant role in conceptual analysis involving the axoneme. Brokaw (1972a) experimented with curvature control functions that could activate force production by providing the necessary time delay from the current state. Miles and Holwill (1971) devised a control function based on drag and elastic strain transmitted along the flagellum to provide the phase delay necessary for curvature control. Lubliner and Blum (1971) developed a model capable of modifying the timing of curvature activation by incorporating external viscosity and internal shear information into the timing function. This method corrected the problems of eliciting appropriate responses to viscosity changes (increased viscous drag) in previous curva-

38

CHARLES 6. LINDEMANN AND KATHLEEN

S. KANOUS

ture control models. Rikmenspoel (1971) insisted that waveform and wave propagation rate were largely governed by external viscosity and flagellar elasticity. He believed that correct viscous load responses could be obtained, even with a standing wave mode of active moment that had no propagating component. His models of bull and sea urchin sperm were able to automatically adjust both waveform and wave propagation to increased viscosity in a life-like way (Rikmenspoel, 1971). However, Rikmenspoel later realized that the ciliary recovery stroke could not be modeled without a substantial traveling component in the Ma term (Rikmenspoel and Rudd, 1973). Also, using a nontraveling Ma did not allow the duplication of the sea urchin sperm response to cold or low ATP concentrations, in which the waveform remains relatively unchanged while the beat frequency drops substantially. Straightforward curvature control also fails to reproduce realistic results under many experimental conditions. In particular, it is nearly impossible to elicit the clear-cut, simultaneous initialization of Ma over the ciliary length (necessary for effective stroke production) without a totally separate control function that can explain the two phases of the ciliary beat. The deviations from observed behavior led Brokaw (1985) to conclude that all schemes based strictly on curvature control were “incompletely specified.” A successful explanation of the force-producing mechanism must be able to reconcile experimental observations that appear on first examination to be contradictory. As experimental investigations continue to fill in more of the details pertaining to axonemal structure and function, flagelladciliary model creators have been quick to incorporate these new concepts into their computer models. Brokaw (1971, 1972b) was the first to convert the treatment of force production into a computable sliding doublet formulation. Lubliner and Blum (1971) expanded this sliding doublet treatment into a model that included a more complete set of the axonemal components. Sugino and Naitoh (1983) produced a three-dimensional model that successfully explored the geometry and timing of the sliding interactions necessary for the helical beat of many cilia. Others have produced detailed descriptions of the three-dimensional operation of cilialflagella (Holwill et al., 1979; Hines and Blum, 1983, 1984, 1985; Woolley and Osborn, 1984; Sugino and Machemer, 1987; Machemer, 1990; Mogami et al., 1992; Teunis and Machemer, 1994). Holwill and Satir (1990) produced the most structurally complete model, incorporating all the known structural details into a threedimensional computer representation that can be used as a basis for functional modeling. Because dynein is the motor of the axoneme, a complete understanding of axonemal functioning demands that close attention be paid to the role of the cross-bridge cycle. Brokaw (1976a,b) pioneered the efforts in this direction by introducing a two-state stochastic treatment of individual

FLAGELLAR MOTILITY

39

dyneins into his flagellar sliding doublet model. The recognition that bridge attachment is a stochastic process at the molecular level that must be treated by alterations in the probability of cross-bridge formation was a novel concept in treating motor protein behavior. Brokaw used curvature control with a time delay function as the basis for modulating the probability of bridge formation in his model of dynein bridge regulation. Murase and Shimizu (1986) proposed that the control of the beat cycle could come from cooperative dynamics of a number of dyneins, resulting in an activation scheme in which dynein exhibits excitable properties in a three-stage crossbridge activation cycle without curvature feedback control. Several elaborations of this view (Murase et al., 1989; Murase, 1990, 1991, 1992) propose that it is the excitable properties of the dynein cross-bridge cycle that lead to coordination of the multiple bridge actions that organize the beat. This represents a novel alternative model concept in which macroscopic behavior is depicted as a direct outcome of cross-bridge cycle dynamics. A conceptually interesting possibility, as currently postulated, this mechanism involving cooperative dynein excitability has thus far had limited success in replicating the natural beat cycle of cilia or flagella.

6.A Physical Model Based on a New/Old Perspective As we have seen, extensive information defining the structural and functional properties of the eukaryotic axoneme has been painstakingly gathered through experimentation. A successful explanation of axonemal functioning must be compatible with this existing body of knowledge. To be of use to the community of scholars currently exploring cilia/flagella, a workable interpretation of axonemal operation must also be detailed enough to have predictive value. Recently, a plausible hypothesis to explain the beating of cilia and flagella has been advanced that is compatible with much of the accumulated experimental data. This concept is based on relatively simple underlying assumptions and has been dubbed the “Geometric Clutch.” It is based on a long-standing observation that spacing between the outer doublets in the circle of nine is somewhat more than that which will permit easy cross-bridging by the dynein arms (Gibbons and Grimstone, 1960; Allen, 1968; Gibbons and Gibbons, 1973; Warner, 1978; Zanetti et al., 1979).The Geometric Clutch hypothesis adopts the simplest assumption; dynein-tubulin cross-bridge formation is limited by the interdoublet spacing. When cross-bridges form, they produce interdoublet force, and some of this force (strain) between the doublets is directed transverse to their longitudinal axis. This transverse force (t-force), which can move doublets closer together or further apart, controls the probability of cross-bridge formation. Two major sources contribute to the development of t-force:

40

CHARLES B. LINDEMANN AND KATHLEEN S. KANOUS

1. The formation of dynein-tubulin cross-bridges contributes some force pulling adjacent doublets together. When a single dynein head acquires sufficient kinetic energy, through Brownian motion, it bridges the gap between doublets and attaches to the binding site on the adjacent doublet. This stretched bridge contributes a small amount of force, pulling the doublets together, as illustrated in Fig. 9, which increases the probability of additional bridge formation. This is supported by the observation that interdoublet spacing and axonemal diameter are decreased in axonemes

R

Actively bent

Transfer FIG. 9 The transfer of force across the axoneme. As depicted in A, when a dynein bridge forms in a resting axoneme, the interdoublet spacing is greater than the length of the inactive dynein arms. Initially, kinetic energy must contribute to dynein stretching to allow random bridge attachment. As an individual dynein gains sufficient energy to form a bridge, it spans the interdoublet space, attaching to the adjacent doublet. The force contributed by each connection acts to pull the doublets closer together, increasing the probability of additional bridge formation. This initiates a cascade of bridge attachments on this side of the axoneme while providing an adhesive force between neighboring doublets. A strong negative t-force is necessary to overcome these resultant adhesive forces. This one-sided dynein bridge attachment impacts the entire axoneme as demonstrated in B. As bridges form on one side, the probability of bridge formation on the opposite side decreases. This is due to the increase in interdoublet spacings on the passive side resulting from the transfer of forces through the interdoublet linkages acting to separate those doublets. Reproduced from Lindemann (1994b) with permission.

FLAGELLAR MOTILITY

41

demonstrating the presence of cross-bridges in electron micrographs (Gibbons and Gibbons, 1973; Warner, 1978; Zanetti er al., 1979). 2. When dyneins induce sliding (and bending) by exerting force on a pair of doublets, the cumulative tension (or compression) creates a transverse force between the two doublets. This force is proportional to the total longitudinal tension (or compression) times the curvature of the flagellum at that location. This component of the transverse force must be countered by the local dynein bridges and/or the structural interdoublet connectors (spokes and nexin links). The cumulative nature of this element of the t-force enables it to become very large, especially near the basal anchor (basal body). This force can either pry doublets apart or move them together, depending on the direction of the curvature. Kamiya and Okagaki (1986) elegantly demonstrated the premise that an episode of cross-bridge activity can be terminated by the resulting interdoublet t-force. Two individual doublets from a frayed Chlamydomonas axoneme were able to set up a repetitive cycle of bending and straightening. The doublets were observed to associate, forming a bend that reached a critical curvature (1 radlpm). This was followed by unbending and splitting into two separate doublets, one forming a loop against the other. Upon straightening, the two doublets again became associated and repeated the cycle. The best explanation for the termination of the sliding episodes in this experiment is that the t-force mechanism acts to pull the doublets apart as the bend develops beyond a critical degree. Elaboration of this very simple idea, that the t-force between doublets coordinates the action of the dynein motors in the axoneme, is the basis of the Geometric Clutch hypothesis. In fact, the possibility that axonemal distortion might be involved in beat coordination has been alluded to in the literature for decades (Summers and Gibbons, 1971; Summers, 1975; Warner, 1978). However, systematic detailed examination of this idea has been neglected until recently. Using this basic conception, it has been possible to construct computer simulations controlling bridge activation/ deactivation by the t-force principle (using the conventions shown in Fig. 10). In its most rudimentary form, the Geometric Clutch mechanism has shown that t-force resulting from tension on the doublets can initiate a complete beat cycle in a simulated flagellum (Lindemann, 1994a). The resultant beat can be made cilium-like if the bridges on one side of the axoneme are designated as easier to engage, and harder to disengage, than those on the opposite side. The modeling mechanism can produce both recovery stroke-like traveling bends and effective stroke-like simultaneous bridge activation over much of the axonemal length. Bends are observed to initiate automatically near the basal end of the simulation and propagate distally.

CHARLES B. LINDEMANN AND KATHLEEN

42

S. KANOUS

Trans. force =

a

Trans. force =

a

-d0= @ ds

Tension =

@

Trans. force =

a

-d0= @ ds

Tension =

0

Trans. force =

L -

I

a

~

Dynein arms FIG. 10 The t-force of passively and actively bent flagella. Triplets are displayed corresponding to three outer doublets in the process of flagellar bending. Both A and C depict doublets in passively bent flagella (bends imposed by an external force), whereas B and D portray doublets induced to bend by dynein action. The longitudinal force on the outer elements is displayed, along with the resultant t-force (bold arrows). Although passive (imposed) bending compresses the axoneme in the plane of bending, active (dynein-induced) bending leads to axonemal distention. Inwardly directed t-forces (resulting in compression) are assigned a "+," whereas outwardly directed t-forces (causing distention) are designated by a " -." The curvature (dOld.7) multiplied by the tension yields the t-force. The corresponding signs of the curvature and tension are shown for each condition to demonstrate the convention used in modeling the flagellum. Reproduced from Lindemann (1YY4a) with permission.

43

FLAGELLAR MOTILITY

A more advanced formulation of the model incorporated a two-state stochastic treatment of individual dynein bridges (Lindemann, 1994b). This improved version was also scaled to cgs units, allowing the simulation to utilize measured values for dynein force, flagellar stiffness, viscous drag, and flagellar dimensions. The stochastic approach enabled those dynein bridges already formed to influence the probability of future bridge attachment. This permits bridge formation to occur in a spontaneous cascade, starting with a straight, passive flagellum. The model can both initiate spontaneous motility and maintain oscillations using physical parameters appropriate for an actual flagellum. Figure 11 displays the computer output for modeling both a 10-pm cilium and a 30-pm flagellum. Figure 12 displays the progression of events occurring during a beat cycle as envisioned in the Geometric Clutch hypothesis. Each individual diagram represents three adjacent outer doublets (corresponding to doublet set 2,3, and 4 or set 7, 8, and 9). The P bridge set consists of bridges forming the

2231 2387

FIG. 11 Computed simulations of a flagellum and a cilium. The output from the Geometric Clutch simulations of both a flagellar and a ciliary beat cycle are displayed. A 30-fim long “flagellum” with a freely pivoting base is displayed in A, showing every 12th iteration of a cycle divided into intervals of 0.0001 s per iteration. A 10-pm “cilium” with an anchored base is exhibited in B, showing every eighth iteration, utilizing the same iteration intervals as that used in the flagellar model. The numbers printed on the output denote the iteration number of the indicated beat position, corresponding to the first and last elements displayed. Note that the cilium clearly exhibits a two-phased heat cycle, with well-defined effective and recovery strokes. The flagellum shows wave propagation tipward from the base in both bending directions. The Geometric Clutch program was capable of producing both patterns of beating without any fundamental change in the switching algorithm. The main determinants of the resultant beat included base anchoring, axonemal length, and assignment of base-level bridge attachment probability of the P and R bridges on opposing sides of the axoneme. From Lindemann (1994b) with permission (The complete modeling parameters for the figure are given in that original report).

44

CHARLES 6. LINDEMANN AND KATHLEEN S. KANOUS P-Bridges Random bridge attachment

Cascade of bridge attachment due to adhesion A

Initiation of detachment

R- Bridges

,

Random bridge attachment

Inhibition due to force transfer from P side

3 ,

L

Delayed attachment due to force transfer

Propagation of detachment

Initiation of attachment

( i j

Propagation of attachment P I A (+>

Delayed initiation due to force transfer

New eDisode of

,

6

,,

Initiation of detachment

FIG. 12 The beat cycle of the axoneme. In this simplified schematic, the events on the P and R sides of the axoneme are illustrated to present, in a stepwise format, the hypothetical mechanism by which the axoneme develops oscillations. I, Starting in the straight position, both bridge sets have a base level of random bridge attachments. 2, A cascade of attachments on one side (usually the side with the higher base level of attachment probability) begins an episode of sliding and (due to force transfer) simultaneously inhibits bridge attachment on the opposing side. 3, As bending increases, a negative t-force develops on the active side of the axoneme and is strongest near the base. Force transfer continues to inhibit the opposite side but becomes weaker as detachment of dynein bridges proceeds. 4, A propagating area of bridge detachment on the P bridge side is accompanied by a positive t-force from passive bending on the R bridge side. and this ensures a cascade of bridge activation on the R side.

FLAGELLAR MOTILITY

45

principal (or dominant) bend, which have the higher initial probability of attachment. On the opposite side, the R bridge set acts to form the reverse bend. The P arrows signify the passive force of stretching elastic interdoublet links, whereas the A arrows represent the active forces of dynein bridges, and the A* arrows depict active force transferred from the opposing side through the nexin links. The events progress in the following stepwise fashion: (i) Both bridge sets undergo random, sporadic bridge formation while the axoneme is initially straight. (ii) A cascade of P bridges are formed due to the higher probability of attachment on that side. This inhibits R bridge formation due to force transfer through the interdoublet links. (iii) As the bending increases, the P bridges experience a negative t-force that is greatest in the basal region. P bridge detachment ensues, whereas the inhibition of R bridge formation decreases. (iv) P-bridge detachment continues, resulting in a positive t-force effecton the R bridges and a subsequent cascade of R bridge attachments. (v) The P bridges become inhibited through interdoublet-link force transfer as the R bridge formations reverse the axonemal curvature. (vi) The increased bending in the reverse direction exerts negative t-force in the flagellar basal end that initiates a chain reaction of R bridge detachment. Simultaneously, the passive links and residual active bridges exert a positive t-force on the P bridges, activating bridge attachment on that side. This sets in motion the events in the initial steps, and all steps then repeat themselves in a cyclical fashion, propagating stable flagellar oscillations. This hypothetical mechanism is dependent on certain crucial axonemal properties. There must exist a small propensity for random bridge attachment, in the absence o f t force,to initiate the oscillatory cycle in a straight, immotile axoneme. In addition, elastic linkages must exist to contribute to bridge engagement during oscillation while acting to restrict the axonemal splaying during bridge detachment. Lastly, longitudinal force must be transferred from opposite sides of the axoneme. Otherwise, bridge attachment probability would increase on the opposing side as soon as the curvature increased (see R bridge Step 2), and bridges would attach simultaneously on both sides of the axoneme. This would restrict the developing bend from reaching the crucial curvature necessary to instigate bridge detachment. The t-force concept illustrates the need

5, Curvature is now reversing due to the R bridge forces. P bridges are temporarily inhibited by force transfer from the R bridge side. 6, The curvature is now favorable for production of a negative t-force near the base on the R bridge side. and deactivation begins there. Meanwhile. a new cycle is beginning on the P bridge side, as positive t-force from passive links and residual active bridges contribute to activation. Arrows labeled P are passive force contributions originating from stretching the passive interdoublet links. Arrows labeled A are active forces from bridges, and A* indicates active force transferred from the opposite side. Reproduced with permission from Lindemann (1994b).

46

CHARLES B. LINDEMANN AND KATHLEEN

S. KANOUS

for side-to-side force transfer between opposing dynein bridges. The probable mechanism for this side-to-side transfer is illustrated in Fig. 9B.

C. t-Force The key to the Geometric Clutch design is the t-force, which acts as the main regulator of dynein-tubulin interaction in this hypothesis. So, what exactly is t-force? Whenever a flexible structure is under tension or compression, it requires some externally applied force to maintain a curved configuration. In the axoneme, flexible rods of tubulin (forming the doublets) are collectively connected at the basal attachment. To help visualize this structural arrangement, imagine two flexible wooden reeds fastened together at one end, as shown in Fig. 13A. If force is exerted from the unattached ends by pushing on one and pulling on the other, the result is that depicted in Fig. 13B. Instead of the two elements bending smoothly,

FIG.13 The mechanism of flagellar bending. A visual demonstration of the principle behind flagellar bend formation is presented. In A, two flexible reeds, anchored at one end to a small wooden spacer with a mechanical clamp, are each held at equal distances from the clamped end. When one reed is pushed baseward (toward the clamp) while the other is pulled tipward (as would be the case in outer doublet sliding), the pushed element buckles outward into an arch and the pulled element remains fairly straight, as shown in B. This separation of the two elements results from the development of t-forces acting to pull the two reeds apart. However, if rubber "linkers" are provided (represented by small rubber bands), these links can bear the outwardly directed t-force such that the same push/pull movement results in the formation of a smooth bend, as can be seen in C . This principle of balancing the t-forces between axonemal doublets using interdoublet linkers is what makes flagellar bending possible.

FLAGELLAR MOTILITY

47

one bows away from the other. A smooth bend of the entire structure can be produced from the applied force only if the two elements arc “linked” together. These connections arc then capable of bearing the outward t-force that would normally cause one of the elements to bow outward, counterbalancing it with the inward t-force developed on the other element, in a kind of force-sharing equilibrium (demonstrated in Fig. 13C). This same stratagem exists in the axoneme. The translocation of one doublet in relation to another can generate bending only if the doublets are linked and share the t-forces in a compensatory manner. Coincidentally, the axoneme contains interdoublet protein connections called nexin “links.” If these links are broken, or digested away, MT sliding within the axoneme produces an effect very similar to that shown in Fig. 13B. This experimentally induced flagellar disintegration has been called axonemal splitting (Satir and Matsuoka, 1989). In fact, it is the removal of the interdoublet connections with trypsin that disrupts the t-force balance, causing splitting due to the weakened axonemal structure. The role of the nexin links in bearing and distributing the t-force in the Geometric Clutch model sets rather specific limitations on nexin’s elastic properties. Life-like simulations of ciliary and flagellar beating are achieved when the nexin elasticity is specified to be within certain limits (0.010.03 dynkm), values given in a recent analysis of predictions derived through the Geometric Clutch model (Lindemann and Kanous, 1995). Within months, Yagi and Kamiya (1995) published an experimentally determined estimate for nexin elasticity, which when converted to cgs units equaled 0.02 dynkm. Although the basic idea of the t-force is conceptually very simple, the interplay between the t-force and the dynein-tubulin motor in the context of a complete axoneme is much more difficult to analyze and predict. The computer simulation is beneficial in revealing the necessary steps to creating the beat cycle, as displayed in Fig. 12. When the model is operating, the t-force can be analyzed through the beat cycle to examine the form the t-force takes in the working simulation with a printout as shown in Fig. 14. Note that the t-force itself develops a traveling component that propagates along the flagellum due to the summation of force from dynein bridges along the doublets. The t-force was observed to reach its maximum amplitude at the flagellar base. The t-force can be designated as positive (favoring bridge formation) simultaneously over a fairly long stretch of the flagellum. This would provide the needed mechanism to create the effective (power) stroke of the ciliary beat cycle, aphenomenon difficult to accommodate with more direct curvature control-based models of dynein activation (Brokaw, 1985). Consequently, the Geometric Clutch mechanism is capable of producing both propagated bending waves and near-simultaneous bridge activation, both using one common switching algorithm.

48

CHARLES 6. LINDEMANN AND KATHLEEN

S. KANOUS

A 2.OE-5 1.OE-5 .w

J

1

0.0 -

a2

k

d -1.OE-53 % -2.OE-5-

0

-3.OE-5

-4.OE-5

1

1 0

I

5

10

5

10

B

15 20 POSITION

25

30

25

30

T

2*0E-5 1 .OE-5

- 1.OE-5 --

B -2.OE-5 -3.OE-5 0

15

20

POSITION FIG. 14 The t-force profiles of the cilium simulation. These graphs are created by plotting the t-force values at six intervals during the beat cycle versus their position along the axoneme. In A, the P bridge side t-forces are displayed, whereas B presents the R bridge side. Each numbered plot represents the values at one 2.6-ms iteration interval of the complete beat cycle. Note the organized t-force propagation, particularly of the negative bridge-terminating effect. This figure graphically explains the location of bridge activity initiation and termination because it is obvious that the t-force values are greatest near the axonemal base and travel tipward. Reproduced from Lindemann (1994b) with permission.

D. The Oscillator An interesting outcome of the Geometric Clutch simulation is the production of stable oscillations with propagating waves by a mechanism that does not specify a phase delay, a timing constant, or a propagation velocity. The t-force algorithm, responsible for organizing the beat, might be considered

FLAGELLAR MOTILITY

49

a subform of a curvature control design based on the curvature term utilized in calculating the t-force. However, because it also includes a force term (equal to the summation of tension on the doublet) there is no absolute curvature threshold necessary for bridge switching in this mechanism. In addition, a fundamental difference exists in the complete departure from the harmonic oscillator concept utilized in most earlier models. This focused on rhythmic, sinusoidal, or periodic application of the Ma driving force. In the Geometric Clutch simulation, the oscillation mechanism can best be described as a relaxation oscillator. There is no mass or inertial term in the equations of flagellar motion because the dissipation due to drag is very high relative to the inertial energy (Hancock, 1953; Machin, 1963; Rikmenspoel, 1966b). However, an inertial mass is a necessary basic component of harmonic oscillations. By contrast, relaxation oscillators develop a cycle of oscillation through two or more cascade events that exhibit hysteresis. That means the events have a different threshold level to start the cascade than to terminate it. In actuality, dynein bridge formation requires only a small t-force to initiate an episode of interdoublet sliding. In fact, just a few randomly attaching dynein heads can start a cascade of additional bridge attachment. Once an episode of sliding has begun, a much stronger t-force is necessary to pry apart the doublets and make the dynein arms release from the binding sites. What contributes to this hysteresis? Most likely it is the adhesive contribution of the dynein bridges themselves! Therefore, a bend must grow fairly large before the curvature and resulting t-force reach the much higher threshold level needed to pull the doublets apart. The feedback to terminate the sliding comes from the force generated by the bridges via the t-force mechanism. Hysteresis in the bridge attachmeddetachment thresholds causes the episode of sliding to proceed far enough to ensure activation of a cascade on the opposing side of the axoneme. Because forces exerted on each doublet will summate toward the basal anchor, Ma is greatest near the flagellar base, and bends will originate there. Consequently, the t-force in the basal region will reach the critical threshold for dynein disengagement first (see Fig. 12). Once these basal dyneins release, the threshold for their more distal neighbors is reduced, sending a wave of dynein disengagement toward the flagellar tip. These events provide the necessary basis for base-to-tip wave propagation in the Geometric Clutch mechanism. The absence of a basal anchor severely affects the key events of the beat cycle. Not only is the formation of a basal bend inhibited but the coordination mechanism to establish repetitive cycles of reciprocation between the two sides of the axoneme is suppressed. Dissected or fractionated axonemal fragments will not typically reactivate to produce coordinated beating (Brokaw and Benedict, 1968; Lindemann and Rikmenspoel, 1972a; Summers, 1975; Woolley and Bozkurt, 1995). How-

50

CHARLES 6. LINDEMANN AND KATHLEEN S. KANOUS

ever, when the cut end of the fragment is manually “reanchored” (Douglas and Holwill, 1972; Woolley and Bozkurt, 1995) beating can be restored. Studies utilizing Ni2’ graphically illustrate the importance of force reciprocation between the two opposing halves of the axoneme in creating a beat cycle. Nickel ion selectively impairs the functioning of certain dynein arms more than others (Larsen and Satir, 1991; Kanous et al., 1993; Lindemann et al., 1995). In bull sperm, Ni2’ blocks the sliding of doublets 1-4 on one side of the axoneme (Kanous et al., 1993). The beating of reactivated bull sperm exposed to a perfusate containing Ni2’ becomes progressively more asymmetric until the flagella ultimately arrest at one extreme curvature of the beat cycle (Lindemann et al., 1995). According to the Geometric Clutch hypothesis, these cells have arrested at the point where the disengagement of the dominant bridge set has initiated in the basal region but has only propagated part way down the flagellum. This is the point where the opposing bridge set would normally start a cascade of attachment to reverse the prevailing curvature, thus completing the beat cycle. However, the cycle stalls because that set of dynein arms has been rendered dysfunctional by nickel ion inhibition. Theoretically, normal beating should resume if the missing motive force is manually supplied in the direction necessary to reverse the prevailing curvature. Experimental examination of Ni2+inhibited bull sperm demonstrated that micromanipulatory bending of the flagellum in the direction opposite to the sustained curvature direction will restore flagellar oscillation as shown in Fig. 7 (Lindemann et al., 1995). This resumption of beating occurs as the imposed bend becomes sufficient to compensate for the force normally supplied by the nickel-inhibited bridges, thus triggering the activation of the functional bridge set. This onesided bridge set induced oscillation fits well within the premises of the Geometric Clutch mechanism. The microprobe substitutes for the dysfunctional bridges, bringing the flagellar curvature to the end point normally controlled by those bridges. This position creates a positive (compressing) t-force that strongly activates the functioning bridges on the opposing side. These working bridges then rebend the flagellum until they reach their own t-force release point. Once they release, the flagellum snaps back to the induced position elastically, and the cycle repeats. The Geometric Clutch mechanism is not only compatible with the observed behavior but it actually predicts this response and explains how the beat is restored.

E. Reflections on the Experimental Data At this point, the Geometric Clutch hypothesis is a rudimentary framework, but one that unifies a large number of experimental observations into one orderly scheme. The conserved geometry of the axoneme might be

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explained by the need to provide just the right spacing to permit dynein bridge formation in response to distortion of the mechanical framework. Elastic interdoublet linkages are vital to the basic organization of the Geometric Clutch and are conserved along with the spacing requirements, even when the axoneme is modified to its most extreme. The necessity of force summation to produce directional dynein attachment episodes is consistent with the universal presence of a basal anchoring structure. In cases in which nature has eliminated the basal centriole, as in mammalian sperm, a replacement anchoring structure (the connecting piece) has been incorporated. The potential for reciprocal activation of two (or more) opposing dynein bridge sets also seems to be vigorously conserved, as would be expected if reciprocation were key to maintaining the oscillatory mechanism. This may define one role of the central partition as organizing the beat through entraining force reciprocation, thereby coordinating opposing bridges over longer distances. Perhaps this is why the partition is sturdiest and most easily observed in mammalian sperm (Lindemann et al., 1992; Kanous et al., 1993), compound cilia (Afzelius, 1959; Tamm and Tamm, 1984), and sea urchin sperm (Sale, 1986), all of which have a long working length. In shorter cilia, the axonemal torsion producing a more helical beat would interfere less with beat cycle coordination, as long as there was still sufficient reciprocation to provide the necessary activation trigger for the opposing bridges. Although axonemal division into two opposing bridge sets is not an absolute requirement of the Geometric Clutch mechanism, activation of particular bridges leading to the subsequent reciprocal activation of opposing bridges is necessary. This is achievable if the t-force resulting from each episode of bridge activity favors the activation of opposing bridge sites. If the axoneme is not bisected by a partition, the result is a more helical beat pattern. The extensive work of Sugino and Naitoh (1983) and Sugino and Machemer (1987, 1988) consistently demonstrates that, even in cilia that beat with a very three-dimensional waveform, there is still a general pattern of reciprocation between two opposed bridge sites. The fundamental nature of the dynein motor also seems particularly well suited for a geometrically gated coordination mechanism. When allowed to directly engage MTs, dynein arms translocate the MTs in a free-run reaction. Nothing like the locally imposed troponin-tropomyosin gating mechanism of skeletal muscle has been identified yet for dynein. The dynein motor is a fast translocator, allowing the rapid sliding rates necessary to power the 10- to 60-Hz beating of most cilia and flagella. Sliding rate modulation has been observed directly in in vitro assays and can be attributed to the axonemal functions controlling the speed or frequency of the beat but not the coordination mechanism (Hamasaki et al., 1991; Satir et al., 1995).

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Given the nature of the isolated motor, understanding the arrest response and calcium response of cilia and flagella may only be possible in the context of the intact axoneme. In the Geometric Clutch mechanism template, the shape of the beat and failure to complete the beat cycle is likely a result of changes in the probability of bridge attachment between the two opposing sets and failure to reach the t-force thresholds necessary for bridge attachment/detachment during beating. In the Geometric Clutch simulation of a cilium (Lindemann, 1994a,b), the asymmetry of the ciliary beat can be greatly enhanced by setting the t-force necessary for bridge engagement lower for the P-bend bridges while setting the t-force cutoff for bridge disengagement more negative. In other words, the dynein bridges on that side are easier to attach and harder to detach. In the intact axoneme, this could be accomplished by any change that increases the likelihood of bridge formation on one side of the axoneme. This could include the presence of a Ca2+-sensitive contractile protein in the nexin (Ohnishi et al., 1982) or dynein stalk/DRC complex (Piperno et al., 1992; LeDizet and Piperno, 1995a). Okuno (1980) analyzed the vanadate arrest of sea urchin sperm, deducing that the partially bent final configuration was due to the flagellum stopping just prior to the commencement of active sliding in the opposite direction. Satir and co-workers ( Wais-Steider and Satir, 1979; Satir, 1985; Satir and Matsuoka, 1989) first hypothesized that arrest responses are logically attributable to switching failure in the reciprocation mechanism of the two bridge sets. In terms of the Geometric Clutch mechanism, this switching failure occurs if (i) the dynein activation cascade is not initiated at the end point of the sliding episode of the opposing bridges or (ii) the generated t-force is insufficient to terminate a sliding episode. The first condition is probably the key in Ni2+-induced flagellar arrest because it has been shown that one set of dynein bridges is selectively impaired (Kanous et al., 1993; Lindemann et al., 1995). The second condition may describe the calcium and vanadate arrest circumstances. It has long been surmised that the flagellum remains under tension during calcium arrest, as if the bridges on one side of the axoneme are locked “on” (Gibbons and Gibbons, 1980). In the case of vanadate arrest, the tension necessary to reach the critical t-force level necessary for switching is probably compromised. Additionally, the ability to initiate a cascade of bridge attachment is negatively affected because vanadate interferes with the dynein cross-bridge cycle, leading to an accumulation of dynein in the unattached state (Sale and Gibbons, 1979;Mitchell and Warner, 1980; Okuno, 1980). If a sliding episode is already in progress, vanadate would be expected to weaken force production. Because t-force switching depends on the product of cumulative force X curvature, vanadate could impair the switching mechanism by interfering with both bridge detachment and reattachment.

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If the arrest patterns observed with Ca2+and Ni2+are a result of switching failure, then manipulation of arrested flagella to help trigger the switching event should induce an active flagellar response. This has been demonstrated in Ni2+-arrested bull sperm (Lindemann et al., 1980, 1995) and Ca2+-arrested Mytilus gill cilia (Stommel, 1986), resulting in a resumption of bend propagation. The defining principle of the Geometric Clutch hypothesis is the role of t-force, which is directly and predictably modified by mechanically repositioning the flagellum/cilium. Therefore, mechanosensitivity is an innate and unavoidable property of the axoneme in the Geometric Clutch paradigm. Disturbing the natural flagellar curvature with imposed vibrations (Gibbons et al., 1987; Eshel and Gibbons, 1989), mechanically imposedhestricted bends (Holwill and McGregor, 1974; Okuno and Hiramoto, 1976), or external fluid flow of sufficient strength and speed (Murase, 1990) all result in adjustments in the phase of beating. This is exactly the expected response if the t-force is responsible for coordinating the switching events in the beat cycle. The curvature of the flagellum is one of the two key determinants of the t-force magnitude. In long flagella with a simple axoneme, such as those of sea urchin sperm, the wave of active bending seems to be defined by a traveling region of uniform curvature. This observation undoubtedly played a key role in the efforts to develop a dynein regulatory model based on curvature control. The Geometric Clutch hypothesis predicts that this curvature uniformity is a result of a consistent switching threshold along the flagellar length, with each traveling bend powered by approximately equal numbers of active bridges. In contrast to sea urchin sperm, mammalian sperm are much stiffer near their base, thereby requiring the development of a greater cumulative force on the doublets to bend these flagella. Therefore, because the product of force times curvature determines the switching threshold, it should be reached at a lesser curvature in these axonemes. Not only do large mammalian sperm exhibit just such a reduced-curvature form of flagellar beating but the propagating bends increase in curvature as they move toward the flagellar tip where the stiffness is reduced (Gray, 1958). The mystery of the simultaneous and traveling components of M a highlighted previously (Rikmenspoel, 1971) becomes less enigmatic in light of the Geometric Clutch concept. The ciliary computer simulation demonstrates that bridge activation along most of the axonemal length can occur almost simultaneously when the t-force algorithm is used to control switching. This requires that the bridges controlling the P bend engage substantially easier than those forming the R bend. If this is the case, then nearsimultaneous activation of the P-bend bridges occurs spontaneously as the R-bend bridges disengage. The events mediating this trick of nature can

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be explained by the peculiar dynamics of t-force. The ciliary recovery stroke is dominated by the P-bend propagation created by the bridges on that side. R-bend bridge activation follows the propagating P bend, reversing the basal curvature. As R-bend bridge action terminates on reaching their switching threshold, there are still working P-bend bridges near the flagellar tip and these contribute a t-force that favors P-bend bridge attachment in the basal region of the flagellum. Normally, the action of one set of bridges works to disengage those bridges (self-terminating). However, because the R-bend bridges have reversed the curvature in the basal half of the flagellum (as can be seen at the end of the recovery stroke in Fig. l l ) , the force contributed by the P-bend bridges at the flagellar tip now promotes, rather than terminates, the engagement of more P-bend bridges near the flagellar base. This simultaneously snaps “on” the P-bend bridges along most of the cilium. This is the only currently devised bridge-switching scheme that logically explains both the ciliary and flagellar beat cycles with one consistent mechanism.

F. Caveats Although the Geometric Clutch idea can accommodate a great many experimental observations into one conceptual framework, it must be noted that it is still in a rudimentary form. The computed simulations are not yet sufficiently detailed to address certain important questions. As pointed out in a previous minireview (Lindemann and Kanous, 1995), the magnitude of the determined t-force is too large to be compensated for by the nexin links alone. Additional structures must also distribute and bear some of the t-force to prevent the axoneme from distorting greatly, or even rupturing, during normal operation. Goodenough and Heuser’s (1985) extensive microscopic reconstruction of the axoneme gives evidence for a set of transient linkages that move along with each dynein head as the doublets translocate. These “B-links” may be the structures that bear some of the t-force as the ATPase sites of the dynein head disengage from the adjacent doublet. The spokes may also serve to bear a considerable share of the t-force in an intact axoneme. The original Geometric Clutch simulations simplified the axonemal structure down to two opposing bridge sets (2,3,4 and 7,8,9) on opposite sides of the axoneme. In actuality, some of the force is also transferred to doublets 5-6 and 1 through the dynein bridges present in a complete axoneme. The spokes would be largely responsible for bearing the t-force acting on these doublets. Like the B-links, spokes have been observed to move along (or jump) as axonemal bending progresses. If the spokes and B-links are assumed to be the axonemal components designed to

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withstand and redistribute t-force, it becomes obvious that no hypothetical switching mechanism would be entirely satisfactory unless the influence of these structures has been incorporated. The intricacy of the organization of the inner arms and dynein regulatory complex suggests that further clarification of the function of these components is necessary. As discussed previously, a flagellum without outer dynein arms can still coordinate a normal (although slower) beat (Kamiya and Okamoto, 1985; Mitchell and Rosenbaum, 1985; Kurimoto and Kamiya, 1991; Hard et al., 1992). This indicates that the inner arms play a key role in the beat cycle events. The inner arms are positioned such that they are closest to bridging the interdoublet gap, and it is likely that they initiate the cascade of bridge attachment required for normal oscillation. Their location also makes the inner arms the ideal site for waveform modification and arrest. Although much more information will be needed to fully understand how the DRC and spokes are involved in beat modulation, a possible explanation is suggested by the Geometric Clutch mechanism. Changes in the configuration of accessory proteins at or near the attachment of the crucial inner arms may influence the ease o r difficulty of dynein bridge engagement and disengagement. This could be accomplished either by changing the orientation of the dynein head to the adjacent doublet or by changing the interdoublet spacing. At present, the Geometric Clutch model incorporates the known traits of the dynein motor in a simplified manner. Refinement of the model will involve a continued demonstration of compatibility with the best functional descriptions of all the axonemal components.

VII. Concluding Remarks The major strength of the Geometric Clutch hypothesis is its ability to make sense of a wide variety of experimental observations on cilia/flagella, consolidating them through one well-defined functional mechanism. The strict conservation of certain geometric and structural axonemal traits, such as interdoublet spacing and nexin links, can be identified as necessary in the axonemal conversion of dynein activity into flagellarkiliary beating. The action of the t-force in orchestrating the beat demonstrates that mechanical sensitivity, curvature control, and arrest phenomena are not exclusive of one another. In addition, simple rules of the Geometric Clutch mechanism allow small differences in t-force thresholds (necessary for bridge attachment/detachment) on opposite sides of the axoneme to have dramatic effects in modifying thc beat. This could form the basis for variations in beating observed in living cilia and flagella, including an explanation for the mechanism

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underlying the calcium response. This view of axonemal functioning is still formative (at present), but further exploration of this hypothesis, including additional experimental evaluation of its predictions, may provide a key to unlocking the mysteries of eukaryotic flagellar motility.

Acknowledgments The authors thank Dr. Esther Goudsmit for valuable input in the preparation of the manuscript. This work was supported by Grant MCB-9220910 from the National Science Foundation.

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Witman, G . B., Plummer, J., and Sander, G. (1978). Chlamydomonas flagellar mutants lacking radial spokes and central tubules. J. Cell Biol. 76, 729-747. Wong, P. Y. D., Lee, W. M., and Tsang, A. Y. F. (1981). The effects of extracellular sodium on acid release and motility initiation in rat caudal epididymal spermatozoa in vitro. Exp. Cell Res. 131, 97-104. Woodward, R., Carden, M. J., and Gull, K. (1995). Immunological characterization of cytoskeleta1 proteins associated with the basal body, axoneme and flagellum attachment zone of Trypanosoma brucei. Pwasitology 111,77-85. Woolley, D. M., and Bozkurt, H. H. (1995). The distal sperm flagellum: Its potential for motility after separation from the basal structures. J. Exp. Biol. 198, 1469-1481. Woolley, D. M., and Fawcett, D. W. (1973). The degeneration and disappearance of the centrioles during the development of the rat spermatozoon. Anat. Rec. 177,289-301. Woolley, D. M., and Osborn, I. W. (1984). Three-dimensional geometry of motile hamster spermatozoa. J. Cell Sci. 67, 159-170. Yagi, T., and Kamiya, R. (1995). Novel mode of hyper-oscillation in the paralyzed axoneme of a Chlamydomonas mutant lacking the central-pair microtubules. Cell Motil. Cytoskeleton 31,207-214. Yagi,T., Kamimura, S., and Kamiya, R. (1994). Nanometer scale vibration in mutant axonemes of Chlamydomonas. Cell Motil. Cytoskeleton 29, 177-185. Yanagimachi, R. (1981).Mechanisms of fertilization in mammals. I n “Fertilization and Embryonic Development In Vitro” (L. Mastroianni, Jr. and J. D. Biggers, Eds.), pp. 81-182. Plenum, New York. Yanagimachi, R., and Usui, N. (1974). Calcium dependence of the acrosome reaction and activation of guinea pig spermatozoa. Exp. Cell Res. 89, 161-174. Yano, Y.. and Miki-Noumura, T. (1980). Sliding velocity between outer doublet microtubules of sea-urchin sperm axonemes. J . Cell Sci. 44, 169-186. Yeung, C. H. (1987). Inhibition of the ATP-induced reactivation of demembranated hamster spermatozoa by the action of free A T P and MgATP2-. J. Reprod. Fertil. 81, 195-203. Yokota, E., and Mabuchi, I. (1994). CIA dynein isolated from sea urchin sperm flagellar axonemes. J. Cell Sci. 107, 353-361. Zanetti, N. C., Mitchell, D. R., and Warner, F. D. (1979). Effects of divalent cations on dynein cross bridging and ciliary microtubule sliding. J. Cell Biol. 80, 573-588.

Basement-Membrane Stromal Relationships: Interactions between Collagen Fibrils and the Lamina Densa Eijiro Adachi,*

Ian Hopkinson,*lt and Toshihiko Hayashit

* Department of Anatomy and Cell Biology, Kitasato University School of Medicine, Sagamihara, Kanagawa 228, Japan; t Wound Healing Research Unit, Department of Surgery, University of Wales College of Medicine, Cardiff CF4 4XN, Wales, United Kingdom; and Department of Life Sciences-Chemistry, Graduate School of Arts and Sciences, The University of Tokyo, Tokyo 153, Japan

Collagens, the most abundant molecules in the extracellular space, predominantly form either fibrillar or sheet-like structures-the two major supramolecular conformations that maintain tissue integrity. In connective tissues, other than cartilage, collagen fibrils are mainly composed of collagens I, 111, and V at different molecular ratios, exhibiting a Dperiodic banding pattern, with diameters ranging from 30 to 150 nm, that can form a coarse network in the extracellular matrix in comparison with a fine meshwork of lamina densa. The lamina densa represents a stable sheet-like meshwork composed of collagen IV, laminin, nidogen, and perlecan compartmentalizing tissue from one another. We hypothesize that the interactions between collagen fibrils and the lamina densa are crucial for maintaining tissue-tissue interactions. A detailed analysis of these interactions forms the basis of this review article. Here, we demonstrate that there is a direct connection between collagen fibrils and the lamina densa and propose that collagen V may play a crucial role in this connection. Collagen V might also be involved in regulation of collagen fibril diameter and anchoring of epithelia to underlying connective tissues. KEY WORDS: Collagen V, pNcollagen 111, Collagen IV, Collagen VII, Collagen XVII, Laminin, Fibronectin, Lamina densa.

1. Introduction The evolution of the coelomata, organisms characterized by separate tissues with specialized structures and functions, was critically dependent on the I,irentorioiial h'wicw of C'sro/og\,,V i d 17.1 lXJ74-7hY6lY7 $?S.lXJ

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Copyright 0 1997 by Academic Press. All rights of reproduction in any form reserved.

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FIG. 1 Quick-freeze, deep-etch electron micrograph showing the architecture of the fibrillar components in the extracellular space of the mouse pancreas. This figure demonstrates four major structures: (i) the stroma-bundles of collagen fibrils (COL) on which periodical ridges and furrows corresponding with the 67-nm periodic banding pattern are observed (ii) the lamina fibroreticularis (F)-collagen fibrils run independently and divide into two collagen fibrils (arrow): Microfibrils are also present, forming a filamentous network: (iii) the lamina

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development of extracellular matrixes (ECMs) that separate these tissues and organs. The structure and composition of different tissues vary but are largely defined by variations in the ratio of cellular to noncellular components. Parenchymal tissues, such as epithelia, endothelia, and neuronal tissues, consist of closely packed cells of different types but largely of a similar embryonic derivation and small amounts of stroma. The acellular stroma represents a morphological structure described by histologists, but studies during the past 30 years have demonstrated that this stroma represents a complex ECM that includes many gene products-the proportions of which define the nature of the tissue (Fawcett, 1968). Recently, it has become evident that the ECM has a complex interactive relationship with cells that it surrounds, influencing cellular morphology, proliferation, and migration (Haralson, 1993; Kreis and Vale, 1993; McMinn, 1967). The basement membrane (BM) represents a specialized ECM that separates tissue compartments, as exemplified by the separation of the epidermis from the dermis by the cutaneous BM, that acts as a selective filtration barrier as in the glomerular BM and modulates cellular proliferation and migration as seen following cutaneous injury. ECMs, both stromal and BMs, are composed of differing elements including fibers such as collagens and elastin and the diffuse “ground substance”-a complex heteropolymer that includes multiple forms of carbohydrate and glycoprotein moieties (Hukins, 1984; Timpl, 1989; Yurchenco, 1994). The BM may be divided into three layers on the basis of morphological studies: the lamina lucida, the lamina densa, and the lamina fibroreticularis (Inoue, 1989; Kefalides et al., 1979). Electron microscopic analyses reveal that the lamina densa, often referred to as the basal lamina, is a sheet-like structure that lies deep to the plane of the parenchymal cell layer (Fig. 1). The lamina fibroreticularis underlies the lamina densa and is considered to be the zone of the BM in closest apposition to the underlying stroma. The lamina fibroreticularis contains fine fibrils that demonstrate a banding pattern along their long axes and that exhibit argylophilia, that is, these fibrils stain black following silver impregnation. The lamina densa, an argylophilic structure, is widely seen in various tissues including simple epithelia, such as that of the duodenum; endothelia, as seen in arterioles; and stratified epithelia, as exemplified by the skin

densa (D)-a meshwork, composed of polygons, that underlies a pancreatic acinar cell (cell); (iv) the lamina lucida (L)-This layer appears as a narrow space between the lamina densa and the basal surface of the pancreatic acinar cell. Anchoring filaments (arrowhead) traverse the lamina lucida lo connect the lamina densa with the cell membrane. Bar = 0.5 pm. Reproduced with permission from Adachi and Hayashi (1994).

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(Montes et al., 1980). Light microscopic examination of the BM zone demonstrates that the lamina fibroreticularis contains collagen fibers and it is possible that these components of the lamina fibroreticularis may physically interact with those in the underlying stromal connective tissue. Studies performed by Plenk (1927), using light microscopy and silver impregnation staining, showed that coarse argylophilic fibers in the stromal matrix merge into a network of fine fibers that underlies epithelia (Fig. 2). This fine network is only poorly visualized by light microscopy but correlates with the basal lamina demonstrated by electron microscopy. Detailed electron microscopic studies of multiple thin sections have demonstrated a paucity of banding fibrils in or under the lamina densa and that the fine fibrils with the 67-nm periodic banding pattern, i.e., reticular fibrils, run parallel to the lamina densa rather than inserting and terminating in this structure (Fawcett, 1968). The physical relationship between the collagenous stroma and the lamina densa, the two major structures in the ECM, is unclear. Detailed analysis of the collagenous components of both of these ECMs has resulted in the description of multiple distinct collagen types while failing to elucidate the nature of the interaction between the stroma and the lamina densa. Collagens I, 111, IV, and V are the major collagenous components of the soft connective tissues; collagens I, 111, and V are categorized as fibrillar colla-

FIG. 2 Light micrograph of reticular fibers (arrowheads) in the small intestine revealed by the silver impregnation method. Blackened fibers are in the stroma of villi approaching and merging with the basement membrane (arrow) underlying the epithelium (epi). Bar = 10 pm.

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gens because of their potential to form banding fibrils, whereas collagen IV forms the meshwork structures that are characteristic of the lamina densa (van der Rest and Garrone, 1991; Weiss, 1984). The markedly different supramolecular aggregates that are formed by collagen IV, compared with those formed by the fibrillar collagens, have rendered the conceptualization of direct physical interactions between these contrasting structures difficult. It has been suggested that collagen V may directly mediate interactions between collagen IV in the BM and fibrillar collagens in the stromal ECM, possibly through direct physical connections, although other investigators have suggested that collagen V may form a second meshwork in the lamina densa together with collagen IV (Roll et a/., 1980). It was originally postulated that genetically distinct collagen types may form one of two possible types of supramolecular aggregate-collagens I, 11, and 111 forming fibrils with defined 67-nm banding patterns and collagen IV forming meshworklike structures. It was proposed that collagen V formed nonbanding fibrils spanning between the lamina densa and collagen fibrils with a 67-nm periodic banding pattern in the stromal ECM on the basis of immunoelectron microscopic findings; however, this remains somewhat unclear because there is some cross-reactivity between the anticollagen V antibody and collagen VI, which may itself form nonbanding fibrils (Ayad el af., 1984; Modesti et al., 1984). A direct consequence of distinct collagen species forming unique supramolecular structures, as described in the previous paragraph, is that there should be some form of direct physical interaction between these different supramolecular aggregates if tissue integrity is to be maintained. This is demonstrated by the network system of anchoring fibrils that underlies the lamina densa of stratified squamous epithelia, such as the skin or the mucosa of the oral cavity and the vagina. In these tissues the interstitial collagen fibrils of the underlying stroma are trapped by the arcade-like structures of anchoring fibrils formed from collagen VII, which has been localized in the lamina fibroreticularis of stratified squamous epithelia (Burgeson, 1993). In 1983, Adachi and Hayashi demonstrated for the first time that genetically distinct collagens could associate in single supramolecular structures, describing heterotypic collagen fibril formation. Initially, they showed that collagen V can form fibrils with a 67-nm periodic banding pattern; however, this was followed by the remarkable finding that collagen V could form heterotypic fibrils with collagen I, as demonstrated by transmission electron microscopy (Adachi and Hayashi, 1985, 1986). These findings demonstrated, for the first time, that heterotypic collagen fibril formation was possible, and as well as observing fibrils consisting of both collagens I and V, it was postulated that heterotypic fibrils of a rather different nature might be formed between collagen IV meshworks and collagen I fibrils. The existence of such collagen IV and V interactions would facilitate our

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understanding of interactions between the lamina densa, the lamina fibroreticularis, and the collagenous stroma. In this chapter, we will discuss the biochemistry and molecular biology of the molecules that are present in the BM zone, with particular respect to the collagenous components, the ultrastructure of the ECM in this zone, and the interactions between epithelial BM and the underlying stroma. The regulation of collagen fibril diameter by heterotypic fibril formation (especially collagen V and pNcollagen 111), the role of collagen IV retaining the structural information to form the meshwork structure of the BM, and the role of the anchoring fibril network and other anchoring systems are also discussed. Heterogeneous aggregates are composed of proteins that are members of different protein families, for example, the lamina densa, which contains collagen IV, laminins, and perlecan. Heterotypic fibrils, on the other hand, represent supramolecular structures that contain two or more members of the same protein family, whereas homotypic fibrils are composed of identical polypeptide (collagen molecules).

II. Molecules Related to Interactions between Collagen Fibrils and Lamina Densa Collagen molecules in ECMs demonstrate many unique features, one of which is the ability to form differing supramolecular aggregates, depending not only on the specific genetically distinct collagen type present but also on the ratio of different genetically distinct collagens present in heterotypic fibrils. The different collagen supramolecular conformations may also interact with other noncollagenous moieties, such as laminin or proteoglycans, that can also modify the properties of these heterogeneous aggregates although these noncollagenous supramolecular structures are not considered in detail in this article. In connective tissues other than cartilage, collagens I, 111, and V are the major collagenous components, forming banding fibrils, whereas collagen IV and laminin are restricted to the lamina densa (Fig. 3).

A. Collagen I Collagen I is the most abundant collagen in vertebrate tissues and represents approximately 70% of the total collagen content of such tissues; indeed, collagen I is the most abundant protein in vertebrates, accounting for approximately 22% of total body protein (Kielty et al., 1993). This typical fibrillar collagen is the major protein component of connective tissues, such

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FIG. 3 Thin section image of collagen fibrils in the dermis. In this tissue, collagens I and 111 are major constituents of the collagen fibrils that show a 67-nm banding pattern along their axes (bracket). Bar = 50 nm.

as tendon and bone, that consist largely of rectilinear arrays of collagen fibrils; however, collagen I is also a major constituent of skin and other complex connective tissues (Uitto et al., 1989). It appears that the content of collagen I is increased relative to that of collagen 111 in fibrotic disorders of the liver (Aycock and Seyer, 1989; Rojkind el al., 1979). We will discuss the structure, biochemistry, and molecular biology of collagen I in some detail and refer back to similarities to, and differences from, this molecule in the discussions of other collagens. Collagen I fibrils and microfibrils (see Section VI,A) are present in various tissues, although their arrangement in different tissues varies markedly: In tendon the fiber units of collagen are arranged in large parallel bundles, in skin the collagen fibrils form a coarse network that runs parallel to the skin surface, in the corneal stroma the collagen fibrils are arranged in orthogonally oriented and regularly packed fibrils (this arrangement is a factor in determining corneal transparency), and in cortical bone the collagen fibrils are arranged in concentric circles (Kielty ef al., 1993). The central role of collagen I in maintaining tissue structure is demonstrated by the widespread, sometimes catastrophic, clinical sequelae of mutations of the collagen I genes that result from

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apparently subtle alterations in the conformation of the collagen I protein (Prockop, 1990, 1995). 1. Molecular Structure of Collagen I

Collagen I isolated from mammalian tissues is a thread-like molecule that is 1.5 nm wide and 300 nm long (Weiss, 1984). Usually, collagen I is found in mammalian tissues as a heterotrimer of two al( I) collagen chains and one a2(I) collagen chain, although other variants have been isolated in disease states. The heterotrimeric collagen I molecule consists of three major structural domains; the amino and carboxyl non-triple-helical (TH) collagenous telopeptide domains and the central T H domain. The telopeptide domains are too small to be clearly visible by rotary shadowing; however, Kobayashi et al. (1986) demonstrated, using electron microscopy, two extensions corresponding with the carboxyl and amino telopeptides at both ends of segment-long-spacing crystals of collagen I, in which the collagen molecules are packed laterally and unidirectionally (Kiihn, 1982; Fig. 4). The thread-like and apparently rather sticky central TH domain of collagen I confers rigidity on the molecule and defines its unique physical proper-

FIG. 4 Electron micrograph of segment-long-spacing (SLS) crystallites of collagen I rotary shadowed with platinum vapor at an angle of 7". Collagen I, thread-like molecules approximately 300 nm in length, align in parallel with their ends in register through the crossbridges between the positively charged amino acids mediated by polyanion, adenosine 5'-triphosphate. The length of the SLS crystallites is identical to that of collagen I when measured crossing their banding pattern corresponding to the local bulkiness of the molecule. The arrow indicates the length of collagen I. N, N-terminal side; C , C-terminal side. Bar = 50 nm. Micrograph kindly provided by Dr. K. Kobayashi, Nagoya University, Japan.

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ties (Kielty et af., 1993). This central domain is composed of a triple helix formed from three polypeptide chains, each containing the characteristic G - X , - X 2repeating amino acid sequence motif, where X I is often proline and X2 is often the posttranslationally modified imino acid hydroxyproline (Bornstein and Traub, 1979); however, recent sequence analysis studies indicate that the proline at the X I position is by no means invariant (D. L. Evans, personal communication). Computer modeling studies have demonstrated that a minimal heptad G - X I - X 2repeat, which corresponds with two complete turns of the super helix, is required to generate the stable collagenous T H structure (Evans et af., 1995).

2. Genes Encoding Collagen I Structurally, the genes encoding the major fibrillar collagens are remarkably similar-consisting of 52 exons (Chu and Prockop, 1993)-although there are subtle differences in their exon : intron arrangement, for example, in the proal(1) collagen gene, exons 33 and 34 are fused. The genes encoding the major fibrillar collagens are composed largely of exons consisting of 54, or multiples of 54, nucleotide exons that are particularly GC rich as they encode a preponderance of glycine and proline residues, with the major, central, triple-helical domain being encoded by 42 exons. Each exon starts with a codon that encodes a glycine residue and each exon encodes a discrete number of G - X , - X 2repeating units. The exon :intron arrangement of the major fibrillar collagen genes is reminiscent of a conserved cassette that has undergone duplication and lateral shuffling to appear in a variety of proteins, as is demonstrated by the appearance of the repeating G - X 1 - X 2collagenous motif in proteins of such diverse function as the fibrillar and meshwork-forming collagens, collectins (Hoppe and Reid, 1994), bacterial pullulanase (Evans and Hopkinson, 1995), and the samirine tumourigenic protein of the saimirine herpes virus (Geck et al., 1990). This pattern of exon :intron structure has essentially been conserved between the genes encoding the different major fibrillar collagens in higher chordates; however, recent molecular phylogenetic analyses have shown that collagens I, 11, and I11 are comparatively recent members of the collagen superfamily that arose in concert with the evolution of the chordatae (Evans et af.,1995). The biosynthesis of collagenous proteins is complex but has been studied in the case of collagen I (Kielty et al., 1993; Kivirikko and Myllyla, 1982, 1985).The genes encoding the human a1( I) and a2( I) procollagen polypeptides are located at 17q21-22 (Solomon et af., 1984) and 7q21-22 (Henderson et al., 1983), respectively, and the cDNA encoding the proal(1) chain is some 4.8 kb in size, whereas that encoding the proa2(I) chain is 4.6 kb in size. Several positive and negative regulatory sequences have been defined in the genes encoding the crl(1) and a2(I) chains, both in an upstream

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location and in the first intron of these genes, although the detailed structure and interrelationships of these elements remain unclear (Chu and Prockop, 1993; Karsenty and Park, 1995; Vuorio and de Crombrugghe, 1990). The recent description of two common cis-acting elements in the upstream promoter regions of both these genes, one a CCAAT pentanucleotide motif that binds a heteromeric CCAAT-binding protein (Karsenty and de Crombrugghe, 1990), and the second centered around a G-rich region that binds the recently described c-krox protein (GalCra el al., 1994), presents a potential mechanistic explanation for the coordinated expression of the al(1) and a2(I) procollagen genes in the appropriate 2 : l ratio. An enhancer element located between 700 and 1300 base pairs (bp) in the first intervening sequence of the proal( I) chain of human procollagen I has been described (Rossouw et al., 1987), as well as an element at 8201093 bp that produces inhibition of transcription (Bornstein et al., 1987). The work on the mouse gene encoding the procul(1) chain has indicated that there is a complex series of positive and negative regulatory elements in the upstream region and first intervening sequence of this gene, including a strong promoter that is located some 250 bp upstream of the transcriptional start site and a site at -97 bp to which the CCAAT-binding protein may bind (Rippe et al., 1989). Three separate cis-acting elements have been delineated in the mouse a2(I) procollagen gene, at -80, -250 and -300 bp from the transcription start site, that are required for optimal activity of this promoter (de Crombrugghe et al., 1990). Recently reported studies from de Crombrugghe’s group, using the introduction of serially deleted upstream sequences of the pro al(1) gene into transgenic mice, have shown that the 3.2-kb upstream region from the transcriptional starting site contains a modular arrangement of at least three separate elements that confer tissue-specific expression on this gene (Rossert et al., 1995).

3. Posttranslational Modification of Collagen I The mature transcripts are translated on polysomes generating the initial preprocollagen polypeptides, which are translocated into the lumen of the rough endoplasmic reticulum where the 25-approximately amino acid residue signal sequence is cleaved (Gierasch, 1989). Within the lumen of the rough endoplasmic reticulum these single polypeptide chains undergo several enzymatically mediated posttranslational modification reactions (Kielty et al., 1993; Kivirikko and Myllyla, 1985). The most completely characterized of these posttranslational modifications is the hydroxylation of proline residues in the 4 position-the synthesis of 4-hydroxyproline being a prerequisite for the synthesis of stable collagen triple helices (Kivirikko et al., 1989). This hydroxylation reaction is catalyzed by the enzyme prolyl4-hydroxylase, a tetrameric enzyme that consists of two catalytically

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active cy subunits ( M , = 640 kDa), that have been completely sequenced from human, rat, and chicken tissues and two p subunits (M,= 60 kDa), that have been sequenced in human and other tissues (Kivirikko et al., 1989) revealing that this polypeptide subunit is identical to the enzyme protein disulfide isomerase that catalyzes the rearrangement of disulfide bonds (Koivu et al., 1987). The hydroxylation of proline residues in the 3 position, catalyzed by the enzyme prolyl 3-hydroxylase, also occurs in the lumen of the rough endoplasmic reticulum (Tryggvason et al., 1979); however, the functional significance of the product within the collagen triple helix and the structure of the catalytic enzyme are less well characterized than those for the 4-hydroxylation. A further enzymatic posttranslational modification of the procollagen polypeptides is the hydroxylation of lysyl residues, which are catalyzed by lysyl hydroxylase, a dimeric enzyme of 92 kDa in size (Myllyla et al., 1989). The hydroxylysine residues generated in this posttranslational modification step are involved in forming intraand interchain bonds as well as acting as sites for the enzymatic attachment of carbohydrates. Within the lumen of the rough endoplasmic reticulum galactosyl residues are linked to hydroxylysine residues in the collagen chains in reactions catalyzed by the enzyme hydroxylysyl galactosyl transferase; the galactosyl hydroxyline then acts as s further attachment sites for glucose that is linked in reactions catalyzed by the enzyme galactosyl hydroxylysyl glucosyl transferase (Kivirikko and Myllyla, 1979). These enzymatically catalyzed reactions may only occur on single collagenous polypeptide chains and are indeed a prelude to chain association that is initiated at the carboxyl termini of the three candidate polypeptide chains (Brass ef al., 1992)-this chain association event completely inhibits the hydroxylation and carbohydrate addition events. The chain association is also accompanied by the formation and rearrangement of intra- and interchain disulfide bonds, particularly at the carboxyl termini of the trimers, stabilizing this structure, which may then act as a nucleus for triple helix formation. It is probable that the enzyme protein disulfide isomerase (the /3 subunit of prolyl 4-hydroxylase) has some as yet ill-defined role in regulating the formation and rearrangement of these disulfide bonds and may be the ratelimiting factor for the chain association step (Koivu and Myllyla, 1987; Fig. 5). 4. Assembly of Collagen I into Fibrils The non-triple-helical amino- and carboxyl-terminal propeptides of the trimeric procollagen polypeptides are secreted into the extracellular space, where they are cleaved by specific enzymes; procollagen N-proteinase (Hojima et al., 1989) and procollagen C-proteinase (Hojima et af., 1985) render the molecules insoluble. These insoluble molecules, i.e., collagen, then

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FIG. 5 Scheme showing the biosynthesis (left column) and self-assembly (right column) of fibrillar collagens. In the rough endoplasmic reticulum, three pro-achains are posttranslationally glycosylated, associate through disulfide bonds in the C-propeptides, and fold into a triple helix in a zipper-like manner. The secreted procollagen is cleaved at each end by the two processing enzymes, N- and C-proteinase, to generate collagen molecules, which then self-assemble to form fibrils in the extracellular space. The collagen molecule is represented as a triple helix intracellularly but as a rectangle in the self-assembly process. Slightly modified with permission from Prockop and Kivirikko (1984).

undergo a spontaneous self-assembly process in which the processed collagen molecules aggregate into fibrils with the characteristic D-periodicity (Kadler et al., 1988; Mould el al., 1990). Approximately 2 h following in vitro cleavage of the propeptides at a temperature over 2YC, fibrillar structures that have one blunt end and one highly tapered end can be detected by darkfield microscopy (Miyahara et al., 1982; Fig. 6 ) . Fibril extension occurred only from the highly tapered ends, but spear-like projections eventually appeared from the blunt ends and these projections acted as initiation points for the growth of fibrils in the opposite direction (Kadler el al., 1990). Extension in both directions resulted in the formation of symmetrical fibrils in vitro. The studies suggest that collagen molecules retain the information that is required for a self-assembly process (Kadler

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FIG. 6 Time-lapse. darkfield light micrographs showing the formation of collagen fibrils in v i m . A mixture solution of pccollagen I (70 mg/ml) and C-proteinase was incubated at 29°C. The collagen fibril was photographed at 3 (a), 3.5 (b). 5 (c), and 6.5 h (d) after incubation. The collagen fibrils were first detected by darkfield microscopy as fibrillar structures with one blunt end and one highly tapered end. (a) Fibril extension occurred only from the pointed ends, but spear-like projections appeared at the blunt ends (b) and acted as new pointed ends for fibril growth in the opposite direction (c, d). Bar = 100 pm.

et af., 1987; Piez, 1982). The assembled structures are rapidly stabilized by the formation of intra- and intermolecular covalent cross-links generated from aldehyde derivatives of lysyl and hydroxylysyl residues in an enzymatic process in which the enzyme lysyl oxidase is involved in the early stages (Eyre, 1987). 5. Disorders of Collagen I

The central role of collagen I in maintaining tissue integrity is demonstrated by the sometimes catastrophic sequelae of even subtle changes in the conformation of the molecule caused by mutations in the genes encoding the a l ( I ) and a2( I ) procollagen polypeptides (Prockop and Kivirikko, 1995). This is most dramatically demonstrated in the osteogenesis imperfecta complex of disorders (Prockop, 1992), which is characterized by mutations in the genes encoding the collagen I chains leading to bone fragility that may be so severe as to be accompanied by intrauterine fractures or neonatal fatality as a result of compression of the fragile skull during vaginal delivery (Kuivaniemi et al., 1991). More commonly, the disorder is manifest as the occurrence of multiple pathological fractures during the teenage years, the

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occurrence of blue-tinged sclerae (due to some ill-defined abnormality of the arrangement of collagen fibrils in this tissue) and skin fragility, and a propensity to heal with abnormal scarring (Prockop et al., 1994).

B. Collagen 111 Collagen 111, another example of the fibril-forming collagens, represents between 5 and 20% of total collagen in mammals and is particularly abundant in large blood vessels (Mayne and Burgeson, 1987). Fibrils containing collagen I11 are thinner than those formed by collagen I, particularly in connective tissues with elastic properties such as the aorta, intestine, and lung (Fleischmajer et al., 1981). Collagen I11 was first isolated from human skin (Miller et al., 1971), although it has subsequently been shown to be present in adult skin as well as having a widespread distribution in other normal adult tissues, such as blood vessels, lung, and the wall of the small and large intestinal tracts, in which it often codistributes with collagen I. Covalent, lysine-derived cross-links have been demonstrated between collagen I and collagen I11 molecules (Henkel and Glanville, 1982), and it was shown that the collagen fibrils in skin include both collagens I and I11 (Keene et al., 1987a). The fine reticular fiber networks, described by histologists, that surround cellular aggregates in parenchymal tissues, such as the liver and the lung, have been shown to consist largely of collagen I11 (Adachi et al., 1991; Konomi et al., 1981). 1. Collagen I11 during Morphogenesis and Tissue Remodeling

Collagen I11 represents a quantitatively important collagen during fetal development and growth and the proportions of collagen I :collagen I11 vary with maturation both in utero and in the infant and adult states. In fetal skin, collagen I11 represents more than 50% of the soluble collagen (Epstein, 1974), but with postnatal maturation the synthesis of collagen I exceeds that of collagen I11 so that in early adult life the ratio of collagen I to collagen I11 in normal skin becomes approximately 6 : 1, as is generally described (Uitto et al., 1985). Interestingly, it appears that the expression of collagen I11 is increased relative to that of collagen I in cutaneous wound healing (Kuhn et al., 1989). This relative increase in collagen I11 expression is particularly marked in the early stages of the healing processes and is consistent with a recently described hypothesis in which it is proposed that tissue repair and tumorigenesis mimic some aspects of embryonic development and that the similarity of these processes may be at least partially defined by the preferential expression of embryonic-like ECM molecules (Evans and Hopkinson, 1996a,b).

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2. Genes Encoding Collagen 111 Structurally, collagen 111is quite similar to collagen I on the basis of electron microscopic analysis. The collagen I11 molecule is 1.5 nm wide and approximately 300 nm long and appears to be rigid like collagen I (Kiihn, 1982); however, unlike collagen I, it is a homotrimer of three identical a l ( I I 1 ) collagen chains [ a l (III)I3. The gene encoding the human al( 111) procollagen polypeptide is located at 2q24-33 (Solomon et al., 1985) and encodes for 1029 amino acids, including 343 repeating G - X , - X , triplets that correspond with a central T H domain that is 15 amino acids longer than that of collagen I (Ala-Kokko et al., 1989). The overall structure of the collagen 111 gene is remarkably similar to that encoding collagen I, although the collagen 111 gene is slightly longer and exons 4 and 5 are fused, unlike in the collagen I genes (Ala-Kokko et al., 1989). The delineation of regulatory elements in the collagen 111 genes is not as far advanced as for collagen I; however, negative regulatory elements have been described in the promoter region some 150 bp upstream of the transcriptional start site for collagen 111 (Mudryj and de Crombrugghe, 1988).

3. Synthesis, Secretion, and Possible Roles of Collagen 111 in Fibrogenesis The biosynthetic pathway that the nascent collagen 111 molecule follows is similar to that for collagen I; however, in the case of collagen 111, three copies of the single gene product aggregate in the lumina of the rough endoplasmic reticulum to form the TH structure prior to secretion into the extracellular space. Unlike collagen I, collagen I11 has a relatively higher 4-hydroxyproline content and contains cysteinyl residues as the carboxylterminal residue of the TH domain (Lillie et a!., 1987). In the extracellular mileu the amino- and carboxyl-terminal propeptides are cleaved in specific enzymatically mediated reactions, rendering the soluble precursors insoluble. A specific procollagen 111 amino proteinase has been described (Halila and Peltonen, 1986), but it appears that cleavage of the carboxyl-terminal propeptide of collagen 111 occurs preferentially compared with that of the amino propeptide because a substantial proportion of the collagen 111 molecules present in fibrils in vivo appear to have retained their aminoterminal propeptides (Fleischmajer et al., 1981). It has been proposed that the retention of the amino-terminal propeptides specifically limits the diameter of collagen I fibrils (Romanic et al., 1991), which in part explains the network of fine fibrils containing collagen I11 seen in relatively elastic connective tissues such as the aorta (see Sections 111,AJ and 111,BJ). The structural importance of collagen I11 in maintaining tissue integrity may be inferred from the sometimes dramatic clinical consequences of

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abnormalities in this protein caused by mutations in the collagen I11 gene as seen in the type IV variant of the Ehlers Danlos complex of disorders (Kuivaniemi et al., 1991; Prockop, 1992; Prockop et al., 1994; Prockop and Kivirikko, 1995). Patients with this disorder may present for the first time with sudden, catastrophic aortic or intestinal rupture, which is a result of instability of these tissues in which the abnormal collagen I11 is a major structural component (Pearl and Spicer, 1981). Similarly, a relationship between mutations in the gene encoding collagen I11 and familial aortic or cerebral aneurysm formation in the absence of the Ehlers Danlos type IV syndrome has been postulated (Kontusaari et al., 1990; Prockop, 1992), but this relationship is currently unclear.

C. Collagen IV 1. Molecular Structure of Collagen IV Collagen IV, composed of three a chains with homologous sequences, consists of three distinct domains; a short T H domain (7s domain) at an amino-terminal region, a major TH domain having multiple interruptions in the central region, and an NC1 domain at the carboxyl-terminal region (Kiihn, 1994; Hudson et al., 1993; Fig. 7a). This molecular structure is still hypothetical because collagen IV molecules cannot be isolated as full-length monomers without oligomers. Isolation of a monomeric protein with a triple-helical conformation would be necessary for direct protein analysis. Most of the findings concerning the chain composition have been derived from the analysis of the isolated NC1 domain trimer (Hudson et al., 1993). Although the chain composition of collagen IV is still controversial, the isoform, containing two al(1V) chains and one a2(IV) chain, is believed to be the major constituent of the collagen IV in tissues. Recently, we obtained the collagen IV samples without NC1 and 7s domains from the acid extract of bovine lens capsules by chymotrypsin treatment (Mukaiyama et al, 1995; Nakazato et al., 1995a). The chymotrypsin-treated collagen IV has a thread-like structure measuring 300 nm in length by electron microscopy. The chymotrypsin-treated collagen IV-derived triple-helical molecules may correspond well with the T H domain predicted from the base sequence. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed y chain (trimer of a chains) in size under nonreducing condition. Under reducing condition, two bands on SDSPAGE were seen with molecular weights of 140 and 115 kDa with an intensity ratio of about 2 :1. The 140-kDa band is composed of the a l ( IV) chain from the reactivity with a monoclonal anti-al( IV) chain antibody. These findings suggest that a major portion of the acid-soluble collagen IV

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FIG. 7 Structures of collagens IV (a, IV), V (b, V), and laminin (c, LN). Rotary shadowing electron micrographs of collagen IV isolated from bovine lens capsule, collagen V isolated from human placenta, and laminin 1 isolated from EHS tumor matrix. Collagens V and IV are thread-like structures that measure approximately 350 (a) and 300 nm (b) in length. Collagen IV appears to be more flexible than collagen V and has conspicuous globules, which are approximately 10 nm in diameter, at its carboxyl terminus. Laminin 1 has a cruciate structure, with three short chains (one of 43 nm and two of 36 nm in length) and a single long arm (77 nm in length). The three short arms have globular domains, which are separated by rod-like segments. At the end of the long arm, there is a globular structure (arrowheads) that corresponds with the G domain. Bar = 50 nm.

molecule in the lens capsule is constructed of two al(1V) chains and one a2( IV) chain. Four other a( IV) chains, namely a3( IV), a4( IV), a5(IV), and a6(IV), have been cloned (Kuhn, 1995). These chains have been classified into the collagen IV family on the basis of the polypeptide amino acid sequence deduced from the nucleotide sequences containing the unique, homologous domains in the al( IV) and a2(IV) chains. Although a3(IV), a4(IV), a5( IV), and a6( IV) chains are distributed in vivo in a tissue-specific fashion, immunohistochemical analysis with monoclonal antibodies against NC1 domains of six a chains of collagen IV revealed that all the collagen IV a chains have the restricted distribution on BMs (Ninomiya et al., 1995; Sado et al., 1995; see Section V,A,2). The collagen IV isolated from the mouse Engelbreth-Holm-Swarm (EHS) tumor matrix (Kleinman et al., 1982), which may consist of major BM components, has been used for biochemical studies on the structure and property of the collagen IV protein for many years. The protein is composed of two al(1V) chains and one a2(IV) chain with molecular

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weights of 185 and 175 kDa, respectively (Timpl el al, 1978). The collagen IV isolated from the bovine lens capsule, in addition to the protein mainly composed of al(1V) and a2(IV) chains, contains small amounts of a3(IV) and a4( IV) chains (Langeveld et al., 1988). However, no positive staining was obtained in the acid-soluble fraction of bovine lens capsule with antibody against the a3( IV), a4(IV), as(IV), or a6( IV) chains, suggesting that these chains were not enriched in the extracted collagen IV (M. Iwata et al., unpublished observation). Many reviews on collagen IV have been published (Inoue, 1989;Kefalides et al., 1979;Merker, 1994; Sage, 1982; Stanley et al., 1982;Timpl and Martin, 1982;Yurchenco, 1994).Therefore, we will focus on the collagen IV protein isolated from bovine lens capsule that contains short al(IV) or a2(IV) chains in this review because the collagen IV from lens capsule was found to reconstitute an architecture distinct from EHS tumor collagen IV in vivo and in vitro. 2. Length Polymorphism of a(1V) Chains

The collagen IV extracted from bovine lens capsule showed a distinct feature from the EHS collagen IV. Immunoblotting analysis revealed two bands with molecular weights of 180 and 160 kDa with monoclonal antial(1V) collagen antibodies in an acetic acid extract from bovine lens capsule. Two other polypeptides with molecular weights of 175 and 155 kDa reacted with a monoclonal anti-a2( IV) collagen antibody (Muraoka and Hayashi, 1993; Iwata et al., 1996). The 160-kDa al(1V) chain isolated from the bovine lens capsule retained the same NC1 domain as the 180-kDa al(1V) chain. The evidence may be summarized as follows (Iwata et al., 1995, 1996): First, relative intensity of protein staining of the 160-kDa polypeptide band to that of the 180 kDa band was the same as those of immunostaining with a monoclonal antibody, H11 (Sado etal., 1995),against the NC1 domain of the human al(1V) chain [positions 1643-1650, near the carboxyl-terminal end of the human al(1V) chain] as well as with a monoclonal JK-132, recognizing the TH region of al(1V) chain. Second, bacterial collagenase treatment of this 160-kDa polypeptide isolated from bovine lens capsule yielded a polypeptide of 30 kDa that retained the immunoreactivity with the H11 monoclonal antibody, corresponding well with those of the carboxyl-terminal NC1 domains of the collagen IV a chains (25-30 kDa). The amino-terminal region or 7 s domain ( M , = 20 kDa) would have been excised from the 180-kDa al(1V) chain to yield the 160-kDa al(1V) chain if the 160-kDa al(1V) chain is a processed form of the 180-kDa al(1V) chain rather than an alternatively spliced product of the al(1V) collagen gene.

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The lens capsule constitutes a specialized BM that is not connected to underlying connective tissue; it appears to support only the lens epithelial tissue (Brinker rt al., 1985), and it is much thicker (2 pm; Hogan et al., 1971) than other BMs (40-120 nm; Inoue, 1994). Other tissues rich in BM components, such as placental and renal tissues, were examined to find out whether they contained the short forms of the collagen I V a chains. Collagen IV, which comprises the skeletal meshwork of the lamina densa, is as highly insoluble as the major fibrillar collagens. No method is available for nondegradative extraction of collagen IV from these structures. After several attempts, nondegradative extracts from human placenta and bovine kidney with a neutral buffer containing SDS and urea or with acetic acid and urea were found to contain immunodetectable amount of short forms of the 160-kDa al(1V) and 155-kDa a2(IV) chains as well as the 180-kDa al(1V) and the 175-kDa a2(IV) chains (Sasaki et al., 1996). The 160-kDa al(1V) chain from human placenta may retain the NC1 domain in the same manner as the 160-kDa al(1V) chain from bovine lens capsule does because relative reactivities between the 180- and 160-kDa polypeptides extracted from human placenta were the same with the H11 monoclonal antibody raised against an epitope of the al(1V) NCl domain and with JK132 monoclonal antibody against the helical portion of a l ( IV) chain. The 175- and 155-kDa polypeptides may have been derived from the a2( IV) chain, as has been described for the collagen IV from the bovine lens capsule, because the 155-kDa polypeptide band as well as the 175 kDa band on SDS-PAGE reacted with H21, a monoclonal antibody raised against the NCI domain of the a2(IV) chain. Alternative splicings of the human a3( IV) (Bernal et al., 1993; Feng et af.,1994) and the a5(IV) chains (Nomura et al., 1993) occurred in the NC1 domains, but no alternative splicing has been reported in the genes encoding the a l ( I V ) and a2(IV) chains. Only a single size of a l ( I V ) transcript has been detected in the cells that produce both the 160-kDa al(1V) and the 180-kDa al(IV) polypeptides in the cell layer (M. Iwata et al., unpublished observation). The 160-kDa polypeptide was found in tissue extracts but not in the culture media of adult bovine lens capsular epithelial cells, although the 180- and 175-kDa polypeptides were present both in the culture media and in organ culture media (Taylor and Grant, 1985). Tokyo Metropolitan Institute of Gerontology-l (TIG-1) fibroblast from human fetal lung was found to secrete and deposit collagen IV in the cell layer with increased reactivity to a monoclonal anti-collagen IV antibody after several days of culture (Takeda et al., 1993). Collagen IV samples prepared from the cell layer contained two at( IV) chains (180 and 160 kDa), whereas the culture medium contained only the 180-kDa a I ( I V ) chain, implying that the 160-kDa al(1V) chain was generated in the extracellular space at a later stage of culture (Sasaki et al., 1996). Because the 175-kDa peptide

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reactive with anti-a2( IV) antibody was found in the media as well as in the deposit of cell culture and the 155-kDa peptide reactive with antia2(IV) antibody was found only in the deposit, the 155-kDa peptide may be derived from the 175-kDa peptide through processing as discussed for the production of the 160-kDa al(1V) chain from the 180-kDa peptide. Thus, the 175- and 155-kDa peptides may well be the a2(IV) chains and correspond with the 180- and the 160-kDa a l ( IV) chains, respectively. From the results described previously, the cell culture showed that the 160and 155-kDa peptides may be processed forms of the 180-kDa a l ( I V ) and 175-kDa a2(IV) chains, respectively. Recently, we found that the TIG-1 culture media and cell layer contained 180- and 175-kDa polypeptides, but no short chains, when the cells were incubated in the culture media without fetal bovine serum (Takahashi et al., 1996).

3. Homotypic Interaction of Collagen IV The collagen IV meshwork is formed and stabilized by various intermolecular interactions, including the formation of tetramers through the 7 s domains, the formation of dimers through the NC1 domains, lateral association between the T H domains, and NC1 domain-TH domain interactions (Siebold et al., 1987; Tsilibary and Charonis, 1986; Yurchenco and Furthmayer, 1984). In addition, collagen IV molecules are linked covalently with one another through disulfide and other covalent bonds (Ries et al., 1995;Weber et al., 1988). Because the interaction involving a combination of tetrameric associations at the 7 s domains and dimeric associations through the NC1 domains is only taken into consideration in the meshwork model of the collagen IV aggregate proposed by Timpl etal. (1981), the meshwork model has a structure of 800-nm sided polygons. This model cannot be directly adopted as a skeletal structure of the lamina densa because the meshwork size is too large in comparison with the fine meshwork found in normal tissues. Another model that incorporates the lateral association between the T H domains of collagen IV has been proposed as a skeletal architecture of type IV collagen in viva However, this model does not explain the fine meshwork of the lamina densa in the tissues. Consequently, the collagen IV and laminin double meshwork model is the currently accepted model (Yurchenco 1994; see Section IV). The increase in the turbidity of a collagen IV solution upon incubation above 28°C has been regarded as being a result of the aggregation or polymerization of these molecules (Veis and Schwartz, 1981; Yurchenco and Furthmayer, 1984). Several studies concerning the collagen IV isolated from EHS tumor indicated that the extracted molecules reassembled under physiological conditions, but that they could form a gel (see Section IV,B) only with other BM components (Kleinman et al., 1986). Yurchenco and

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Furthmayr (1984) reported that the association between the TH domains of collagen IV was thermally driven, as is the case with collagen I, to form a three-dimensional meshwork with branching filaments. Schwartz and Veis demonstrated that an acid extract of the bovine lens capsule, which contained collagen IV as a major component, formed aggregates under physiological conditions (Schwartz and Veis, 1980; Veis and Schwartz, 1981). They reported that a collagen IV solution remained soluble at 4°C at a neutral pH, but it gradually became turbid within 30 min after warming to 28°C. Turbidity increase of the solution depends on the temperature during incubation, protein concentration, and ionic strength. We revealed that the unchanged turbidity of the collagen IV did not indicate the absence of selfassembly. Recently, contrary to the previous consensus that collagen IV stays in solution as dispersed molecules at a low temperature, we found that collagen IV in a neutral pH solution maintained at 4°C became viscous. Furthermore, the continuous incubation at 4°C caused the lens capsule collagen IV to form meshworks that are quite similar to the skeletal structure of lamina densa in vivo (Adachi et al., 1996; Nakazato et al., 1996). The 160-kDa polypeptide was incorporated into the collagen IV meshwork reconstituted from the isolated bovine lens capsule collagen IV. Collagen IV molecules containing the 160-kDa polypeptide may differentially contribute to the intermolecular interactions in comparison with the molecules containing only the 180-kDa polypeptide and, hence, they influence the formation and structure of the meshwork. The NC1 domain of collagen IV has versatile specificity in the interactions of collagen IV molecules, presumably depending on environmental conditions. The NC1 domains may bind to each other to form end-to-end associations. Lateral association of the T H domains was initiated by the binding of NC1 domains to the triple-helical domain of a neighboring collagen IV molecule at several sites (Tsilibary and Charonis, 1986). The 160-kDa (rl(1V) chain can be dissociated from the bovine lens capsule in mild conditions, presumably due to the lack of about 144 residues of the 7 s domain. The 180- and 175-kDa a( IV) chains cannot dissociate under the same condition (Iwata et al., 1996) because the 180- and 175-kDa polypeptides were covalently linked through 7 s domains. The existence of the collagen IV molecules containing two 160-kDa a l ( IV) polypeptide chains and one trimmed a2(IV) chain is theoretically possible, and short molecules might be involved in the formation of the NC1 dimer, in the NCI-TH domain interaction, and in lateral association between TH domains but be free from the restraint of tetrameric associations through the 7 s domains. Incorporation of such trimmed collagen IV molecules may help loosen the architecture of the lamina densa, increase the flexibility of the collagen I V meshwork, or contribute to the formation of fine pore-sized meshwork. The presence

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of short a(1V) chains in the molecules may affect the interactions with other ECM molecules, particularly collagen V (see Section V,A,3).

4. Collagen IV Gel

Kleinman et al. (1982,1986) reported that extracts from EHS tumor matrix containing collagen IV formed a gel under physiological conditions. The major components, other than collagen IV, required for gel formation included laminin, heparan sulfate proteoglycan, and nidogen. These components polymerized in a defined proportion, implying that they interacted in a particular manner to form a lamina densa-like structure in a neutral phosphate buffer (Grant et al., 1989). Yurchenco and Furthmayr (1984) demonstrated that the collagen IV purified from EHS tumor in a neutral phosphate buffer increased its viscosity upon incubation at 28"C, presumably due to the self-assembly of the collagen IV molecules. However, the gel formation was not referred to. Recently, we observed that acid-soluble collagen IV from the bovine lens capsule formed gels in 2 M guanidine-HC1 and 50 mM dithiothreitol (DTT) in the absence of the other BM components (Muraoka et al., 1996). This is peculiar in that these conditions are generally believed to be dissociative for macromolecular complexes. In these experiments, the collagen IV solution dialyzed against 2 M guanidine-HC1 became viscous and eventually formed a gel by addition of 10 mM DTT.This implies that the interacting property of collagen IV cannot be extrapolated from a general consensus about protein-protein interactions. Indeed, these reagents were employed for the extraction of collagen IV from the EHS tumor (Kleinman et al., 1982). A summary of the comparison between the gel formed from bovine lens capsule collagen IV and Matrigel formed from EHS tumor components (Kleinman et al., 1986) is shown in Table I. Matrigel consists of laminin, collagen IV, heparan sulfate proteoglycan, and nidogen. Laminin accounts for almost 60% of the material, collagen IV for 30%, heparan sulfate proteoglycan for less than 3%, and nidogen for less than 6%. The gel formed from bovine lens capsule collagen IV is rigid and tough to mechanical, distorting force, whereas Matrigel is fragile and likely to be disrupted during manipulation. Recently, we found that a neutral pH solution of acid-soluble bovine lens capsule collagen IV became viscous and formed a gel in 150 mM NaCl at 4°C. The gelation' of collagen IV under physiological pH and NaCl raised a possibility that collagen IV forms fine meshworks of lamina densa, determining the physical structure and property in vivo (Nakazato et al., 1996).

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BASEMENT-MEMBRANE STROMAL RELATIONSHIPS TABLE I Comparison of Guanidhe-HCI-DT Gel and Matrigel

Guanidine-HCI-DTT gel Starting material Extraction with

Bovine lens capsule 0.5 M acetic acid

Purification Gelation conditions

DEAE-Sephacel and dialysis

Components

Matrigel EHS tumor 2 M urea, 50 mM Tris-HCI, pH 7.4

2 M guanidine-HCI, 10 mM DTT, 50 m M Tris-HC1, pH 8.1 at 4°C

0.15 M NaCI, 50 mM Tris-HCI, pH 7.4 at 35°C

Collagen IV

Laminin (60%), collagen IV (30%), heparansulfate proteoglycan (3%), nidogen (5%)

Note. Adapted from Muraoka et af. (1996) with permission.

" DTT. dithiothreitol.

D. Collagen V Collagen V is classified as a member of the fibrillar collagen family based on its primary structure and its potential to form banding fibrils (Adachi and Hayashi, 1985; van der Rest and Garrone, 1991; Fig. 7b). The tissue distribution and the amino acid sequence of collagen V tempted us to speculate that collagen V constitutes fibrillar aggregates for connecting lamina densa with collagen fibrils in the stroma (Adachi and Hayashi, 1994; Konomi et al., 1984). Immunohistochemical studies have demonstrated that collagen V is a constituent of collagen fibrils with a D-periodic banding pattern in cornea (Birk et al., 1988; Nakayasu et al., 1986), spleen (Adachi et al., 1987), and liver (Adachi et al., 1991; Schuppan et al., 1986). Studies involving a targeted mutation in the COL5 A2 gene suggested that collagen V has a regulatory role in ECM assembly (Andrikopoulos et al., 1995). Initially, collagen V was considered to consist of two a l ( V ) chains and one a2(V) chain (Burgeson et al., 1976). A third collagenous polypeptide chain was found in the copurified fraction from human placenta with these two chains (Sage and Bornstein, 1979). The third chain was originally described as the a C chain (Sage and Bornstein, 1979) and later renamed the a3(V) chain (Bornstein and Sage, 1980). Certain tissues, such as placenta (Madri et al., 1982; Sage and Bornstein, 1979), skin (Brown et al., 1978), and the synovial membrane (Brown et al., 1978), contain a relatively small amount of the a3(V) chain in comparison with the a l ( V ) and a2( V) chains. The complete primary sequences of human pro-al(V) chain (Greenspan et al., 1991; Takahara et al., 1991) and pro-a2(V) chain (Myers et al,, 1985;

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Weil et al., 1987) have been determined. The pro-al(V) chain, consisting of 1838 amino acid residues, is longer than the pro-a2(V) chain, which consists of 1496 residues. About one-third of the sequence of collagenous domain of pro-a3(V) chain has been determined (Mann, 1992). Another a chain was obtained from chick embryo tendon as the a4(V) chain but was renamed the a l f ( V ) chain because the mobility of the a4(V) chain after pepsin digestion on SDS-PAGE was similar to that of the a l ( V ) chain (Fessler et af., 1985a,b). The al(X1) transcript was proved to be present in chick embryo tendon and the a l ’ ( V ) chain is likely to be the al(X1) chain (Mayne etaf.,1996).The amino acid sequences of the alchains of collagens V and XI are homologous; particularly high homologies exist between the a l ( V ) , al(XI), and a2(XI) chains and between the a2(V) and a3(XI) chains.

1. Molecular Structure of Collagen V N-Terminal Region, TH Region, and C-Terminal Region Collagen V a chains, a l ( V ) and a2(V), undergo processing prior to incorporation into supramolecular aggregates in tissues (Fessler and Fessler, 1987) as collagens I, 11, and 111. However, collagen V is unique in that a ( V ) chains in tissue retain globular domains at the N terminal or at both N and C terminals. Sizes of the remaining globular domains of the a l ( V ) and a2(V) chains in tissue remain controversial (Bachinger et af., 1982; Fessler and Fessler, 1987; Moradi-Ameli et af., 1994; Niyibizi and Eyre, 1993; Niyibizi et af., 1984). On the other hand, there are no reports found concerning the tissue size of the a3(V) chain. The domains of procollagen V are designated as follows corresponding with homologous domains of collagen XI (Zhidkova et af.,1993); an NC domain (NC1) near the carboxyl end, a central collagenous T H domain (COLl), and the amino-terminal region, which is subdivided into an NC domain (NC2) , a short collagenous domain (COL2), and another NC domain (NC3). a. N-Terminal Region of Collagen V: NC2, COL2, and NC3 Domains The primary structure of the N-terminal propeptide region of procollagen V is similar to those of other fibrillar procollagens in that the domain organization of NC3, COL2, and NC2 is conserved. The overall sizes of the N-terminal region of procollagens V and XI are greater than those of the other fibrillar procollagens I, 11, and 111. The following subdomains of the NC3 domain of collagen V have been designated from the amino terminus: the signal peptide and the cysteine-rich and tyrosine-rich subdomains. The tyrosine residues in the NC3 domain are often sulfated (Fessler et af., 1986). The COL2 domain of the d ( V ) chain is interrupted at two sites, forming three small triple-helical domains composed of 5, 17, and 4

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triplets in the pro-al(V) chain. The corresponding structure consists of the 4, 18, and 2 triplets in the pro-a2(V) chain. Intensive studies, as reviewed by Fessler and Fessler (1987), have demonstrated that newly synthesized procollagen V molecules undergo more than one processing reaction step prior to deposition in tissues. Niyibizi and Eyre (1993) reported that the amino-terminal residue of the tissue form of the a1 (V) chain was alanine, which may correspond with Ala436 in the human a1 (V) chain (Ayad et al., 1994). The residue Ala436 is the eighth residue from the border of the COL2 domain. Processing of the bovine a2(V) chain appeared to be at Glu28 of the human pro-a2(V) sequence from the peptide sequencing of fetal bovine bone and skin. On the other hand, Linsenmayer et al. (1993) have proposed that the N-proteinase cleavage site is at Pro249-Gln250 of chick a1 ( V ) ,which corresponds to the Pro252-Gln253 of human al( V ) (Ayad et al., 1994). Furthermore, Moradi-Ameli et al. (1994) suggested that the putative cleavage site of human a l ( V ) chain is further down toward the C terminus from the finding that the epitope recognized by the antibody (residues 284-293) is not present in the mature collagen V protein. Although the COL2 domain of collagen V contains G-Xl-X2 sequences, the formation of a T H structure has not been proved. Using heparin column chromatography, which can separate the a l ( V ) chain from the a2(V) and a3(V) chains by the high affinity of the former chain, the existence of at least two major different sizes of the a l ( V ) chain was demonstrated in mouse embryos (K. Mizuno and T. Hayashi, manuscript in preparation). This is consistent with the report by van der Rest and colleagues (Moradi-Ameli et al., 1994) on two different tissue forms of the a I ( V ) chain in human tissues such as bone, umbilical cord, and chorioamniotic membrane. The functions of the NC domains at the N terminal are not known. The homotypic interaction and the association with other ECM molecules might be affected by the NC regions at both ends of collagen V.

6. The COLZ Domain The a l ( V) and a2( V) chains contain the COLl domain, which is composed of 1014 amino acid residues-that is, 338 uninterrupted G - X I - X , triplets. The portion of the T H region of the a3(V) chain sequenced has a higher homology with the a l ( V ) collagen chain than with the a2(V) chain (Mann, 1992). We should keep in mind that the amino acid composition and the distribution of residues in the a-chains of collagen V are most different from those of collagen I, a typical collagen among fibrillar collagen family. The similarity of collagens IV with V with respect to the amino acid composition of the major T H domain should be noted. The content of alanine residues in the a l ( V ) and a2(V) chains is less than that of the a(I) chains, whereas it is similar to that of the corresponding TH regions of collagen IV (Kresina and Miller, 1979; MacWright et al.,

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1983;Sage et aZ., 1979).Furthermore, the contents of bulky hydrophobic residues, such as leucine, isoleucine, phenylalanine, and tyrosine, in the triplehelical domain of the collagen V molecule with a chain composition of [a1(V)I2a2(V) are approximately 1.4 fold higher than that of the collagen I molecule and are similar to those of collagen IV. The number of posttranslationally modified lysine residues, such as hydroxylysine, galactosyl hydroxylysine, and glucosyl galactosyl hydroxylysine, in the a1( V) and a2( V) collagen chains is also higher than that in the a(1) chains and is close to that in the a(1V) chains (Brown etaZ., 1978;Hashimoto etal., 1988;Sage and Bornstein, 1979).The chemical similarity of T H domains of collagen IV and Vmolecules raises a possibility that the two types of collagen T H regions can give rise to heterotypic lateral association in tissues (see Section V,A,2 and V,B, 1). Because we have observed that pepsin-solubilized collagen V that does not have the NC domains can reduce the diameter of the reconstituted heterotypic fibrils of collagens I and V, we hypothesize that the T H domain of collagen V has the regulatory role in determining the fibril diameter, which is in contrast to the well-known hypothesis by Birk and colleagues (Birk and Linsenmayer, 1994;Linsenmayer et al., 1993) that the NC domain of collagen V is responsible for limiting the fibril diameter. Reconstituted fibrils of collagen V derived from pepsin-treated human placenta (Adachi and Hayashi, 1985) and of collagen XI derived from pepsin-treated bovine cartilage (Smith el al., 1985) exhibited fibrils with a diameter of approximately 30-50 nm and a D-periodic banding pattern, although the banding pattern may be less conspicuous than that of reconstituted fibrils of collagen I. The periodic distribution of hydrophobic amino acid residues has been described for the al(1) chain (Hulmes et al., 1973), but such hydrophobic periodicity is not found in the a l ( V ) and a2(V) chains (Y. Imamura, personal communication). Restriction in lateral association resulting in limiting of the collagen fibril diameter and a decrease in staggered arrangement of the fibrils involving collagen V may be related to the biochemical characteristics of the T H region of collagen V. There are no interruptions in the G-Xl-X2 triplet sequence in the major T H domain of a l , a2, and probably a 3 chains of collagen V. The frequency of proline residue in the COLl domain of the a ( V ) chains is about the same as that of the a(I ) chain-approximately 20% of the total amino acid residues. However, the distribution of proline in the a l ( V ) chain is uneven compared to that of the al(I) chain. Proline and hydroxyproline are thought to contribute to the stability of the triple-helical structure essentially through the entropic restraint by pyrrolidine ring and/or interchain hydrogen bonds (Privalov, 1982). The imino acid-deficient region of the collagenous triple-helical structure is suggested to be flexible (Veis and George, 1994; Paterlini et al., 1995). In the al(V)chain, proline residues are missing in a run of 7-triplet sequence together with two runs of 5-triplet sequences

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and six runs of 4-triplet sequences without proline residues. Some of the corresponding triplet sequences of the a2( V) chain also have a lower proline content. The regions devoid of proline are suggested to be less stable, and other regions rich in proline might be more stable. Then, at least the major subtype of collagen V containing two a l ( V ) chains in a molecule, [a1(V)12a2(V), might have a heterogeneity in the helical stability along the molecule. In fact, the subtype of [al(V)jza2(V) is reported to be susceptible to trypsin below physiological temperatures and the trypsin treatment gave rise to large fragments (Morris et al., 1990). Because the experimental data suggest that the subtype with the chain composition of a1 (V)a2( V)a3( V) has less stability than the subtype of [ a l ( V)],a2( V ) with respect to the temperature as monitored by CD spectrum change and the susceptibility to trypsin (Morris et al., 1990; K. Mizuno and T. Hayashi, unpublished observation), the subtype of a1 (V)a2( V)a3( V) might also have a different flexibility in the major T H domain. The native collagen V can be cleaved by the 72-kDa gelatinase (MMP-2) (Murphy et al., 1981; Sage et al., 1981) and the 92-kDa gelatinase (MMP-9) (Niyibizi et al., 1994). On the contrary, the TH domain of collagen V is not susceptible to cleavage by a mammalian collagenase (MMP-1) (Sage and Bornstein, 1979) that readily cleaves collagens I, I1 and I11 at a quarter length of the molecule from its carboxyl terminus. These results also suggest that the property of the TH domain of collagen V is distinct from that of fibrillar collagens I, 11, and 111. Pepsin-solubilized collagen V, which is composed almost entirely of the TH domain, has also been shown to interact with other proteins such as thrombospondin (Mumby et al., 1984), insulin (Yaoi et al., 1991), decorin and biglycan (Whinna et al., 1993), DNA (Gay et al., 1985), and heparin (LeBaron et al., 1989; Yaoi et al., 1990). The a chains of XI, al(X1) and a2(XI), also have the heparin binding sites in COLl domains as the a l ( V ) chain. However, none of the a2(V), a3(V), and a3(XI) chains have a heparin binding site. The peptide sequence responsible for heparin binding is well conserved in the al(X1) and a l ( V ) chains. Although the one-third of the amino acid sequence of the a3(V) chain sequenced has 73% homology with the a l ( V ) chain (Mann, 1992), the a3(V) chain has no affinity for heparin (Mizuno and Hayashi, 1996).

c. The C-Terminal Region of Collagen V The organization of the Cterminal domain is highly conserved among the fibrillar collagens. Because the folding of fibrillar collagens would initiate from the association of three a chains at the C-terminal domain and proceed to the N terminus, the high homology in the C-terminal domain between the al(X1) and a l ( V ) chains suggests a possible formation of heterotypic collagen V/XI molecules. Indeed, the possibility was demonstrated by the finding that [al(XI)12a2(V)

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collagen was isolated from the vitreous humor of the eye (Mayne et al., 1993) and from cultured rhabdomyosarcoma cells (Kleman et al., 1992). 2. Collagen V Subtypes

The chain composition of the collagen V molecule still remains to be elucidated, but the major triple-helical form of collagen V has the chain composition of [al(V)12a2(V) in human placenta and other noncartilaginous tissues (Fessler and Fessler, 1987). The a3(V) chain was originally isolated from human placenta (Sage and Bornstein, 1979). The chain has an amino acid composition similar to the a l ( V ) and a2(V) chains (Brown and Weiss, 1979; Sage and Bornstein, 1979). An al(V)a2(V)a3(V) form of collagen V is present in human placenta (Niyibizi et al., 1984; Rhodes and Miller, 1981) together with the [al(V)I2a2(V) form. The subtype accounts for approximately 45% of the total collagen V in placenta (Rhodes and Miller, 1981). On the other hand, the existence of other collagen V subtypes with the chain compositions [a1(V)I3 (Haralson et al., 1980,1984; Kumamoto and Fessler, 1980; Moradi-Ameli et al., 1994) and [a3(V)13 (Madri el al., 1982) has been suggested for different tissues.

a. Preparation of Collagen V Subtypes Different subtypes of collagen V molecules have been separated by using phosphocellulose chromatography (Rhodes and Miller, 198l), ammonium sulfate fractionation in 0.5 M acetic acid (Niyibizi et al., 1984), selective precipitation in phosphatebuffered saline (Hashimoto et al., 1988), sulfonate-column (Fractogel EMD SO3-; Merck Co. Ltd.) chromatography (Sato et al., 1995), or heparin column chromatography (Mizuno and Hayashi, 1996). The separation of [ ( ~ l ( v ) ] ~ c ~ 2from ( V ) al(V)a2(V)a3(V) can be accomplished by any one of these methods. The bound fraction of thermally denatured collagen V on a heparin column contains the a l ( V) chain, whereas the flowthrough fraction contains two components resolved on SDS-PAGE: a fast migrating a2(V) chain and a slow migrating a3(V) chain. The affinity of the a2(V) chain for heparin was slightly higher than that of the a3(V) chain (Mizuno and Hayashi, 1996). When the collagen V in native conformation was applied to the heparin column with the solvent of 20 mM phosphate buffer, pH 7.2, containing 2 M urea and 150 mM NaCl, the flowthrough fraction contained the al(V), a2(V), and a3(V) chains in an almost equal proportion. The bound fraction, which could be eluted from the column with 350 mM NaC1, contained no a3(V) chain but contained a l ( V ) and a2(V) chains with a ratio of 2 : 1. The differential heparin affinity of the two fractions could be ascribed to the number of a l ( V ) chains in the collagen V triple-helical molecule. The [a1(V)I2a2(V) subtype with two a l ( V )

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chains has roughly twofold heparin affinity of the al(V)a2(V)a3(V) subtype with respect to the required NaCl concentration for prevention of the binding (Mizuno and Hayashi, 1996). b. Possible Functions of Collagen V Subtypes The [a1(V)I2a2(V) and the al(V)a2(V)a3(V) subtypes essentially have a potential of forming the fibrils with a 67-nm periodicity at a temperature between 31 and 34°C. However, the fibrillar aggregates of the al(V)a2(V)a3( V) subtype reconstituted at 37°C tended to lose the periodicity, probably due to the weak association in lateral or in D-staggered array of this subtype, distinct from that of the [al(V)],a2(V) subtype (K. Mizuno et al., manuscript in preparation; see Section V,A,3). Therefore, we speculate that the a l ( V)a2( V)a3( V ) subtype is one of the candidate molecules that directly interact with collagen IV through the T H helical regions.

E. Collagen VII Collagen VII is a large, multidomain molecule that is restricted to a subBM distribution in stratified squamous epithelia such as those of the skin, cervix, and oral mucosa. This collagen was first described by Bentz et al. (1983) and was shown to be the unique molecular component of the anchoring fibrils that stabilize subepithelial BMs on the underlying stroma (Sakai et al., 1986). Collagen VII is synthesized predominantly by keratinocytes; however, dermal cells of a mesenchymal origin have also been shown to synthesize this molecule (Lunstrum et al., 1986). In recent years, intensive investigations have focused on the gene structure, protein structure, and macromolecular aggregation of collagen VII into anchoring fibrils, particularly in the context of the skin, because mutations in the gene-encoding collagen VII have been shown to cause some forms of the epidermolysis bullosa dystrophica complex of disorders (Burgeson, 1993;Uitto and Christiano, 1992).

1. Molecular and Supramolecular Structures of Collagen VII Collagen VII is a homotrimer, [a1(VII)I3, with a central TH domain that is approximately one and a half times as long as that of collagen I. Native, pepsin-digested collagen VII monomers are rod like and 470 nm long; however, the molecule is usually isolated as a dimer some 800 nm in length in which two monomers associate with an overlap at the globular carboxyl termini (Burgeson et al., 1990). Collagen VII is larger than the fibrillar procollagens ( M , = 1000 kDa) and has a large non-TH amino (NC1)- and a smaller non-TH carboxyl (NC2)-terminal domain. The central TH domain

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contains 19 non-TH discontinuities and it is thought that these interruptions, particularly the large discontinuity toward the center of the TH domain, confer some flexibility on the molecule (Bachinger et al., 1990). The large amino-terminal domain contains several sequences that encode for domains associated with cell matrix adhesion molecules including a motif that is homologous to the cartilage matrix protein sequence, nine fibronectin type I11 repeats, a von Willebrand factor domain, an R G D sequence, and a cysteine proline-rich domain (Christiano et al., 1994). It has been postulated, with some experimental support, that these adhesion sequences in the NC1 domain of collagen VII may mediate interactions between the anchoring fibrils and the BM and the underlying stroma (Burgeson et af., 1990). The smaller carboxyl-terminal domain contains a Kunitz-type proteinase inhibitor sequence. Type VII procollagen monomers associate into antiparallel dimeric assemblies that overlap at their carboxyl termini to form dimers in the extracellular space, which then aggregate by lateral association. These aggregates, which are stabilized by intermolecular disulfide bonds, represent anchoring fibrils with large globular NC1 domains at each of their ends (Morris et al., 1986). Such anchoring fibrils form a tightly packed fibrous structure, with their amino-terminal domains being inserted into the lamina densa at one end and into the collagen IV and laminin containing anchoring plaques in the sub-BM stroma at the papillary dermis in the case of skin (Burgeson, 1993; Keene et al., 1987b; Uitto and Christiano, 1992). Some anchoring fibrils have a U-shaped structure, both ends of which insert into the lamina densa-thereby entrapping the banded collagen fibrils-in the dermal connective tissue stroma. These anchoring fibrils form an extensive scaffold with a sub-BM distribution that together with the laminin 5 (kalinin/ epiligrin) containing anchoring filaments (Carter et al., 1991; Rousselle et al., 1991; Uitto and Christiano, 1992) generates a complex arrangement of anchoring fibrillar structures that stabilizes the attachment of the cutaneous BM on the dermis (see Section V1,C).

2. Gene Structure of Collagen VII The gene encoding human collagen VII, which is located at 3p21.3 (Parente et al., 1991), is approximately 31 kb in length and contains 118 exons (Christiano et al., 1994; Parente et al., 1991)-the largest number of exons described for any gene. The collagen VII gene encodes a 16-amino acid residue signal sequence, the large amino-terminal NC1 domain, the large T H domain, and the smaller carboxyl-terminal NC2 domain. The NC1 domain contains 1253 amino acid residues yielding a predicted chain size of 133,802Da, which is slightly smaller than the size predicted by electrophoretic analysis, but this is probably the result of posttranslational modification, including the putative covalent attachment of carbohydrate residues in N-glycosylation reactions. The central TH domain consists of approximately

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1530 residues with a total of 19 interruptions in the G-Xl-X2repeating sequence. One cysteine residue was identified in the TH domain and it has been postulated that it may form intermolecular disulfide bonds with cysteine residues in the NC2 domain of other collagen VII molecules. The carboxyl-terminal NC2 domain consists of 161 amino acid residues and contains eight conserved cysteine residues that are likely to be involved in the formation of disulfide bonds that stabilize the antiparallel association of two collagen VII molecules during the assembly of the anchoring fibrils. The complete sequence of collagen VII also includes a total of four R G D sequences that may be involved in the integrin-mediated interaction of collagen VII with cells. Interestingly, recent molecular phylogenetic studies have demonstrated that collagen VII represents an extant precursor for the major fibrillar collagen grouping (Evans et al., 1995) and this has facilitated the testing of the hypothesis that collagens with ancestral structural characteristics may be preferentially expressed in human tissue repair and disease states (Evans and Hopkinson, 1996a,b). Preliminary data indicate that collagen VII is indeed relatively overexpressed in human cutaneous wound healing (Anglin et af,, 1995), and studies are currently under way in our laboratories to analyze the expression of collagen VII in other human disease and developmentally associated states. 3. Roles of Collagen VII in Vivo The functional significance of collagen VII in maintaining the stability of the BM zone is highlighted in patients with various forms of epidermolysis bullosa dystrophica, which are caused by mutations in the collagen VII gene (Burgeson, 1993; Uitto and Christiano, 1992). The cardinal clinical feature of this disorder is the formation of blisters at the epidermal-dermal junction, which initiate at the level of the anchoring fibrils either spontaneously or following minimal trauma. The clinical severity of these disorders in individual patients may, to some extent, be related to the location of the causal mutation in the collagen VII gene and the predicted consequences of such mutations on the structure of the protein (Christiano et af., 1994). The detection of disease-causing mutations in the collagen VII gene, together with the subsequent mapping of the resulting abnormalities in the protein and the determination of the relationship between the protein abnormalities and disease severity, represents an elegant, dramatic advance in human molecular genetics. F. Collagen XVll 1. Molecular Structure of Collagen XVII

Collagen XVII was originally characterized as the 180-kDa bullous pemphigoid antigen (BPAG2; Westgate et af., 1985), a component of the hemides-

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mosomes (Uitto and Christiano, 1992), by using anti-BP180 autoantibodies isolated from patients with bullous pemphigoid and herpes gestionis to identify cDNA clones in human keratinocyte expression libraries (Giudice et al., 1992). Bullous pemphigoid and herpes gestionis are two of a group of autoimmune skin-blistering diseases in which IgG autoantibodies react with protein components of the hemidesmosomes-attachment structures located at the basal keratinocyte-lamina lucida junction in stratified squamous epithelia. The predicted amino acid sequence of the human BP180 protein (Giudice et al., 1992) contains several TH domains and this together with the recent determination of the complete primary structure of its murine counterpart has resulted in this protein being renamed collagen XVII (Li et al., 1993). Collagen XVII is an integral membrane protein that has been shown to be conserved in many species (Giudice et al., 1992; Li et al., 1993) and it is likely that its major function is the anchoring of epithelial cells to the underlying stroma. Molecular phylogenetic analyses have demonstrated that collagen XVII represents an extant progenitor for the entire collagenkollectin superfamily (Evans et al., 1995) and it is one of the few examples in which an extant progenitor molecule has been identified.

2. Gene Structure of Collagen XVII The human collagen XVII gene is located at 10q24.3 (Li et al., 1991) and the complete primary sequence of human collagen XVII has been determined by the sequencing of overlapping cDNA clones (Giudice et al., 1992). The protein is an integral membrane protein with its carboxylterminal collagenous domains projecting into the extracellular space. Human collagen XVII is 1497 amino acid residues in length and shares 80% identity with its mouse homolog. The collagenous portion of collagen XVII is located in the 1007-residue extracellular domain and includes 15 short TH segments separated by short non-TH interruptions, which range from 8 to 60 residues in length. Analysis of the cDNA-derived amino acid sequence of human collagen XVII predicts the existence of a transmembrane domain, a most unusual structure for a collagen, which due to steric constraints is the most highly conserved domain in the chick, mouse, and human collagen XVII sequences (Giudice et al., 1992; Li et al., 1993). The murine collagen XVII gene is the best characterized of the collagen XVII genes (Li et al., 1993) and spans some 4299 nucleotides between the translation initiation and termination codons, encoding a polypeptide of 1433 amino acids with a predicted molecular mass of 144 kDa. Regulatory sequences in the collagen XVII gene are poorly understood; however, a consensus TATA box has been located 26 nucleotides upstream of the transcriptional start site and two inverted repeat sequences, (T)13AA and

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7T(A),I , have been described, which may facilitate RNA secondary structure formation with some possible role in the translational regulation of gene expression. The amino-terminal region consists of a noncollagenous segment of 573 amino acids-the NC1 domain that does not include any previously described signal sequence; however, four closely neighboring cysteine residues, sites for phosphorylation, a leucine zipper pattern, and an R G D cell adhesion sequence are located in this domain. Computeraided analyses predict the existence of at least one, if not two, membraneassociated sequences in the NC1 domain of murine collagen XVII, indicating that this is a transmembrane protein-one of only two transmembrane collagens so far described, the other being collagen XI11 (Kielty et al., 1993; Prockop and Kivirikko, 1995). The adjacent collagenous sequence of 860 residues, which represents the carboxyl-terminal portion of collagen XVII, includes 13 separate domains with the G - X I - X zrepeating triplet motif, varying in size between 12 and 242 residues, separated by noncollagenous interruptions of 9-57 residues in size. Six putative N-glycosylation sites have been identified in the entire collagen XVII sequence: four within the NCI domain and two in noncollagenous interruptions in the collagenous part of the molecule. The presence of the extracellular interrupted collagenous sequence in collagen XVII may be related to its function as a hemidesmosomal protein in that the extracellular collagenous segment may physically interact with other components of the BM such as integrins or lamina lucida proteins. It has been predicted, on the basis of comparisons with other collagens with multiple interruptions in their TH domains such as collagens IV and VII, that the non-triple-helical interruptions may confer some flexibility to the molecule. These structural features of collagen XVII are consistent with its proposed function in maintaining some direct physical interaction between the two other components of the hemidesmosomesthe 230-kDa bullous pemphigoid antigen and the a6P4 integrin-and epithelial cells overlying the BM.

G. Laminins Laminin, first isolated as a major noncollagenous protein from EHS tumor matrix (Timpl et al., 1979), was shown to exhibit unique cell-attachment properties and was considered to be involved in defining the structural and functional characteristics of the lamina densa (Timpl and Brown, 1994). This protein was initially believed to localized to the lamina lucida (Courtoy et al., 1982; Foidart et al., 1980); however, extensive studies using the immunoferritin technique have shown that laminin is distributed throughout the whole depth of the lamina densa and that it is integrated into the meshwork arrangement of this structure (Inoue, 1989).

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1. Molecular Structure of Laminins Initially, laminin was considered to represent a single molecular species that was restricted to a lamina densa distribution; however, it has become apparent that laminins are members of a family of disulfide-linked heterotrimers composed of three different polypeptide chains designated a,0, and y. There are multiple variants of each of the laminin chains-a ( a l , a2, a3, and a 4 variants), j3 (j31, j32, and j33 variants), and y (yl and y2 variants)-and of estimated molecular sizes-a chains, 200-400 kDa (heavy chains), and j3 and y chains, 150-200 kDa (light chains) (Burgeson ef al., 1994). The various molecular isoforms of laminin are derived from a combination of one a,one j3, and one y chain that twist around each other to form a long arm, as demonstrated by rotary shadowing electron microscopy. Ten laminin isoforms, each with a tissue-specific distribution, have been characterized to date: laminin 1 (alj3lyl), which is present in all lamina densa apart from those in skeletal muscle; laminin 2 (a2j3lyl), present in skeletal muscle and peripheral nerves; laminin 3 (alj32yl), present in the neuromuscular junctions; laminin 4 (a2j32yl), present in the myotendinous junctions; laminins 5 ( ( ~ 3 0 3 ~and 2 ) 6 (a4j3lyl), which are thought to be located at the epidermal-dermal junction, although this latter localization remains the subject of some debate; and laminin 7 (a3j32yl), present in the amnion and the fetal skin (Aumailley and Krieg, 1996; Engvall, 1993). Rotary shadowing electron microscopy has revealed that laminin 1 has a cruciform structure (Fig. 7c), with three short arms (one of 43 nm and two of 36 nm in length) and a single long arm (77 nm in length). The long arm of laminin 1 is composed of a coiled-coil structure of three chains; however, two of the short arms of laminin 1 have two globular and two rod-like domains corresponding with the single j3 and y peptide chains and one arm that corresponds with the globular domains of an a chain (Engel, 1993). Laminin 5 does not assume this cruciform structure, but rather it adopts a dumbbell-shaped form, a result of the 0 3 and y2 chains being shorter than the j3 and y chains of laminin 1 (Rousselle et al., 1991).

2. Roles of Laminin Superfamily in Vivo Laminins are intimately involved in many physiological phenomena such as cell attachment and neurite extension through interactions with specific integrin receptors. Specific immunofluorescence staining for laminins was observed prior to the formation of the lamina densa during the branching morphogenesis process in salivary glands and the renal glomeruli when the distal parts of the long arms of laminin 1 interacting with a601 are considered to have a central role in this process (Kadoya et al., 1995; Klein er al., 1988; Sorokin el al., 1992).

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The a6P4 integrins, which are present on the stromal side of epidermal basal cells (Sonnenberg et al., 1991), have an important role in maintaining the structural integrity of the skin, because they are a component of the hemidesmosomes that stabilize the epithelial side of the basement membrane through a network of cytoskeletal filaments (Carter et al., 1990; Marchisio et al., 1991). Recently, we have observed immunospecific labeling for laminin 5 on the anchoring filaments traversing the lamina lucida of the skin, which is consistent with laminin 5 representing an anchoring filament-like structure in the lamina lucida that interacts with the a6P4 integrin component of the hemidesmosomes. The hemidesmosome is a complex structure that contains several polypeptides of varying molecular weights, ranging from 120 to 500 kDa on electrophoretic analysis (Owaribe et al., 1991). The major cytoskeletal elements in epidermal cells are the tonofilaments, which are largely composed of members of the keratin superfamily of gene products (type I, 9-17; and type 11, 1-6). Keratin filaments associate to form bundles that span between desmosomes and electrondense regions of hemidesmosomes, indicating that there is clear structural continuity between cytoskeletal filaments in the cytoplasm and anchoring filaments in the ECM (Moll et al., 1982). The interaction between integrins and laminins is a central determinant of cellular behavior. Recent data from our laboratory have demonstrated that laminin 5 promotes the migration of keratinocytes and triggers the assembly of the lamina densa by keratinocytes harvested from synthetic dermal-equivalent tissues (Adachi, 1996). The intermediate filaments, in turn, would be connected to the nuclear envelope, which contains B-type lamins, suggesting some alteration in nuclear structure (Georgatos and Maison, 1996). 3. Interactions between Laminins and Other ECM Molecules Laminins and collagen IV interact with each other directly (Ohno et al., 1991) or through nidogen (see Section II,H), a 150-kDa glycoprotein that is found in the lamina densa (Paulsson et al., 1987a). Yurchenco and O’Rear (1994) have proposed that structurally independent homotypic aggregates of laminin and collagen IV, stabilized by nidogen, may be superimposed on one another to form the three-dimensional network of the lamina densa. This model is in some respects unconvincing, because it is unclear whether two separate superimposed meshworks are indeed present in the lamina densa, because domain IV [the domain most intimately involved in the self-assembly of laminin 1 (a1/3171)] is not well conserved in the laminin a3 and p3 chains and is absent from the y2 chain. We have also observed anchoring filaments spanning between the basal surface of keratinocytes and collagen fibrils reconstituted from collagen I in skin equivalent tissues (Adachi and Tsunenaga, 1994); moreover, it has been shown that collagens

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VII and XVII can interact with a3, p3, and y2 chains of laminins indicating that these molecules are likely to have some role in maintaining the stability of the BM zone in epithelial tissues (Aumailley and Krieg, 1996). H. Other Components: Nidogen, Perlecan, and Osteonectin The lamina densa is an undulating sheet-like structure, formed from heterogenous extracellular aggregates, that associates with epithelial, endothelial, and some mesenchymal cells and has a central role in the regulation of cellular migration, morphology, and proliferation and may also act as a physical and electrostatic filtration barrier for macromolecules. Several macromolecular aggregates are involved in the formation of the lamina densa and define its supramolecular architecture-local variations that can confer specific cellular signals on restricted portions of this structure (Kleinman et al., 1986; Timpl, 1989). The major structural components of the lamina densa, other than collagen IV and laminin, are nidogen, perlecan or low-density heparan sulfate proteoglycan, and osteonectin. Nidogen is a sulfated glycoprotein composed of a single polypeptide of approximately 150 kDa with globular domains at each end of the molecule flanking a rod-like central domain as seen by electron microscopic rotary shadowing examination (Carlin et al., 1981; Timpl et al., 1983). The central (G2)and carboxyl-terminal (G3) globular domains of nidogen are involved in binding with the rod-like segments of the short arms of laminins and with collagen IV, respectively. These binding activities of nidogen are considered to have a central role in stabilizing the heterogeneous aggregates that are involved in the assembly of the lamina densa (Fox et al., 1991). Nidogen is exclusively distributed in the lamina densa and has been shown to be expressed as early as the 8-16 cell stage of embryonic development in the mouse (Dziadek and Timpl, 1985). Perlecan is a large proteoglycan consisting of a core protein (M, = 400-450 kDa) with three heparan sulfate side chains (M,= 30-60 kDa) attached to its amino-terminal end. The core protein of perlecan is 83 nm long when viewed by rotary shadowing electron microscopy and contains five or six globular structures, ranging from 5 to 15 nm in diameter, that form a tandem array resembling a necklace of pearls. Perlecan has been shown to be localized to the lamina densa in various tissues and it is thought to be an integral membrane protein that interacts with laminins and collagen IV (Hassell et al., 1980; Paulsson et al., 1987b). The negative electrostatic charge of the perlecan molecule is largely derived from the heparan sulfate side chains and this is likely to have a role in the process of charge-selective filtration at the glomerular BM. It is also likely that perlecan may also be involved in multiple cellular functions, which include cell attachment

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(Clkment et al., 1989), tissue development, and neoplastic growth (Iozzo, 1994) and the assembly of macromolecules in the lamina densa (Laurie et al., 1986), in particular anchoring antithrombin (Pejler et al., 1987) and basic fibroblast growth factor at the locations predicted by other studies (Laurie et al., 1993; Rouslahti and Yamaguchi, 1991). Osteonectin was first isolated from periosteal tissues as a noncollagenous protein that is related to the process of mineralization (Romberg et al., 1985) and was then shown to be identical to a novel 40-kDa protein that was subsequently isolated from a 6 M guanidine-hydrochloride extract of EHS tumor matrix. The results of further studies suggest that osteonectin is produced in many soft connective tissues and has a broader function than was originally envisaged in a variety of developmental, tissue repair, and remodeling processes (Dziadek et al., 1986).

111. Regulation of Collagen Fibril Diameter by pNcollagen 111 and Collagen V The diameter of collagen fibrils is different in various tissues, for example, 120 nm in tendon, 80 nm in the dermis, and less than 50 nm in cornea (Kielty et al., 1993; Fig. 8). Prior to the isolation and characterization of collagen I1 in hyaline cartilage (Miller and Matsukas, 1969), the variation in collagen fibril diameter in different tissues was attributed to differences in the microenvironment where fibrillogenesis had occurred because collagen I fibrils that were apparently composed of a single molecular species were the only banded fibrils that had been described. The isolation and characterization of further genetically distinct collagens, particularly collagens I1 and 111 (the other two major fibrillar collagens), was followed by the description of banded fibrils of varying diameters in tissues (Bornstein and Sage, 1980). It was assumed that each genetically distinct collagen would form distinct fibrillar structures containing that collagen alone, that is, homotypic fibrils, and that the fibrils formed by each individual collagen type would have their own characteristic features, e.g., collagen I would form thick fibrils and collagen 111 would form distinct thin fibrils even in the same tissue, because it had been shown that several genetically distinct types of collagen could be isolated from a single tissue. In recent years, it has become evident from the results of many studies using a combination of rigorous isolation techniques, immunohistochemical techniques at both the light and electron microscopic level, immunoblotting analyses, epitope-specific antibodies, and various in vitro fibril assembly models that individual collagen fibrils of varying sizes are in fact composed of more than a single collagen type. This is seen clearly in the case of banded collagen fibrils in the skin, in

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FIG. 8 Cross-sectional profiles of collagen fibrils in the dermis and the cornea. The average diameters of the collagen fibrils were approximately 80 nm in the dermis and 40 nm in the cornea. Collagens I and I11 are major constituents of collagen fibrils in the dermis, whereas collagens I and V are the major constituents of those in the cornea. The diameter of the collagen fibrils depends mostly on the collagen types involved and the ratio of different collagen types in a single heterotypic fibril. Bar = 50 nm.

which individual fibrils have been shown to stain with specific anticollagen I, 111, V,and XI1 antibodies, indicating that these fibrils do in fact include several collagen types and represent heterotypic aggregates that associate to form banded fibrils (Adachi and Hayashi, 1986; Birk et al., 1986; Keene et al., 1987a; Lapikre et al., 1977; Mendler et al., 1989). Although some authors showed that the microenvironment, such as the pH range of 7.2-8.0, may be necessary for the formation of collagen fibrils (approximately 20 nm in diameter) in organ culture of chick corneas (Bard et al., 1993), pNcollagen I11 and collagen V would be the most responsible molecules to inhibit the growth of collagen fibrils.

A. Collagen V and pNcollagen 111 Involved in the Regulation of Collagen Fibril Diameter in viva Variation in collagen fibril diameter and composition is clearly seen in human skin. Histologically, the skin consists of three compartments: the epidermis superficially, the dermis, and the subcutaneous tissue-the deep

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layer. The dermis is further subdivided into two layers: the papillary layer, which lies immediately deep to the lamina densa, and the reticular layer that corresponds with the deeper, underlying stroma in other tissues. In the reticular layer, the bundles of collagen fibrils, which are 80-100 nm in diameter, interact with one another to form a network, whereas in the papillary layer the collagen fibrils (40-60 nm diameter) run independently to form a separate reticular structure (Holbrook and Smith, 1993). The most obvious difference between the two collagen fibril systems in the reticular and papillary dermis is the diameter of the individual fibrils: Collagen fibrils in the reticular dermis, corresponding with the deep stroma, are thick and tend to form bundles, whereas those in the papillary layer next to the lamina densa are thin and run independently with little tendency to form bundles. Currently, two distinct collagens are considered to have a major role in determining the diameter of heterotypic collagen fibrils: collagen V and pNcollagen 111, that is, collagen 111 from which the carboxyl-terminal propeptide has been cleaved but that retains the amino-terminal propeptide. Collagen V and pNcollagen 111are similar in shape, particularly with respect to the large globular domains that are present at their amino termini, and it is considered that these similar domains may inhibit the lateral association of collagen molecules into fibrils, thereby limiting fibril diameter (Fleischmajer et al., 1985; Fig. 9; see Section III,B,2). The major difference between collagen V and pNcollagen 111, in this context, is the potential for cleavage of the amino propeptide of pNcollagen 111 by the collagen 111-specific Nproteinase, which could potentially occur following the incorporation of pNcollagen 111 into fibrils because the N-proteinase is present and active in the extracellular mileu where it is secreted by the collagen-synthesizing cells (Halila and Peltonen, 1984; Nusgens et al., 1980).

1. pNcollagen 111 on Fine Collagen Fibrils Fine fibrils are also widely distributed in embryonic tissues in which substantial amounts of collagen 111 and pNcollagen 111 (collagen I11 that has retained its amino-terminal propeptide) are found (Fessler et al., 1981). It has been demonstrated that the average diameter of the fine collagen fibrils in embryonic skin in vivo is inversely proportional to their pNcollagen 111 content (Fleischmajer et al., 1985); collagen 111 itself was not considered to regulate fibril diameter because collagen 111 fibrils of various sizes have been identified in tissues such as skin (Keene et al., 1987a). The relationship between fibril composition and fibril diameter is of particular interest in embryonic tissues because the size of collagen fibrils changes dramatically during both embryonic development and extrauterine maturation of skin and tendons. pNcollagen 111 has also been shown to be distributed in the

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FIG. 9 Immunoperoxidase staining of collagen fibrils in the human placenta using antibodies against pNcollagen I11 (pNIII) and collagen V (V). Dense reaction products are seen on collagen fibrils (arrows) under the lamina densa of endothelial cells (end) and in placental villi, but they are not present on microfibrils. The diameter of these collagen fibrils is between 30 and 40 nm. Bar = 0.5 pm.

small (40-50 nm diameter) collagen fibrils of the hepatic sinusoids, in which preliminary investigations indicate that it is present at an approximately equimolar ratio to completely processed collagen I11 (Geerts et al., 1986). Collagen fibrils of 30 nm diameter, approximately one-third that of the fibrils in normal control ligaments, have been detected in the rat medial collateral ligament during the process of repair following experimentally induced division of the ligament followed by autologous transplantation of ligament fragments. These fibers were also shown to stain positively for pNcollagen I11 (Frank et al., 1992; Nakamura et al., 1996; Sato er al., 1986), indicating that pNcollagen I11 may be incorporated into the newly formed small diameter collagen fibrils during the rapid phase of ECM synthesis that is associated with tissue repair processes. The observations described in this section would be consistent with pNcollagen 111, rather than collagen 111, having a central role in regulating the diameter of collagen fibrils in viva

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2. Collagen V on Fine Collagen Fibrils Electron microscopic studies have revealed a reticular network of fine fibrils in tissues such as the liver, spleen, and lymph nodes. A similar reticular network of fine fibrils is also seen in the lamina fibroreticularis deep to the lamina densa in the BM zone of tissues including stratified squamous epithelia. The fibrils that constitute these reticular networks exhibit a Dperiodic banding pattern, but because their diameter was less than 50 nm and they demonstrated argylophilic staining properties these fibrils were thought to be different from those in connective tissues (Snodgrass, 1977). Recent studies have shown that the distribution pattern of these argylophilic fibers is similar to that of collagen V in the spleen and lymph nodes, as demonstrated by immunofluorescence staining, and that the staining pattern of collagen V in the liver sinusoids was identical to that of the argylophilic fibrils in this tissue (Adachi et al., 1991). Collagen V shows two major distribution patterns on light microscopic examination-deep to the lamina densa and in the stroma of the oral mucosa, arterioles, liver, and intestine-whereas immunoelectron microscopic examination reveals that collagen V is distributed on fibrils less than 40 nm in diameter with a D-periodic banding pattern in the spleen (Adachi et al., 1987). liver (Adachi et al., 1991), and cornea (Birk et al., 1988). When collagen V was first described as the fifth type of collagen molecule, it was considered to be a second component of the lamina densa and several authors postulated that collagen V represented a member of a new family of collagens that were involved, together with collagen IV, in the formation of the lamina densa meshwork (Roll et al., 1980). In contrast, other authors postulated that collagen V was a fibrillar collagen on the basis of its association with banding fibrils (Adachi et al., 1987). Immunoperoxidase staining also demonstrated that collagen V could be identified as interstitial punctate staining in the connective tissue stroma deep to the lamina densa in the interstitial space in the kidney (Martinez-Hernandez et al., 1982), whereas further studies showed that collagen V was distributed on 12-nm nonbanding filaments deep to the lamina densa of the amniotic membrane (Modesti et al., 1984). The punctate distribution of collagen V in the stroma and its identification in conjunction with nonbanding filaments are now considered to be artifacts caused by the cross-reactivity of polyclonal anticollagen V antibodies with collagen VI (Ayad et al., 1984). The staining of banding fibrils in the liver, spleen, and human placenta was, however, consistently demonstrated after the anticollagen V antibodies were absorbed with collagen VI prior to staining. On the basis of these findings it has been shown that collagen V is the major component of the collagen fibrils in the reticular fibers in the spleen, liver, and lymph nodes as well as the argylophilic fibers seen in the lamina fibroreticularis in epithelial tissues (Adachi and Katsumata, 1997).

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B. Regulation of Collagen I Fibrils in Wtro 1. pNcollagen III and the Growth of Collagen Fibrils in Vitro To confirm the postulated role of pNcollagen 111 in regulating the diameter of collagen I fibrils, it would be important to demonstrate that smalldiameter fibrils could be reconstituted from a mixture-solution of collagen I and pNcollagen 111. Collagen 111 can form banding fibrils in vitro, and pNcollagen I11 in isolation cannot form any kind of aggregates in vitro but can be incorporated into aggregates with collagen I, yielding small-diameter heterotypic collagen fibrils. When mixtures of procollagen I11 and pCcollagen I were incubated with C-proteinase at 37"C, fibrillar structures were generated spontaneously in v i m , all of which stained positively with an anticollagen 111N-propeptide antibody, as observed by fluorescence microscopy (Fig. 10). Electron microscopic examination of these fibrils demonstrated that they were of a smaller diameter than those generated from a mixture of pccollagen I and C-proteinase (Fig. 11). The diameter of the reconstituted fibrils was shown to depend on the procollagen 111 content

FIG. 10 Darkfield (a) and immunofluorescence (b) micrographs of collagen fibrils formed from a mixture solution containing pccollagen I (35 mg/ml), procollagen 111 (50 mg/ml), and C proteinase at 37°C for 24 h. All the fibrils, demonstrated by darkfield microscopy, are intensely labeled with FITC-labeled polyclonal anticollagen 111 N-propeptide antibody. This image suggests that all the fibrils are heterotypic, containing collagen I and pNcollagen 111. Bar = 100pm.

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FIG. 1 1 Immunogold (5 nm) labeling of collagen fibrils as shown in Fig. 10 using anticollagen 111 N-propeptide antibodies. The collagen fibrils are heavily labeled with the anticollagen 111 N-propeptide antibody. It is clearly shown that pNcollagen 111, partially processed collagen 111, is incorporated into banded fibrils. Bar = 50 nm.

of the mixture with the diameter of the fibrils formed from an equimolar mixture of procollagen I11 and pccollagen I being approximately half those of pure collagen I fibrils (Fig. 12). These data indicate that pNcollagen I11 can be incorporated into the same fibrils as collagen I and can inhibit the lateral growth of fibrils (Romanic ef al., 1991). The most likely explanation of the mechanism by which pNcollagen 111 can restrict the lateral growth of heterotypic fibrils is that the N-propeptide may cause steric hindrance of the lateral association of the molecules. Chapman (1989) proposed that the globular domain at the amino terminus of pNcollagen I11 may occupy the surface circumference of the fibrils, inhibiting the further lateral association of the collagen molecules. Cleavage of the N-propeptide of pNcollagen I11 is slower than that of the C-propeptide (Peltonen et al., 1985) and we have preliminary data that show that the N-propeptide of collagen I11 can be cleaved by the collagen I N-proteinase following fibril formation (A. M. Romanic, personal communication). All these data, when combined,

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_I T

0

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FIG. 12 Regulation of the fibril diameter by pNcollagen 111. The diameter of the collagen 1 fibrils was 180 nm on average. The average diameter of the collagen fibrils generated in a mixture solution of equimolar concentrations of collagen I and pNcollagen I11 was reduced to 97 nm. Increasing the procollagen I11 content of the initial mixture resulted in a progressive decrease in fibril diameter. Adapted with permission from Romanic et a/. (1991).

indicate that pNcollagen I11 can transiently limit the growth of collagen fibrils, a finding consistent with the high relative content of pNcollagen I11 in early embryonic skin, which contains a preponderance of fine fibrils, and the decrease in the pNcollagen 111 content of this tissue in later embryonic stages, which are associated with the presence of thicker fibrils (Fleischmajer et al., 1981, 1990).

2. Collagen V and the Growth of Collagen Fibrils in V i m Collagen V, which has essentially the same domain structure as that of the other fibrillar collagens (Fessler and Fessler, 1987; Schuppan et al., 1986), can form banding fibrils (approximately 38 nm in diameter) that fray out at both ends to form subfibrils that may or may not demonstrate a banding pattern (Adachi et al., 1989). The appearance of the banding pattern on collagen V fibrils is extremely sensitive to alterations in the conditions, particularly in terms of temperature and contamination by other proteins, in which the collagen was isolated and the in vitro incubation conditions used prior to preparation for electron microscopic examination. This accounts for the somewhat variable appearance or nonappearance of banding patterns on collagen V fibrils. The most important factor in maintaining the appearance of the banding pattern along the axes of collagen V fibrils has been demonstrated to be the incubation temperature used prior to preparation (Adachi and Hayashi, 1985).

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It was shown by Adachi and Hayashi (1986) that heterotypic fibrils could be generated from mixtures containing varying ratios of collagen V and collagen I solutions (Figs. 13 and 14) and that the average diameter of the heterotypic fibrils thus formed was less than 50 nm when the concentration of collagen V exceeded that of collagen I in the starting mixture (Figs. 15 and 16). These findings indicated that collagen V can interact with other fibrillar collagens, such as collagen I or collagen 111, and limit the growth of collagen fibrils in a concentration-dependent manner (Adachi and Hayashi, 1986, 1987). These findings were subsequently confirmed by Linsenmayer et a[. (1993). The other characteristic features of collagen V fibrils are the presence of frayed ends in these fibrils (Fig. 17) and branching of the banded fibrils (Adachi et al., 1989; Fig. 18).Branching collagen fibrils have been frequently observed in the lamina fibroreticularis of the BM zone (Fig. 1) and it is suggested that the function of collagen V in the lamina fibroreticularis is to restrict fibril diameter by a combination of branching and inhibiting the lateral association of fibrillar collagen molecules. The network of collagen

FIG. 13 Negative staining images of collagen fibrils formed from pure collagen I ( I ) , a mixture solution of collagens I and V at an equimolar ratio ( V : I), and pure collagen V (V). The samples were incubated at 37°C for 2 h prior to being stained with 0.5 % uranyl acetate. All the fibrils show 67-nm periodicity (brackets); however, a major repeating pattern, a black and white repeat, tends to be discernible when collagen V exceeds 50% of the collagen content of the mixture solution. Eight or more transverse lines (Hodge and Schmitt, 1960) are discernible at comparable positions along the three fibrils in each 67-nm period. Bar = 50 nm. Modified with permission from Adachi and Hayashi (1986).

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Ratios of Collagen V to I 0:l 1:l l : o + + +

+ +

ca

cl b2 bl

a4

+

a1

+

el

+ d +

ca

FIG. 14 Schematic representation of collagen fibrils formed from solutions containing collagens V and I at 0: 1, 1 : 1, and 1:0 ratios. Minor periodic bands are observed at identical positions designated al, a4, bl, b2, cl, c2, d, and el (Chapman, 1984). Collagen V and heterotypic fibrils show comparable banding patterns, suggesting that there may be similarities between the amino acid sequences of collagens V and I. Adapted with permission from Adachi and Hayashi (1987).

8

3 *

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Collagen V : I = 1 : 1 40

x 30

3

-

.-P

9

20

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0 0

SO

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FIG. 15 Histogram illustrating the distribution of the diameters of fibrils formed from solutions of collagen V and I and a mixture of collagen V and I at equimolar ratios (shown in Fig. 13). The distribution curve of heterotypic fibrils formed from a mixture of collagens V and I has only one peak, at about 40 nm. This finding suggests that collagen V directly interacts with collagen I to form heterotypic fibrils in vitro and in vivo. Modified with permission from Adachi and Hayashi (1986).

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-I

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70

100%

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FIG. 16 The limitation of fibril diameter by collagen V. When the proportion of collagen V is increased in the mixture solution, the average diameter of the collagen fibrils is reduced markedly. When the proportion of collagen V is greater than SO%, the average diameter of collagen fibrils is less than SO nm. This finding indicates that collagen V can limit the diameter of heterotypic collagen fibrils in vitro and in vivo. Adapted with permission from Adachi and Hayashi (1 986).

fibrils described in the lamina fibroreticularis could be formed through the branching and anastomosing of collagen fibrils, including heterotypic fibrils that include collagen V (Adachi and Hayashi, 1994). Collagen fibrils are usually tapered at both ends, both in vivo and in v i m , when examined by electron microscope (Birk et af., 1989; Kadler etal., 1990); however, collagen V fibrils have unusual frayed ends. Collagen fibrils with frayed ends have also been described in the ECM of the pancreas when examined by the quick-freeze, deep-etching technique (Fig. 19). These findings indicate that collagen V demonstrates less of a tendency to form fibrils than other fibrillar collagens, but it has been shown that collagen V in the lamina fibroreticularis functions as an intermediate structure that mediates the interaction of collagen fibrils with the lamina densa, possibly through a direct physical connection (Adachi et al., 1989). The most important difference between collagen V and pNcollagen I11 is that the globular amino propeptide of pNcollagen I11 may be cleaved, possibly following fibril formation, by the processing enzyme N-proteinase, whereas the globular amino-terminal domain of collagen V may not be cleaved or the triple-helical domain of collagen V may limit the fibril diameter. Therefore, the incorporation of collagen V into collagen fibrils limits their diameter until the collagen V is removed from the fibrils, whereas the incorporation of pNcollagen I11 into fibrils may transiently limit fibril diameter and represent an intermediate biosynthetic step and

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FIG. 17 Immunogold (15 nm in diameter) labeling of reconstituted collagen V (V) fibrils using an anticollagen V antibody. (a) The ends of the fibrils frequently fray forming subfibrils that may or may not have a banding pattern. Collagen V fibrils often divide into two branches, which are heavily labeled with colloidal gold. Bar = 1 pm. (b) Higher magnification of the frayed ends of collagen V shown in a. These subfibrils also label with an anticollagen V antibody and the frayed ends were also observed in the lamina fibroreticularis as shown in Fig. 19. Bar = 100 nm. Modified with permission from Adachi and Hayashi (1989).

confer some plasticity on collagen fibril formation (Adachi and Hayashi, 1986; Linsenmayer et al., 1993; Romanic et al., 1991).

IV. Collagen IV and the Skeleton of Lamina Densa A. Meshwork Structure of Lamina Densa The lamina densa is revealed as a continuous, undulating sheet that is about 50-100 nm thick in thin sections. At higher magnification, the lamina densa was shown to be fuzzy and to consist of irregular material that was ordered into strands and it was believed that these strands formed the three-

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FIG. 18 Higher magnification of the inscribed area shown in Fig. 17. The collagen V fibril ( V ) splits into two branches, which exhibit a banding pattern (arrowheads) and label with colloidal gold (15 nm in diameter). This branching pattern was also observed in the lamina fibroreticularis, as indicated by the arrow in Fig. 1. Bar = 50 nm.

dimensional meshwork that defined the structure of the lamina densa. In the lamina lucida, the zone of the BM that is in closest apposition to the membranes of the neighboring cells, there is a complete lack of the meshwork arrangement described in the lamina densa, although occasionally thin filaments-the anchoring filaments-are detected that span the region between the cell membranes and the lamina densa (Inoue, 1989). It has been argued that the lamina lucida represents an artefactual gap that is formed following aldehyde fixation prior to electron microscopic examination, as a result of the findings obtained when freeze-substitution fixation was used rather than aldehyde fixation, because it was shown that the space corresponding with the lamina lucida appeared to be electron opaque (Chan et al., 1993a). These findings remain somewhat controversial and it is not at all clear whether the electron-dense zone corresponding with the lamina lucida seen after freeze-substitution represents a similar meshwork structure to that found in the lamina densa (Goldberg, 1986; Inoue, 1995). In our opinion the lamina lucida that corresponds with the gap between epithe-

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FIG. 19 Quick-freeze, deep-etch electron micrograph of collagen fibrils in the lamina fibroreticularis of the mouse pancreas. Collagen fibrils (asterisks) are frayed out into two or more subfibrils (arrowheads). This image is similar to the frayed ends of reconstituted collagen V fibrils as shown in Fig. 17. These findings indicate that collagen V may be involved in the branching and fraying of collagen fibrils. Bar = 50 nm.

lial cells and the lamina densa when examined electron microscopically following the quick-freeze deep-etching preparation technique is a consistent and indisputable finding (Adachi and Hayashi, 1994; Gotow and Hashimoto, 1988; Leblond and Inoue, 1989; Merker, 1994). It is generally accepted that the lamina densa of different tissues mainly consists of a three-dimensional meshwork composed of collagen IV, laminin, nidogen, and perlecan (Kleinman et al., 1982; Timpl, 1989). Such meshworks composed of polygons with sides that are less than 20 nm in length have been described in the lamina densa of various tissues such as heart muscle (Frank and Beydler, 1985), Schwann cells (Ushiki et al., 1990), the glomerular BM (Kubosawa and Kondo, 1985; Takami et al., 1991), the pancreas (Adachi and Hayashi, 1994), and the lens capsule (Barnard et al., 1992). Despite the diversity of the components of the lamina densa and tissue-specific variations in the proportions of these components, the essential supramolecular structure of the polygonal skeleton of the lamina densa is well conserved in different tissues and in different species. The lamina

123 fibroreticularis is present in most epithelial tissue and around muscle fibers (the endomysium) but is lacking in some tissues such as the glomerular BM formed by endothelial cells and podocytes in the renal glomeruli (Inoue, 1989). The lamina fibroreticularis is characterized by an extensive network of small-diameter collagen fibrils rather than the well-defined meshwork seen in the lamina densa and it appears that meshworks composed of polygons, with sides of less than 20 nm, are restricted to the lamina densa (Chan et al., 1993b; Yurchenco and Ruben, 1988). BASEMENT-MEMBRANE STROMAL RELATIONSHIPS

6 . Formation of Meshwork in the Absence of Other Macromolecules in W t m The exact nature of the minimal molecular components that are required for the formation of the skeletal architecture of the meshwork found in the lamina densa is the subject of some debate; however, collagen IV is considered to be the most likely candidate for this role. In 1981, Timpl et al. reported that collagen IV prepared from EHS tumor matrix formed dimers through interactions at their carboxyl-terminal ends and tetramers through associations at the 7s domain and proposed a model for the collagen IV meshwork that described a four-sided structure with sides that were approximately 800 nm in length corresponding with two collagen IV molecules. The validity of this model is questionable, particularly because the thickness of the lamina densa in normal tissues ranges from 15 to 150 nm and it seems somewhat unlikely that this structure could include a meshwork with sides of approximately 800 nm in length, even if it was to undergo extensive folding and rearrangement (Inoue, 1994). A further model for the collagen IV network, which was based on the finding that collagen IV molecules may associate in a lateral manner comparable with that shown by fibrillar collagens, was proposed by Yurchenco and Furthmayr (1984). According to this model, each segment of the meshwork polygons consisted of two or more T H regions of collagen IV, which would confer some rigidity on this skeletal structure (Yurchenco et al., 1986). Later, this same group reported that the average meshwork interval length in EHS tumor matrix is approximately 41 nm, which is comparable with that of reconstituted collagen IV aggregates from EHS tumor matrix. Collagen IV isolated from EHS tumor matrix contains two a chains of M, = 185 and 175 kDa by SDS-PAGE analysis. A meshwork with 44-nm-sided polygons was reconstituted from these fractions, suggesting that collagen IV can form a meshwork structure through lateral associations, as postulated by Yurchenco and Ruben (1988). The size of the polygons formed by the EHS tumor matrix is greater than twice that of the meshwork found in the lamina densa in normal tissues, including skeletal muscle, Schwann cells, the pancreas, and

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the lens capsule. In order to circumvent these discrepancies Yurchenco proposed an alternative model in which the fine meshwork of the lamina densa in normal tissues may be composed of two independent meshworks-a collagen IV meshwork of 44-nm-sided polygons and a laminin meshwork of 31-nm-sided polygons (Yurchenco et al., 1992)-and that nidogen may bridge and connect these two meshworks. It is difficult to envisage that this superimposition of these two disparate meshworks of collagen IV and laminin could form the polygonal skeletal meshwork of the lamina densa with its characteristic consistent regularity (Yurchenco, 1994; Yurchenco and O’Rear, 1994). Recently, we identified a collagen IV polypeptide of M , = 160 kDa from bovine lens capsule as a short al(1V) chain that had not been described in collagen IV derived from the EHS tumor matrix (Iwata et al., 1995). The collagen IV preparation from bovine lens capsule containing the 160-kDa polypeptide was shown to form a meshwork with 14-nm-sided polygons in reconstitution experiments (Fig. 20). The reconstituted meshwork showed three- to six-sided polygons constituted of filaments that were an average of 6.7 nm in width with branching intervals of 21.6 2 11.3 nm (mode = 14 nm) on electron microscopic examination following the quick-

FIG. 20 Quick-freeze, deep-etch electron micrograph of the reconstituted meshwork of collagen IV prepared from the bovine lens capsule. Isolated collagen IV was dialyzed against PBS at 4°C and then incubated at 37°C for 2 h prior to quick-freezing. The aggregates show a meshwork with extensive lateral splitting or anastomosing of collagen IV filaments (arrows). The mean length between branching points is 21.6 nm (mode = 14 nm), comparable with the meshwork dimensions of the lamina densa reported by others (Inoue, 1989). Globular structures (arrowheads), corresponding with the NC1 domain of collagen IV, are also observed on the collagen IV filaments. Bar = 50 nm.

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freeze, deep-etch technique. We were then able to demonstrate a similar meshwork, with branch intervals of 20.1 2 7.7 nm (mode = 18 nm) statistically identical to those of the reconstituted meshwork, in the lamina densa of mouse pancreas using the same preparative technique. The major difference between the two meshworks was the thickness of filaments that formed the polygonal structures in that those in the reconstituted bovine lens capsule collagen IV were 6.7 2 1.6 nm thick and those in the pancreatic (in vivo) meshworks were 10.6 t 2.7 nm thick (Adachi et al., 1997). Inoue et al. (1983) described comparable meshworks composed of filaments 1012 nm in length in Reichert’s membrane and the basal lamina of the rat yolk sac and also demonstrated that the dimensions of the polygons in the meshwork in Reichert’s membrane were unchanged by plasmin digestion, which preferentially degrades laminins. On the basis of these findings, Inoue (1994) proposed that collagen IV formed the skeleton of the meshwork found in the lamina densa by the lateral association of collagen IV molecules over a short distance, usually 12 nm, which then formed polygons. It was suggested that other molecules, including laminin and perlecan, could then associate with the collagen IV framework. Observations from our laboratories show that the filaments that constitute the meshwork of the lamina densa in tissues are approximately one and a half times thicker than those in the reconstituted meshworks; however, this may be explained in terms of the decoration of the filaments that constitute the meshwork in vivo by other components of the lamina densa, such as laminin, nidogen, and perlecan, consistent with Inoue’s proposal (Inoue, 1989). On the basis of all the findings described in this section, it is apparent that collagen IVislikely to represent the centralmolecular component of the meshwork that defines the structural and functional properties of the lamina densa in most, if not all, tissues.

V. Interactions between Collagen Fibrils and Lamina Densa A. Connections between Collagen Fibrils and Lamina Densa 1. Connections between Collagen Fibrils and Lamina Densa at the Light Microscopic Level

The nature of the structures that are involved in the physical interactions between epithelia and underlying connective tissues has been the subject of intensive study and debate for many years. Prior to electron microscopic studies argylophilic fibers, revealed by silver impregnation and light micros-

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copy, were thought to represent the major structure that physically connected the BM to collagen fibers in the underlying stroma (Bargmann et al., 1936). Recently, electron microscopic studies have demonstrated the existence of the lamina fibroreticularis, a specialized structure within the underlying stroma and closely apposed to the lamina densa, that on light microscopic examination appears to be an integral part of the BM. The lamina fibroreticularis contains small-diameter collagen fibrils that run parallel to the deep surface of the lamina densa and it has been observed that some of these fibrils appear to leave the lamina fibroreticularis and merge into collagen bundles in the deeper stroma (Ham and Cormack, 1979). These findings appeared to refute the possibility of a direct connection between collagen fibers in the stroma and the basement membrane because collagen fibrils in the stroma that could possibly interact with the BM seemed to merge directly with fibrils that ran parallel to the lamina densa but did not insert directly into it. Such fibrils that run parallel to the deep surface of the lamina densa in skeletal muscles are specifically referred to as the endomysium, which is continuous with the perimysium (thin septa extending from the epimysium) and epimysium (dense connective tissue surrounding the muscle), which are composed of bundles of collagen fibrils (Fawcett, 1968). In other words, muscle cells are ensheathed in a lamina densa that is covered with an extensive network of collagen fibrils, which has been shown to include collagen V by immunofluorescence microscopy (Duance et al., 1977; Schuppan et af., 1986). Functionally, traction forces generated by the intracellular actin-myosin filament system in muscle are transmitted to the surrounding connective tissues, such as tendon through the lamina densa. Cells of epithelial tissues that are exposed to the external environment, such as the skin, intestine, and urinary tract, are also connected to the underlying connective tissues through a continuum of macromolecules that extends from their cytoplasm into the ECM. These direct physical interactions between cells and the ECM have been studied extensively in the stratified squamous epithelia of the skin, oral mucosa, esophagus, and vaginal mucosa, and it has been postulated that there is a direct physical linkage of molecules stretching from the intracellular intermediate filaments to the anchoring fibrils in the extracellular space involving hemidesmosomes and the lamina densa (see Section 11,E). The nature of such direct physical intracellular-extracellular connections is uncertain in epithelial tissues that do not express collagen VII, the molecule that constitutes the anchoring fibrils, because this molecule is usually preferentially expressed only in stratified squamous epithelia (Burgeson et al., 1990; Kawanami et al., 1978; Uitto and Christiano, 1992).

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2. Codistribution of Collagens IV and V in Vivo The lamina densa is an extracellular structure that appears as a continuous sheet underlying the basal surface of epithelia from which it appears to be suspended by anchoring filaments that span the lamina lucida (Holbrook and Smith, 1993). Collagen IV represents the only collagen that has been identified in the lamina densa and has been described previously (see Section 11,C). This molecule is distinct from the fibrillar collagens, particularly with respect to its potential to aggregate into meshwork-like structures rather than the heterotypic banding fibrils that are formed by the fibrillar collagens (Kuhn, 1994). It is likely that the self-assembly of collagen IV is independent of the fibrillar collagens in the stroma and heterotypic aggregates of collagen IV and other collagens have not been described in the tissues of adult animals; however, collagen IV immunoreactivity was demonstrated on banding collagen fibrils in the developing avian cornea (Fitch et al., 1991). We have also demonstrated collagen IV immunoreactivity on fibrillar collagen structures in the cirrhotic liver; although a distinct lamina densa has not been described in normal liver sinusoids, the presence of collagen IV has been shown immunohistochemically (Yoshida et al., 1992). We have also demonstrated, with immunoperoxidase staining, that collagen V is widely distributed on small-caliber collagen fibrils in the liver sinusoids and it is possible that in the fibrils described in the cirrhotic liver collagen IV may be associated with collagen V (Adachi et al., 1991). In other tissues that contain a reticular network, such as the spleen (Adachi et al., 1987), lymph nodes (Karttunen et al., 1986; Konomi et al., 1981), and skeletal muscles (Gulati, 1985), collagens IV and V have been colocalized at the light microscopical level on reticular fibers, specifically collagen fibrils, of less than 40 nm in diameter (Konomi et al., 1984). These observations may indicate that collagens IV and V in fact form heterotypic aggregates. 3. Direct Physical Connection of Collagen Fibrils with Lamina Densa In epithelial tissues, collagens IV and V appear to form independent supramolecular structures. Therefore, four connecting systems between the collagen fibrils and the underlying BM have been proposed: microfibrils composed of fibrillin or fibronectin; beaded fibrils composed of collagen VI aggregates; and anchoring fibrils composed of collagen VII aggregates; and microthreads composed of proteoglycans that are stained by alcian blue or ruthenium red (see Section VI,D). We have reported that collagen fibrils merge directly into the lamina densa of the mouse pancreas, human placenta, and the oral mucosa of the macaque monkey. In most epithelia, if

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not all, the deepest cells lie directly over the lamina densa connected by anchoring filaments that bridge between them (Fig. 21). Electron microscopic examination of quick-freeze, deep-etch preparations of the mouse pancreas were performed in order to graphically demonstrate the merging of collagen fibrils and the lamina densa because embryologically the pancreas is considered to be a derivative of the intestinal epithelium. Pancreatic exocrine cells lie on a lamina densa that is not associated with an anchoring fibril arcade structure; therefore, in this case it is difficult to envisage direct connections between collagen fibrils and the lamina densa. Collagen fibrils in the lamina fibroreticularis of the mouse pancreas, recognized by virtue of the periodic ridges and furrows along their long axes that correspond with the overlap and hole regions of the 67-nm periodic banding pattern, were small in diameter (less than 40 nm) and either divided into two branches or had frayed ends as exhibited by

FIG. 21 Quick-freeze, deep-etch electron micrograph showing the en face view of the lamina densa in the mouse pancreas. The lamina densa (D) is a polygonal structure formed by filaments that range from 5.9 to 19.9 nm in width. The average distance between the branching points is approximately 20.1 nm (mode = 18 nm). Anchoring filaments (arrow) traverse the lamina lucida (L) to connect the lamina densa to the deep surface of pancreatic acinar cells (cell). Collagen fibrils (asterisk) appear to flatten as they approach the acinar cell, divide into two subfibrils (arrowheads), and merge with the meshwork of the lamina densa. Filaments in the lamina densa anastomose and /or branch to form a meshwork. Bar = 100 nm. Modified with permission from Adachi and Hayashi (1994).

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reconstituted collagen V fibrils. Approximately 3 of 100 collagen fibrils were shown to approach the lamina densa of the mouse pancreas and these fibrils tend to be flat and lose the periodical ridge and furrow appearance, divide into two or more subfibrils, and then finally merge with the meshwork of the lamina densa (Adachi and Hayashi, 1994). Anchoring fibrils composed of collagen VII appear to originate from, or terminate in, the stromal side of the lamina densa, forming arcade-like structures that seem to separate collagen fibrils in the stromal ECM from the stromal aspect of the lamina densa. Most of the collagen fibrils in the lamina fibroreticularis of the oral mucosa run parallel to the lamina densa separated from it by a gap of approximately 50-100 nm; however, occasionally collagen fibrils tend to change their course and approach the lamina densa through the anchoring fibril arcade structure (Fig. 22). These collagen fibrils always fray out into at least two subfibrils, which are of a width that is comparable with that of the structures forming the polygonal meshwork, prior to merging with the lamina densa. Based on

FIG. 22 Thin sectional image of the oral mucosa showing the epidermal-dermal junction. The lamina densa (D) is approximately 80 nm in width and underlies the basal surface of keratinocytes (K). Anchoring fibrils (arrow), originating from the lamina densa, form a network in the lamina fibroreticularis. Collagen fibrils run through the network of anchoring fibrils and appear to merge with the lamina densa (arrowhead). Bar = 0.5 Fm. Modified with permission from Adachi and Hayashi (1994).

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these morphological criteria it is likely that these fibrils contain considerable amounts of collagen V (Adachi and Hayashi, 1994; Adachi et al., 1989). The chemical characteristics of collagen V are similar to those of collagen IV with respect to amino acid composition and carbohydrate content (see Section I1,D) and it is possible that collagen V in the subfibrils may associate laterally with segments of the collagen IV polygons that form the meshwork of the lamina densa. Recently, we have isolated collagen V with a chain composition of al(V)a2(V)a3(V) from a human placental preparation by heparin affinity chromatography, and it was shown to form fibrillar aggregates with extensive branching at the ends of the fibrils at 37°C (see Section II,D,2,a). Collagen V with a chain composition of al(V)a2(V)a3(V) is a candidate molecule that may be involved in the heterotypic association between collagen IV and collagen V. The intermediate structure and/or characteristics of the al(V)a2(V)a3(V) chain, with respect to the formation of nonbanding fibrils, may be related to its localization between the lamina densa and the underlying collagenous stroma; however, it is still possible that other molecular components of the BM zone may be required for the association of collagens IV and V. Similar collagen fibrils that merged with the lamina densa have been described in scanning electron microscopic studies performed by other investigators (Campbell el al., 1989) when it was shown that collagen fibrils were also distributed in the lamina lucida of the human placenta. These findings were explained as representing collagen fibrils piercing the lamina densa and it was considered that placental epithelial cells may secrete fibrillar collagens that form fibrils in the lamina lucida; however, we have not observed collagen fibrils in the lamina lucida of normal tissues that we have examined, including the pancreas, oral mucosa, and the kidney. The width of the lamina lucida in the pancreas is less than 25 nm and it seems unlikely that collagen fibrils of at least twice this diameter could run in this structure, and it is possible that another interpretation of the quick-freeze, deep-etch image shown in Fig. 21 is that collagen fibrils in the lamina fibroreticularis would merge with the lamina densa.

B. Possible Functions of Minor Fibrillar Collagens in vivo

1. Significance of Collagen V in Vivo Collagen molecules assemble into heterotypic aggregates that define the biological and mechanical properties of most, if not all, tissues and organs. Fibrillar collagens, such as collagens I, 11, 111, V, and XI, can be divided into two groups: the major fibrillar collagens (collagens I, 11, and 111) and the minor fibrillar collagens (collagen V and XI). The major fibrillar

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collagens form fibrils, which are involved in the maintenance of tissue architecture and rigidity (van der Rest and Garrone, 1991). The selfassembly of collagen I has been well studied, and mutations in these molecules have been shown to cause various types of inherited connective tissue disorder, particularly the osteogenesis imperfecta and Ehlers Danlos complex of disorders (Prockop, 1990). A potential role for the minor fibrillar collagens is the regulation of fibrillogenesis, largely by limiting the diameter of fibrils formed from major collagens (Adachi and Hayashi, 1986; Francomano, 1995). It is also likely that, as well as having this regulatory function, collagen V may also be involved in anchoring collagen fibrils in the stromal ECM to the lamina densa through interactions between collagens IV and V. A decrease in the expression of the a2(1) gene in transgenic mice did not affect the formation of collagen fibrils, and banded collagen fibrils observed in embryonic organs indicated that collagens 111 and V could functionally replace collagen I in the process of organogenesis (Kratochwil etal., 1986); therefore, it is conceivable that alterations in the minor fibrillar collagens may alter the process of fibrillogenesis, with consequent malformation of affected tissues or organs. The recent generation of transgenic mice with abnormal a2(V) procollagen chains (Andrikopoulos et al., 1995) resulted in the occurrence of skin fragility, spinal lordosis, and disorganized collagen fibrils in the cornea, and it was suggested that such deformities, which appeared in the homozygous mutant, were caused by some abnormality of fibrillogenesis and that this phenotype shared some common characteristics with the Ehlers Danlos syndrome in humans. These observations are consistent with the idea that collagen V may play an important role in maintaining tissue integrity by acting as a connecting structure between the connective tissue stroma and the lamina densa as well as in the regulation of fibril diameter.

2. Architecture of Fibrillar Collagens in Tissues We propose a model that describes the architecture of collagen fibrils in the extracellular space in the BM zone (Fig. 23) in which collagen fibrils tend to form bundles in the stroma that then branch or anastomose to form a network in the lamina fibroreticularis. Some collagen fibrils can originate from, or terminate in, the lamina densa, acting as a device that anchors the epithelium to the underlying connective tissue. Collagen V is a candidate for this function because it is widely distributed in a peri-BM distribution in all connective tissues other than cartilage and bone (Adachi and Hayashi, 1994). This model seems to be particularly useful for understanding the relationship between the so-called reticular fibers and the glomerular BM in the kidney (Fig. 24). As described previously (see Section V,A), coarse reticular fibers were labeled with antibodies against collagens I, 111, and

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+

-Collagen Ill + pNII1 V or Collagen I + I l l + pNll1 V

+

-Collagen 1 + 111 + pNIl1 + V or Collagen I 111 pNIIl

+

+

FIG. 23 Putative model of the anchoring of the lamina densa (D) to underlying collagen fibrils derived from observations from studies of reconstituted collagens IV and V, by quickfreeze, deep-etching rotary replication. Collagen fibrils in the stroma are composed of collagen I, which forms collagen bundles. Collagen fibrils in the lamina fibroreticularis are small, 3050 nm in diameter, and are composed of collagens I, 111, and V. The fibrils run individually and form a network by branching and anastomosing with each other. In some tissues, particularly those of embryonic origin, large amounts of pNcollagen 111 may be incorporated into banded fibrils to form small-caliber collagen fibrils. The frayed ends of collagen fibrils can merge with the lamina densa to anchor collagen fibrils to epithelia. The interaction between the lamina densa and collagen fibrils may be connected to the cytoskeleton through anchoring filaments and the hemidesmosome (H) complex. Modified with permission from Adachi and Hayashi (1994).

V, but the finest reticular fibers were only labeled with antibody against collagen V in the spleen (Adachi et al., 1987). It is considered that the glomerular BM is formed by the fusion of the vascular and epithelial BMs, with collagen I, 111, and V containing fibrils excluded from the space formed between these two BMs during glomerular morphogenesis (Sariola et al., 1984) so that the absence of the collagen fibrils from the glornerular BM is consistent with the model that we propose.

VI. Other Systems Involved in the Anchoring of Collagen Fibrils t o Lamina Densa Interactions between epithelia and underlying connective tissues are known to have a crucial role in organogenesis, the maintenance of tissue integrity

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C B

A FIG. 24 Schematic representation of reticular fibers and the glomerular BM formed from the scheme shown in Fig. 23. Two epithelial tissues are apposed on the stromal side of the BM (A). Reticular fibers are mainly composed of collagens 111, IV, and V and are defined as banded fibrils that are embedded in amorphous material that appears to be similar to those in the lamina densa on electron microscopic examination (B). The collagen fibrils between the two epithelial layers are displaced, leaving the fused BMs, as seen in renal glomeruli (C).

and tissue repair. Currently, four types of epithelial-connective tissue stroma interactions have been described that are mediated by four different supramolecular structures: (i) microfibrils, (ii) beaded fibrils, (iii) anchoring fibrils formed from collagen VII, and (iv) microthreads that are probably formed from proteoglycans. All these supramolecular structures are considered to have a role in connecting the lamina densa to underlying fibrillar macroaggregates such as collagen fibrils and elastic fibers. The anchoring fibrils and the microthreads form complex networks under the lamina densa and directly interact with collagen fibrils, leading to fibril trapping and the stabilization of the BM zone. It is apparent that the anchoring fibrils and the microthreads are interacting with the collagen fibrils at the level of the supramolecular aggregate in contradiction to the interaction of collagens I, 11, and V in heterotypic collagen fibril formation; however, it is uncertain whether microfibrils are involved in the formation of heterogeneous aggregates with molecules that are present in the lamina densa.

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A. Microfibrils

Microfibrils, in morphological terms, include all fibrillar elements in the ECM that are thinner than collagen fibrils (Low, 1962),that is, of a diameter of between 10 and 15 nm and that do not exhibit any banding pattern (Inoue and Leblond, 1986). These microfibrils are homotypic, or heterogeneous aggregates with a fibrillar character, that may be formed from several distinct molecular components such as fibrillin and fibronectin, of which those formed from fibrillin have been studied the most extensively (Sakai et al., 1991).

1. Fibrillin Fibrils Recently, two structurally related microfibrillar gene products have been characterized: fibrillin-1and fibrillin-2. Fibrillin-1 is expressed later in development and for a longer period than fibrillin-2,which is expressed predominantly during the earlier phases of morphogenesis. Fibrillin-1 is probably assembled into microfibrils, which act to provide tensile strength to tissues and contribute load-bearing properties to tissues; whereas fibrillin-2 regulates the onset of elastic fiber formation (Zhang and Ramirez, 1995). Fibrillin microfibrils were originally described as fibrils surrounding elastic fibers and were considered to be a normal constituent of connective tissues (Cawlik, 1965). Microfibrils are usually involved in the formation of elastic fibers in which the microfibrillar protein surrounds the surface of elastin supramolecular aggregates, but they may also independently form bundles of microfibrils that, histologically, have been classified as oxytalan fibers (Low, 1962). Elastic fibers consist of a central amorphous core of elastin surrounded by a coat of tubular-appearing microfibrils that measures approximately 10nm in diameter. Both elastic fibers and microfibrils stain positively with Weigert’s resorcin fuchsin stain. In the most superficial layer of the skin microfibrillar thin fibers radiate perpendicularly toward the epidermaldermal junctions in a dendritic (brush-like) pattern originating from a plexus of elastic fibers in the dermis. Electron microscopic studies demonstrated parallel bundles of microfibrils that are oriented toward, and appear to terminate in, the lamina densa (Cotta-Pereira et al., 1976; Kobayashi, 1968), which is also easily visible in quick-freeze, deep-etch micrographs generated in our laboratories. We have recently observed that bundles of microfibrils (oxytalan fibers) can directly associate with the strornal surface of basal keratinocytes cultured on synthetic dermal equivalent systems, with the implication that the elastic fiber network in the connective tissue stroma may be involved in the maintenance of tissue integrity in epithelial layers mediated by adhesive interactions between microfibrils and the lamina

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densa (Essner and Gordon, 1984; Mecham and Davis, 1994; Smith et af., 1982). The arrangement of elastic fibers, collagen fibers, and smooth muscle confer integrity, distensibility, and elasticity to arteries and veins and an extensive network of microfibrils is located in the perivascular space of capillaries and terminates in the lamina densa of the endothelium. A microfibrillar structure, histologically described as anchoring filaments, runs perpendicular to the walls of lymphatic capillaries and it has been suggested that these microfibrillar structures around the capillaries anchor the lamina densa of endothelial cells and have some role in maintaining capillary potency (Ichimura and Hashimoto, 1984; Leak and Burke, 1966). 2. Fibronectin Fibrils Fibronectin has also been implicated in the formation of microfibrillar structures and is localized on collagen fibrils, elastic fibers, and the lamina densa (Couchman et al., 1979; Fleischmajer and Timpl, 1984). Therefore, fibronectin fibrils may connect the lamina densa with collagen fibrils and/ or elastic fibers in the stroma. Fibronectins are high-molecular-weight ( M , = 500 kDa) glycoproteins that are synthesized by hepatocytes and fibroblasts distributed in the lamina densa, the connective tissue stroma, and circulating plasma. The fibronectin isoform that is distributed in the ECM is insoluble and stabilized by disulfide bonds, whereas the plasma form of fibronectin is soluble in body fluids (Hynes, 1989). Fibronectins polymerize to form fibrillar aggregates (Peters el al., 1990) that act to provide a solid-phase substrate for cellular attachment and retain the cellular signals that stimulate the proliferation of a wide variety of cells including fibroblasts, myoblasts, keratinocytes (von der Mark and Goodman, 1993), and hepatocytes in an attachment-dependent fashion. These fibronectin aggregates have also been implicated in the process of neural crest migration (Rouslahti and Pierschbacher, 1987). The proposed mechanism by which fibronectin influences cellular processes includes initial adhesion of the cell to fibronectin by one of several cell adhesion motifs of which the GRGDS sequence is the best characterized. This initial adhesion event is followed by cell spreading and the formation of focal adhesion plaques in which the fibronectin-containing fibrils and actin filaments become aligned along the cell membrane (Herman, 1987; Singer et d., 1985). Actin filaments converge into the adhesion plaques, where they bind to talin and a actinin and then associate with the /3 subunit of the fibronectin receptor (a5pl) (Marcantonio and Hynes, 1988). Recently, we have demonstrated that plasma fibronectin can form fibrillar aggregates in Tris-buffered saline upon incubation with dithiothreitol. The fibrillar structure without a banding pattern was detected with an electron microscope (L. Sakai et

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al., manuscript in preparation). Fibrillar aggregates may be closely related to the fibronectin matrix that may converge into the adhesion plaques in the ECM.

6.Beaded Fibrils Collagen VI consists of three a chains-al(VI),a2(VI), and a3(VI)-and is 125 nm long with two globular domains. Collagen VI molecules may assemble into tetramers intracellularly because only tetramers could be detected in the culture media of human lung fibroblasts and muscle cells (Engvall et al., 1986). Immunogold labeling with monoclonal antibodies against collagen VI revealed that 100-nm beaded fibrils, 20-35 nm in width, comprise the tissue form of collagen VI fibrils (Bruns etal., 1986). Collagen VI fibrils formed an irregular network around collagen fibrils in the matrix of human femoral head and costal cartilage, in human skin (Keene et al., 1988), and in rat testis (Sawada and Yazawa, 1994). The beaded fibrils were also concentrated in the vicinity of peripheral nerves, blood vessels, and adipocytes, which are ensheathed by the lamina densa. On the contrary, the network of beaded fibrils was reduced in the lamina fibroreticularis of the skin. These findings suggest that beaded fibrils, containing collagen VI, connect collagen fibrils with the lamina densa surrounding cells in the stroma such as endothelial cells, Schwann cells, and adipocytes.

C. Anchoring Fibrils Palade and Farquar (1965) described short striated fibrils in amphibian skin that were considered to represent specialized collagenase-sensitive fibrils (Kobayashi, 1977). Similarly, striated fibrils, described as anchoring fibrils, were also described in normal human skin, where they were shown to form arch-like structures with both of their ends inserted into the lamina densa in the BM zone (Briggerman and Wheeler, 1975). Immunohistochemically, these anchoring fibrils have been shown to consist of collagen VII (Burgeson et al., 1985; Keene et al., 1987b; Sakai et al., 1986). Anchoring fibrils that were initially described in skin have subsequently been described in various other tissues, such as the vas deferens (Clermont and Hermo, 1988), the respiratory tract (Kawanami et al., 1978,1979), and oral mucosa (Susi et al., 1969). In this section we summarize the current understanding of the formation of collagen VII supramolecular aggregates in order to augment our previous discussion of collagen VII (see Section 11,E). Anchoring fibrils, which may be regarded as representing supramolecular aggregates of collagen VII, are 0.5-0.6 pm in length, fusiform in shape,

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and exhibit a symmetrical banding pattern labeled dl-d5 in amphibian (Palade and Farquhar, 1965). Collagen VII is a thread-like molecule approximately 424 nm long that forms antiparallel dimers in tissues with an overlap of 60 nm at the carboxyl termini of the collagen VII monomers, resulting in a dimer of length 785 nm; however, in vivo these structures always appear to be somewhat shorter, implying that either they are not fully extended in tissues or their extremities are inserted deeply into the lamina densa (Clermont and Hermo, 1988). The arcade-like architecture of the anchoring fibrils revealed that anchoring fibrils entrap collagenous fibrillar elements of the lamina fibroreticularis (Briggerman and Wheeler, 1975; Kawanami et al., 1979; Keene etal., 1987b), forming an extensive interlacing network deep to the lamina densa (Fig. 22). Dense plaques, which were shown immunohistochemically to contain collagen IV and the carboxyl-terminal region of collagen VII, were demonstrated deep to the lamina densa and it was suggested that these plaques may act as sites into which the anchoring fibrils insert to form a complex interlacing architecture (Gerecke et al., 1994). Anchoring plaques have been described in the mouse uterus (Rowlatt, 1969), rat vas deferens (Clermont and Hermo, 1988), and human cornea and foreskin (Keene et al., 1987b). It has been shown, with the exception of the human foreskin, that both ends of the anchoring fibrils insert into the lamina densa in most regions of human skin (Kielty et al., 1993). It is likely that these anchoring plaques represent islands of the lamina densa that result from oblique cutting of the tissue sections, as originally described by Palade and Farquer (1965). Similar perpendicular protrusions of the lamina densa are frequently seen in wrinkled skin and are well recognized to create difficulties in the three-dimensional interpretation of twodimensional images of the lamina fibroreticularis. We propose that anchoring plaques may be restricted to highly extensible epithelial tissues or that they may represent interstitial deposits of lamina densa components that are associated with supramolecular structures, such as collagen fibrils, elastic fibers, and microfibrils that are found in the stroma. These issues may be resolved by the scanning electron microscopic and quick-freeze, deep-etch studies currently under way in our laboratory that will generate threedimensional images at the ultrastructural level.

D. Microthreads Microthreads represent another type of supramolecular structure that may be involved in the interactions between collagen fibrils and the lamina densa and have been described in the rat trachea (Wasano and Yamamoto, 1985) and in the developing submandibular gland (Kadoya and Yamashina,

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1992). It is difficult to visualize microthreads in conventionally fixed tissue by electron microscopy because of their low electron density; however, they are visualized after cationic dye staining using ruthenium red or alcian blue. Microthreads are 20-40 nm in diameter and form an extensive weavelike structure deep to the lamina densa and in the interfibrillar space. Granules, ranging from 15 to 65 nm in diameter, are deposited alongside the threads on the stromal side of the lamina densa and on the banded fibrils in the lamina fibroreticularis at regular intervals of approximately 55-65 nm. On the basis of their susceptibility to digestion by heparitin sulfate lyase and chondroitin lyase, these granules, which are associated with the lamina densa and with collagen fibrils, may be heparan sulfate proteoglycan and chondroitin A or C or dermatan sulfate proteoglycan, respectively (Wasano and Yamamoto, 1985). Some microthreads appear to insert into the granules neighboring the lamina densa and others appear to be anchored onto the granules that lie alongside collagen fibrils. These observations suggest that the microthreadwoven sheet may connect the lamina densa to collagen fibrils through the proteoglycan granules. It is possible that the interaction between microthreads and collagen fibrils may play an important role in the early stages of morphogenesis because anchoring fibrils were detectable 21 days following xenografting of human keratinocyte sheets onto athymic mice (Germain et al., 1995).

VII. Concluding Remarks Various macromolecules, which are constituents of the ECM, are intimately involved in several fundamental biological processes including organogenesis, the maintenance of tissue integrity in adult tissues, and the tissue response to injury (Olsen, 1989; Prockop, 1995). Many studies have been performed to elucidate the mechanisms involved in the interactions between cells and the ECM; however, most such studies focused on the relationships between the cell membrane and macromolecules in the ECM. There have been extensive studies concerned with the ultrastructural relationship between the cell membrane and the lamina densa using enzymatic degradation, quick-freeze, and immunocytochemical approaches (Adachi ei al., 1987; Adachi and Hayashi, 1994; Inoue, 1989;Yurchenco and Ruben, 1988). but, with the exception of the anchoring fibril network (Gerecke et al., 1994; Uitto et al., 1992), the structural relationship between the lamina densa and the underlying connective tissue stroma has been little studied. In this article, we focused on the interactions between the lamina densa and collagen fibrils in the lamina fibroreticularis as demonstrated by the

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quick-freeze, deep-etch technique. It is well established that the fibrillar collagens (collagens I, 111, and V) can form heterotypic fibrils in vivo and in vitro;however, it is generally accepted that collagen IV forms the structural meshwork of the lamina densa and that it does not form heterotypic aggregates. Here, we have extensively discussed potential interactions between collagens IV and V in view of similarities in the amino acid composition, sequence, and fibril morphology of these two collagens (Adachi and Hayashi, 1994). However, the detailed nature of any such direct physical interaction is poorly understood, although the al(V)a2( V)a3(V) isoform of collagen V is a putative candidate for mediating this interaction. We have also reviewed the four major types of interaction between the lamina densa and the connective tissue stroma (the two defining structures in the extracellular space) mediated by (i) microfibrils, (ii) beaded fibrils, (iii) anchoring fibrils, and (iv) microthreads (proteoglycans). Microfibrils may mediate interactions between elastic fibers and the lamina densa, particularly in the skin and around blood vessels, as supported by their well-described association with elastic fibers. Beaded fibrils, composed of collagen VI, connect collagen fibrils with the lamina densa surrounding endothelial cells, Schwann cells, and adipocytes in the stroma. Anchoring fibrils form arcs or networks with anchoring plaques deep to the lamina densa, which entrap collagen fibrils, stabilizing the BM zone, particularly in stratified squamous epithelia such as those of the skin, oral mucosa, and oesophagus. Proteoglycans appear as a filamentous microthread network in the interstitial space and as granules distributed on the stromal side of the lamina densa and along collagen fibrils. This proteoglycan network may be involved in the connection of collagen fibrils to the lamina densa. The stability of the BM zone, as defined by the epithelial-lamina densa-stromal interactions described in this article, is a major determinant of tissue integrity, but it is also a major determinant of dynamic processes such as cellular migration and proliferation. We are continuing our studies to further elucidate the detailed nature of all these interactions that are required for the maintenance of normal tissue integrity but that also have central functions in tissue repair and disease processes. Acknowledgments The authors express their appreciation to Mr. Osamu Katsumata and Ms. Aya Nakagawa for their technical assistance, and to Dr. Koichi Nakazato and Dr. Kazunori Mizuno for supplying us their experimental data to complete this review. We are grateful to Professors Yutaka Fukuda, Hisao Fujita, Kenjiro Wake, Darwin J. Prockop, and Shohei Yamashina for constant encouragement. Original work in this review was supported by research Grants 0345438, 04670015,08231213,08243219,07558249,and 07807003 from the Ministry of Education, Science. Sports, and Culture of Japan, and also supported by Shiseido Co. Ltd. and Nippi Co. Ltd.

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The Role of Endoxyloglucan Transferase in the Organization of Plant Cell Walls Kazuhiko Nishitani Department of Biology, College of Liberal Arts, Kagoshima University, Kagoshima 890. Japan

The plant cell wall plays a central role in morphogenesis as well as responsiveness to environmental signals. Xyloglucans are the principal component of the plant cell wall matrix and serve as cross-links between cellulose microfibrils to form the cellulose-xyloglucan framework. Endoxyloglucan transferase (EXGT), which was isolated and characterized in 1992, is an enzyme that mediates molecular grafting reaction between xyloglucan molecules. Structural studies on cDNAs encoding EXGT and its related proteins have disclosed the ubiquitous presence in the plant kingdom of a large multigene family of xyloglucan-related proteins (XRPs). Each XRP functions as either hydrolase or transferase acting on xyloglucans and is considered to be responsible for rearrangement of the cellulose-xyloglucan framework, the processes essential for the construction, modification, and degradation of plant cell walls. Different XRP genes exhibit potentially different expression profiles with respect to tissue specificity and responsiveness to hormonal and mechanical signals. The molecular approach to individual XRP genes will open a new path for exploring the controlling mechanisms by which the plant cell wall is constructed and reformed during plant growth and development. KEY WORDS: Cell wall, Cellulose, Endoxyloglucan transferase (EXGT), Xyloglucanrelated protein (XRP), Plant hormone, Molecular grafting, Xyloglucan.

1. Introduction The plant cell wall is an extracellular supermolecular structure equipped with self-reorganizing machinery, and it provides plant cells with distinguishing features in terms of morphogenesis and responsiveness to environInrrrrirrrio,iul R r ~ w . nof~ Cvnilr~gv,V d 171 lW174-769hi97 $25 (X)

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mental factors. Despite its biological importance in various aspects of the life of a plant, remarkably little is known about the mechanism by which the wall architecture is constructed and maintained during cell expansion and differentiation, the processes essential for regulation of plant growth and development. The first visualization of the plant cell wall can be traced back to the mid-seventeenth century when Robert Hooke observed cells in the cork layer of an oak tree under the then newly invented microscope. His observation of the “cell” was roughly comparable with minute anatomical studies on cell wall frameworks in plant tissues by Marcello Malpighi and Nehemiah Grew. These pioneers described the plant cell wall as a rigid housing that solely partitioned individual cells in plant tissues (Preston, 1974). This early static view of the cell wall has been repeatedly revised. The predominant view now is that the cell wall is a thick but dynamic structure with various physiological roles in a wide spectrum of biological functions (Carpita and Gibeaut, 1993; Bolwell, 1993; Fry, 1995). First, the wall contains various types of enzymes responsible for its own construction and organization, and it undergoes drastic changes in its molecular architecture in such a way that allows controlled cell expansion and hence morphogenesis. Second, it functions as the frontier defense system in plants against various types of microbial pathogens and abiological stresses from the surrounding environment. The plant cell wall also plays an active role in cell-to-cell communication with microbes, particularly in symbiotic interactions. Finally, transportation and uptake of nutrients as well as intercellular transduction of chemical signals are often achieved through the cell wall space called apoplast. These versatile functions of the cell wall are mediated by a wide range of molecules ingeniously organized in the supermolecular architecture of the cell wall. Extensive structural studies in the past three decades on cell wall polysaccharides have revealed that the wall is composed of a three-dimensionally arranged framework of crystalline microfibrils that are interwoven by at least two classes of noncrystalline polysaccharides, particularly xyloglucans and rhamnogalacturonans (Darvill et al., 1980a; Keegstra et al., 1973;Carpita and Gibeaut, 1993; Talbott and Ray, 1992a; Fry, 1986;McCann and Roberts, 1994). This unique supermolecular architecture provides the wall with its distinguishing viscoelastic properties. Although the static aspect of the plant cell wall has been well studied in terms of polysaccharide chemistry, the molecular processes, particularly the enzymatic reactions responsible for the construction and modification of the overall architecture of the cell wall, had remained poorly understood until very recently. It was not until 1991 that enzyme activity potentially capable of rearranging the interwoven framework structure of the cell wall was first suggested to be present in plant cell walls (Smith and Fry, 1991; Nishitani and Tominaga,

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1991). The enzyme itself was purified from the cell wall space of azuki bean (Vigna angularis Ohwi et Ohashi) seedlings and termed endoxyloglucan transferase EXGT (formerly abbreviated as EXT) (Nishitani and Tominaga, 1992). The abbreviation EXT will be changed to EXGT in order to avoid confusion with the Ext genes for extensins, well-known extracellular proteins also found in plant cell walls. EXGT is an enzyme that catalyzes the molecular grafting reaction between xyloglucan cross-links among cellulose microfibrils and thereby mediates key steps in the rearrangement of the cell wall architecture in plant tissues. Recent isolation and characterization of cDNAs encoding EXGT and its structurally related proteins have disclosed the ubiquitous presence in the plant kingdom of a fairly large multigene family, each member of which is involved in the xyloglucan metabolism required for the construction as well as rearrangement of plant cell walls. Section I1 of this review will discuss outlines of the molecular organization of the plant cell wall that have been revealed to date. In the remaining sections, attention will be paid to the molecular functions of EXGT and related proteins, which constitute the xyloglucan-related protein (XRP) family, and their physiological implications for plant cell wall organization-a currently expanding area in the field of plant cell biology.

II. Overview of Cell Wall Architecture in Plants Plant cell walls have been classified into two categories from the physiological point of view: the primary walls and the secondary walls. The former is designated as the walls synthesized in cells that are in the early stage of cell differentiation and are still expanding o r able to expand in response to a developmental program and environmental signals. The secondary wall, on the other hand, is characterized as thicker walls with enriched cellulose microfibrils laid down on the inner surface of the primary wall after the cell expansion has terminated. The deposition of the secondary wall is particularly conspicuous in tracheary elements in vascular tissues (Hogetsu, 1991;Suzuki etal., 1992) and sclerenchyma cells in ground tissues. In dermal tissues, the secondary wall is found in trichome and guard cells. Due to the thick layers of highly ordered crystalline cellulose, secondary cell walls usually show strong birefringence under polarized light microscopy (Potikha and Delmer, 1995). Epidermal cell walls in the dermal tissue are classified as primary walls because of their ability to extend drastically in the course of plant growth. However, they become thicker and contain as much cellulose as typical secondary walls, while still being capable of elongation (Bret-Harte and Talbott, 1993). The borderline between the

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two cell wall types is sometimes vague, and there is no point in trying to strictly distinguish them in this review, in which our attention will be focused on the roles of EXGT in both types. To help the reader visualize the plant cell wall, let us first magnify the plant cell wall by means of electron microscopy followed by molecular dissection of the dynamic aspect of the wall architecture in terms of the proteins and genes involved in its organization.

A. Basic Architecture of the Plant Cell Wall 1. Architectural Models of the Plant Cell Wall

Early electron microscopic observation indicated that the wall construction of plant tissues is achieved by progressive deposition of new lamellae of cellulose microfibrils on the inner surface of the primary wall. T o explain the mechanism by which the lamellae are deposited on the inner surface of the wall during cell expansion, Roelofsen and Houwink (1953) proposed the multi-net growth hypothesis, which states that the microfibrils in the newly deposited lamellae on the inner surface of the wall are oriented transversely along the growth axis of the cell. As the cell elongates, the lamellae become stretched and extend longitudinally in relation to the growth axis. During this time, additional lamellae are laid down transversely on the inner surface of the wall. Consequently, the microfibrils on the innermost surface of the cell wall are likely to show a transverse orientation, whereas those in the outer surface tend to be oriented at random or longitudinal to the growth axis of the cell. The essence of this hypothesis is presented in Fig. 1A (Preston, 1982). Although the multi-net growth hypothesis is applicable to several types of cells, particularly parenchyma cells, it cannot be simply applied to the walls of other cells, such as epidermal walls, that consist of several layers of cellulose microfibrils oriented alternately longitudinally or transversely to its growth axis (Chafe, 1972). This type of wall organization is termed the crossed polylamellate wall structure (Fig. 1B). In epidermal cells of azuki bean (V. angularis Ohwi et Ohashi) epicotyls, the microfibrils on the innermost surface of the outer tangential walls are laid down parallel to each other, and the overall direction of the microfibril orientation differs from cell to cell irrespective of cell age. O n average, however, transversely oriented microfibrils predominate in young cells and longitudinally oriented ones predominate in aged, nongrowing cells (Takeda and Shibaoka, 1981). The fast-freeze, deep-etch, rotary-shadow replica technique made possible the visualization of the spatial relationships of cell wall polymers in the primary cell wall of onion parenchyma (McCann et al., 1990). In this

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B

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C

outer inner

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FIG. 1 Schematic representation of microfibriliar organizations of plant cell wall with lamellae peeled off successively. In these drawings the innermost face of the cell wall is toward the observer. (A) Multi-net growth wall structure represented by the parenchyma cell wall. At the innermost layer of the cell wall, cellulose microfibrils are preferentially oriented transversely or helicoidally to the growth axis. As cell elongation proceeds, the wall becomes extended and the orientation of the microfibrils shifts gradually from the transverse to a random or longitudinal one. (B) Crossed-polylamellate wall structure represented by an outer tangential cell wall of a growing epidermal cell. The orientation of microfibrils in each lamellae differs from layer to layer. At the innermost surface of the wall of young growing cells, transversely oriented microfibrils predominate. As cell growth proceeds, proportions of oblique and longitudinal microfibrils increase. In nongrowing cells, longitudinal microfibrils predominate (Takeda and Shibaoka. 1981).(C) Helicoidal cell wall as represented by a root hair cell wall. The microfibrils within a single lamellae lie approximately parallel to each other. The orientation rotates with respect to its neighbors at a small and constant angle (Emons and Kieft, 1994).

observation, the wall specimen had been prepared by progressive extraction with solvent to specifically remove certain cell wall component. These studies disclosed microfibrils running roughly parallel in a single lamella plane at the same depth in the wall, thus offering evidence to support the polylamellate model of wall architecture. Thin-section electron microscopic observation of the internodal cell walls of Nitella, a giant unicellular algae, revealed the initial microfibrils to be deposited in a helicoidal pattern. The helicoid consists of stacks of thin layers or lamellae, each containing microfibrils oriented parallel to one another. The orientation of microfibrils in neighboring lamellae is regularly and successively rotated by small angles (Levy, 1991). The helicoidal structure is found in some higher plants and is considered to be a specific version of the polylamellate wall. In shoot apices of Petunia hybrida and Vinca

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major, a gradual shift in orientation was observed between lamellae. O n this basis, it is proposed that the orientation of cellulose microfibrils in individual lamellae to be deposited alternates progressively by small angles (Fig. 1C) (Wolters-Arts et al., 1993). These microscopic observations have raised a difficult biochemical question regarding the molecular events by which cellulose microfibrils within the lamellae are reorganized so as to change their orientation as the cell wall expands. 2. Cellulose Microfibrils

In most plant species, including all vascular plants and several algae such as Chlorophyta, Charophyta, Phaeophyta, Chrysophyta, and Rhodophyta, the microfibrils are composed of cellulose I, which is an extended rod composed of several dozen high M , P-lp-glucan chains connected by means of intramolecular hydrogen bonds (Kuga and Brown, 1988, 1991). This crystalline structure renders the microfibrils stable with respect to enzymatic degradation as well as chemical reactions. The width of the microfibrils derived from higher plants was estimated to be 5-10 or 12 nm by rapidfreezing and deep-etching techniques, respectively (McCann et af., 1990; Itoh and Ogawa, 1993). X-ray diffraction analyses of the cellulose crystallites predicted that the cross-section of the crystalline area of the microfibril is a rectangle with dimensions of the 3 X 5 nm (Frey-Wyssling, 1954) likely composed of 36 glucan molecules parallel to each other (Sugiyama et af., 1991).The crystalline core is surrounded by a paracrystalline or amorphous molecular layer of P-l,4-glucans, which might play an important role in interactions with matrix polymers (Talbott and Ray, 1992a; Hayashi, 1989) (Fig. 2). The degree of polymerization of a @-1,4-glucan molecule ranges from 600 to 25,000 (Hayashi, 1989; Kokubo et af., 1991;Timpa and Triplett, 1993; Kuga and Brown, 1991), depending on the plant species and the cell wall type. If a single glucosyl unit in the cellulose crystal is 0.515 nm, the length of ordinary P-1,Cglucans ranges from 0.25 to 0.5 pm, which is much shorter than the usual cell length in plants. Individual glucan molecules are thought to start at different points within a single microfibril so that a single microfibril can be much longer than an individual glucan molecule (Carpita and Gibeaut, 1993). This view is supported by the fact that the end of a microfibril is seldom observed by electron microscopy. Microfibrils are regularly spaced 20-40 nm apart from each other (McCann et af., 1990) in onion bulb cell walls or 18 t 5 nm in suspension-cultured popular cell walls (Itoh and Ogawa, 1993) and are laid down in the cell wall enclosing a whole cell. 3. Cellulose-Xyloglucan Framework Cellulose microfibrils are cross-linked via matrix polysaccharides with divergent structural features. In most flowering plants (except for Poaceae),

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FIG.2 Hypothetical presentation of a cellulose-xyloglucan framework. A cellulose microfibril (CMF) consists of a rectangular crystalline cellulose (cC) core and a paracrystalline cellulose (pC) at the peripheral region. Some xyloglucans (XG) are intercalated into the crystalline cellulose (cC-XG) and others (pC-XG) are hydrogen bonded to the paracrystalline cellulose. The xyloglucan molecule is flexible and long enough to attach to two or more microfibrils, thereby forming a network structure of the cellulose-xyloglucan framework.

xyloglucans (Bauer et al., 1973), rhamnogalacturonans (Talmadge et al., 1973), and glucuronoarabinoxylans (Darvill et al., 1980b; Nishitani and Nevins, 1989) are the three major polysaccharides of the cell wall matrix. Cell walls of Poaceae differ from many other angiosperms in that they contain glucuronoarabinoxylans and /3-1,3-1,4-glucans as the major components (Kato and Nevins, 1986) but have relatively smaller amounts of xyloglucans and rhamnogalacturonans. These matrix polysaccharides are all composed of repeating structural units (Bauer et al., 1973; Kato and Matsuda, 1976; Nishitani and Nevins, 1991; Lau et al., 1985; An et al., 1994; Colquhoun et al., 1990; Schols et al., 1990). Xyloglucans are ubiquitous in the cell wall of seed plants and are characterized by a cellulose-like p-1,4-glucan backbone with frequent a-xylosyl side chains attached at the 6 - 0 position of the glucosyl residues of the backbone (Fig. 3). The basic repeating structural unit consists of four consecutive glucosyl residues with three successive single xylosyl side chains. Xylosyl side chains (R2 and R3 in Fig. 3) are occasionally further substituted side chain (York et with p-1,2-galactosyl or a-1,2-fucosyl-~-1,2-galactosyl al., 1995). In some cases, arabinosyl and/or xylosyl residue is glycosydically linked at C-2 of the glycosyl residue (R1 in Fig. 3) at the nonreducing end of the repeating unit (Kiefer et al., 1990). Fucosylation is common to most cell walls derived from gymnosperms (Acebes et al., 1993) and angiosperms, again except for Poaceas. Xyloglucans from Poacea cell walls contain lesssubstituted xyloglucans without fucosylation. Xyloglucans are tightly attached to cellulose microfibrils to form an immobilized cellulose-

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non-reducing t. 2 reducing t. (1*4)-p-D-Glcp(l+ 4 ) - P ~ G l c p1(+~ ) - P - D - G I C ~ ( ~ - ~ ) - P - P G1--c ~C 4)-b~( 6 6 6

R1:

Hp-0-xylp-1 a-L-Araf -1 .+ a-L-Araf- (1 +3)+

R2:

-- -

HP-D-Galp-1 a-L-Fucp (1

2)- P-o-Galp-1

HP-D-Galp-1 ~ - L - F u c(1 ~

2)- P-D-Galp -1-

-

R3:

P-~-Xylp-I+

-

FIG. 3 Basic structure of xyloglucan. A typical xyloglucan molecule is composed of a repeating structural unit that consists of four glucosyl residues with three successive substitutions with single xylosyl side chains. This basic structure is occasionally modified by further substitution by monosaccharide or disaccharide at R1, R2, and R3.

xyloglucan complex and constitute the major component of the plant cell wall (see refs. in Hayashi, 1989). A 1-cm-long epicotyl section of 6-day-old azuki bean seedlings, for example, contains 62 p g cellulose (Nishitani and Masuda, 1979) and 20 p g xyloglucan (Nishitani and Masuda, 1983), which together occupy up to 55% of the dry weight of the wall polymer. The first structural model for the plant cell wall, which was advanced by the Albersheim’s group in 1973, envisaged the entire length of the xyloglucan molecules to be bound to the surface of cellulose microfibrils by hydrogen bonding between the backbone of xyloglucan and the P-1A-glucans located at the surface of the cellulose microfibrils (Keegstra et al., 1973). Some of the xyloglucan molecules are further linked covalently through their reducing ends to other matrix polymers such as rhamnogalacturonans. Consequently, a single cellulose microfibril coated with xyloglucan molecules would be indirectly interconnected to two o r more microfibrils. These interconnections, repeated frequently among cellulose microfibrils, would form a single supermolecular framework structure (Keegstra et al., 1973). Although various new findings have challenged the details of the original model, its essence has, broadly speaking, withstood the test of time (Talbott

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and Ray, 1992a; Hayashi, 1989; Fry, 1986). It is now widely accepted that the cellulose-xyloglucan framework is the basic framework of the cell wall (Carpita and Gibeaut, 1993; McCann and Roberts, 1994).

4. Interaction between Xyloglucans and Cellulose Microfibrils a. Fucosylution The idea of a cellulose-xyloglucan complex arose from the fact that xyloglucans can bind to purified cellulose by noncovalent interactions (Aspinall et al., 1969; Bauer et af., 1973). Xyloglucan fragments consisting of 4-1 1 repeating units exhibited much higher adsorption activity on Avicel crystalline cellulose than polymeric xyloglucans of higher M , (Vincken et al., 1995). This suggests that cellulose is a porous matrix and high M , xyloglucans are too large for the small pores, whereas small-sized xyloglucans can penetrate them. In stem section of etiolated pea seedlings, 2,6-dichlorobenzonitrile (DCB), a potent inhibitor of cellulose biosynthesis in plants, severely inhibited the incorporation of radioactive glucose into cellulose by 80435% and enhanced elongation of the section by 34-65% during 25 h of incubation. Under these conditions, DCB did not affect either the incorporation of radioactive arabinose into xyloglucan or the ability of the newly synthesized radioactive xyloglucan to bind t o the cell wall (Edelmann and Fry, 1992). This clearly indicates an ample capacity for preexisting cellulose microfibrils to bind newly synthesized xyloglucans and implies that portions of xyloglucans are not directly hydrogen bonded to cellulose microfibrils but rather are held indirectly to them via other xyloglucan molecules. The cellulose-xyloglucan interaction seems to be structurally specific because the affinity of xyloglucan for cellulose does not interfere with other glucans with different glycosidic linkages, such as p-1,2, p-1,3, and p-1,6 linkages (Hayashi ef al., 1987). Computer-assisted calculations of the potential energy of xyloglucan oligomers revealed that the side chain of xyloglucan possesses a tendency to fold onto one surface of the backbone in its fucosylated region, thus stabilizing interactions between the fucosylated side chain and the backbone. Assessment of the side chain flexibility of xyloglucan oligomers by Metropolis Monte Carlo simulations also indicated that the fucosyl-galactosyl-xylosyl side chain is less mobile than the xylosyl side chain. These computer simulation studies strongly suggest that the fucosylated side chains play a role in the binding of xyloglucans to microfibrils (Levy et al., 1991). The fucosylated xyloglucan derived from pea cell walls has a higher adsorption constant for cellulose than nonfucosylated xyloglucan extracted from nastutium (Hayashi et al., 1994). An Arabidopsis mutant murl has impaired fucosylation probably due to its inability to convert GDP-D-mannose to GDP-L-fucose and is deficient in L-fucose in its shoot cell walls. This mutant plant shows dwarfism in its

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morphology and considerably reduced mechanical strength of the shoot (Reiter et al., 1993). Because replacement of L-fucose by L-galactose did not alter the biological activity of the xyloglucan nonasaccharides as measured by inhibition activity toward the 2,4-dichlorophenoxyaceticacidinduced elongation of pea stem segments, the phenotype of the rnurl mutant might reflect alteration of mechanical properties of the cell wall with less fucosylated xyloglucans (Zablackis et al., 1996). Thus, it is quite likely that the mechanical properties of the cellulose-xyloglucan framework depend on the side chain of the xyloglucan and can be regulated by modifying the side chain structure (Hoson, 1993). If fucosyl residues are required to maintain the cellulose-xyloglucan framework, then a-fucosidase, which is found in pea (Pisum sativum) and capable of acting on xyloglucans, may play an important role in regulating the cell wall expansion process (Augur et al., 1995).

b. Two Forms ofXyloglucan Concentrated alkali solutions, such as 24% potassium hydroxide solution or 17.5% sodium hydroxide solution, are often used to extract xyloglucans from the cell wall. Although a small portion (less than 2%) of xyloglucans from the epicotyl cell wall of azuki bean seedlings could be extracted with 4% potassium hydroxide solution without urea, about 40% of the wall xyloglucans was extracted with 4% potassium hydroxide solution containing 8 M urea. The remaining xyloglucans (58%) were extracted by subsequent extraction with 24% potassium hydroxide solution (Nishitani and Masuda, 1983). Concentrated potassium hydroxide solutions of more than -17-18% can disrupt the crystalline structure in native microfibrils (cellulose I), thereby converting them to a crystalline structure termed cellulose 11. On the other hand, lower concentrations of alkali, e.g., 4% potassium hydroxide solution, cause swelling of the amorphous structure at peripheral regions of the cellulose microfibrils (Okano and Sarko, 1985). These findings indicate that xyloglucans exist in two forms, one being bound to the paracrystalline surface of cellulose microfibrils simply by hydrogen bonds and the other one being intercalated into crystalline cellulose microfibrils in such a way as to be resistant to 4% potassium hydroxide extraction. The presence of two types of xyloglucan fractions with divergent M , distribution profiles has also been reported for pea cell walls, in which xyloglucans were resolved into two components by gel permeation chromatography (Talbott and Ray, 1992a). How they are intercalated into the crystalline core of cellulose microfibrils at the cell surface remains an unanswered question. Baba et al., (1994) prepared a macromolecular complex composed of xyloglucan and cellulose by removing pectic substances and glucuronoarabinoxylan from cell walls derived from pea epicotyls. They disclosed xyloglucans as gold particles by means of a negative staining technique coupled

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with immunogold-labeling procedures using xyloglucan-specific polyclonal antibody. Their observation revealed that most of the antigen was widely distributed in between cellulose microfibrils as well as on the microfibrils. This offers further evidence for the occurrence of two forms of xyloglucans in m u m

5. Pectic Polysaccharide Network Another separate framework based on pectic polymers has also been proposed (McCann and Roberts, 1994; Wells et d.,1994). A cellulose-synthesis inhibitor, DCB, inhibits construction of the cellulose-xyloglucan framework. Because DCB did not inhibit formation of the pectic framework in the cell walls, the adaptation of suspension-cultured cells to this inhibitor offered a unique opportunity to produce cell lines with markedly reduced levels of the cellulose-xyloglucan framework (Shedletzky et af., 1990). In the DCB-adapted tobacco BY-2 cells, the cellulose and xyloglucan contents were reduced to 9 and 20% of the nonadapted ordinary cells, respectively. Although the tensile strength of the celluloseless wall was reduced to about 30% of the nonadapted cell, it was still resistant to lysis, and wall porosity was not altered (Shedletzky et al., 1992). These DCB-adapted cells were, however, lysed upon treatment with aqueous solution of cyclohexane-trans1,2-diaminetetraacetate. This agent serves as a potent chelator for calcium ions and removes the ions between polygalacturonans in the pectin framework, thereby causing disintegration of the pectic framework (Ryden and Selvendran, 1990). Fourier transform infrared microspectroscopy equipped with a polarizer was introduced to analyze the orientation of particular functional groups in the pectic frames and wall proteins in rnuro. In elongating carrot cells, these particular functional groups showed an orientation transverse to the growing cell axis (McCann et al., 1993). Based on this information, it is speculated that the pectin framework is oriented roughly parallel to the cellulose and thereby serves as a compression-resistant framework structure that fills in the spaces in the cellulose-xyloglucan framework (McCann and Roberts, 1994).

6.Dynamic Aspects of the Cell Wall Construction Biochemical analyses of cell wall polysaccharides during cell growth and differentiation revealed that the wall undergoes drastic metabolic turnover that enable the structural changes responsible for cell expansion and deformation to take place. This is a process that involves both deposition and rearrangement of the interwoven structure of the cell wall. Thus, the plant

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cell wall is better considered as a vital organelle located outside the plasma membrane. Early in the nineteenth century, von Mohr and von Nageli proposed apposition and intussusception theories, respectively, to explain the mechanism by which the wall is deposited during cell growth. Apposition theory stated that new wall material is deposited consecutively as successive layers one upon the other at the inner surface of the preexisting cell wall, whereas the intussusception theory assumed that new wall materials are incorporated into the preexisting wall to increase its surface area (Ray, 1987). Modern electron microscopic observations clearly indicated the occurrence of an apposition-type construction process even in the primary wall, whereas tracer experiments indicated that new wall material is actually deposited throughout the entire wall, suggesting the occurrence of an intussusception type of wall construction. Although our understanding of the mechanism of cell wall deposition remains primitive, a common assumption is that both apposition and intussusception reactions are involved in the wall construction of growing plant cells. The molecular process underlying these concepts is the main issue in the study of plant cell walls today.

1. Synthesis of Cellulose Microfibrils The site for cellulose synthesis was long thought to be located on the plasma membrane (Robinson and Preston, 1972; Bowles and Northcote, 1974). Using the free fracture technique, Brown and Montezinos (1976) found a highly ordered, rod-shaped structure at the elongating tip of the microfibril on the plasma membrane and named it a terminal complex (TC). This complex was considered to be the site for polymerization, crystallization, and orientation of cellulose microfibrils and was found as a linear- or rosette-shaped structure in various plant species (Giddings et af., 1980). Seed plants have rosettes with sixfold symmetrical structures (Mueller and Brown, 1980). The hexametrix appearance of the rosette may imply that each subunit is involved in the synthesis of six P-l,4-glucan molecules, thereby producing microfibrils composed of 36 glucan chains (Delmer and Amor, 1995). These observations suggest that cellulose synthase consists of several proteins with different functions. Acetobacter xylinum, a prokaryote that can synthesize cellulose at its cell surface, was successfully employed to identify catalytic subunits for the cellulose synthase by use of photoaffinity labeling procedures (Lin et af., 1990). Meanwhile, two operons (acs and bcs) for cellulose synthase were cloned independently from the prokaryote (Saxena et al., 1990; Wong et al., 1990). Two homolog genes, AcsAB and BcsA, are derived from the two operons encoded with catalytic subunits of the synthase complex. In addition to the catalytic subunits, other types of subunits with distinct structure and function were found. The BcsC gene

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is considered to code for a pore-forming protein that has an active role in guiding the polymerized @1,4-glucan chain out of the synthase complex, whereas the BcdD gene might be responsible for crystallization of the microfibrils (Saxena et al., 1994). Although the plant genes responsible for cellulose synthase have not yet been cloned, evidence is accumulating that a similar machinery might exist in seed plants (Amor et al., 1991). A plasma membrane fraction solubilized from cotton (Gossypium hirsutum) fiber with 0.05% digitonin exhibited a cellulose I synthesizing activity in vivo (Kudlicka et al., 199.5). By use of direct photo labeling with [32P]UDP-glucose,Delmer et al., (1991) isolated from cotton fibers an 84-kDa polypeptide that can bind to UDP-glucose. Structural analysis of this peptide showed it to be a membrane-bound sucrose synthase (Amor et al., 1995). O n this and other circumstantial evidence, Delmer and colleagues have proposed that the sucrose synthase exists as a subunit of the cellulose synthesizing complex and mediates the reverse reaction (sucrose + UDP + UDP-glucose + fructose), thereby generating UDP-glucose from sucrose in the vicinity of the catalytic subunit of the cellulose synthase (Delmer and Amor, 199.5). The orientation of cellulose microfibrils-in other words, the path of movement of TC on the fluid plasma membrane-has long been considered to be regulated by cortical microtubules either directly or indirectly through some interaction with TC (Ledbetter and Porter, 1963). This hypothesis has been verified by two lines of evidence. First, parallelism in the orientation between nascent cellulose microfibrils and microtubules is frequently observed in various plant species. Second, agents that specifically inhibit polymerization of microtubules also cause disordered orientation of cellulose microfibrils. Microtubule orientation is, in turn, regulated by several phytohormones, particularly gibberellins (Shibaoka, 1994). The mechanism by which individual phytohormones govern reorientation of cortical microtubules has not been fully elucidated. Emons el al. (1992) hypothesized a geometrical model for the cellulose microfibrils. It supposes that microfibril deposition occurs on the spheroidal surface between the plasma membrane and cell wall under space-limiting conditions. According to this model, microtubules are not required as a guiding structure for T C movement on the plasma membrane (Emons and Kieft, 1994). 2. Modification of Wall Architecture during Cell Wall Expansion

a. Cleavage Model for Cell Wall Loosening The essence of the cell wall model proposed by Albershiem’s group (Bauer et al., 1973) is that cellulose microfibrils are interconnected by noncovalent linkages through matrix

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polysaccharides. The biological implication of this model is that breakage of certain load-bearing molecules between microfibrils is a prerequisite for the cell wall modification that leads to wall expansion or construction. In order to gain insight into the mechanism of cell wall modification, the search for both the load-bearing molecule and the enzyme responsible for hydrolysis of the linkages has been conducted using plant materials that elongate or expand rapidly in response to auxin, a typical phytohormone (Masuda, 1990). Yamamoto et al. (1970) developed a stress-relaxation analysis to evaluate the rheological properties of the cell wall and showed that auxin-induced cell expansion is closely correlated with alteration of a rheological parameter of the cell wall, designated as the minimum stressrelaxation time (Yamamoto, 1971; Sakurai, 1991). Upon application of auxin, the minimum stress-relaxation time of the cell wall decreases within 15 min. This change in the mechanical properties of the cell wall is followed by cell elongation exerted by turgor pressure. According to a Maxwell viscoelastic model, the minimum stress-relaxation time was defined as q/ G, where 77 and G represent viscosity and elasticity components of the cell wall model, respectively. Auxin-induced cell wall modification as detected by the changes in the rheological parameter is considered to be correlated with a decrease in the molecular weight of cell wall polymers (Masuda, 1990). A biochemical approach revealed that auxin promoted the liberation of xyloglucan from the cell wall of pea epicotyl sections that had been fed ['4C]-glucose (Labavitch and Ray, 1974a). This occurred within 15 min of the hormone application, with the degree of xyloglucan liberation increasing in proportion to the hormone-induced cell elongation (Labavitch and Ray, 1974b). Furthermore, acidic pH, which can induce cell expansion growth in many plant tissues, also promoted the xyloglucan metabolism (Jacobs and Ray, 1975). In epicotyl sections of azuki bean (V. angularis), M , of xyloglucans in the cell wall decreased during the auxin- and acidic pHinduced cell extension growth (Nishitani and Masuda, 1981,1982a). Similar M , changes in xyloglucans were observed in monocotyledonous plants such as oat (Avena sativa) (Inouhe et al., 1984) and rice (Oryza sativa) (Revilla and Zarra, 1987) and gymnosperms (Lorences and Zarra, 1987). Hoson and colleagues (Hoson, 1991; Hoson et al., 1991,1993) inhibited xyloglucan breakdown in epicotyls of azuki bean by adding a fucose-binding lectin or the polyclonal antibodies raised against xyloglucan heptasaccharides. Such inhibition of the xyloglucan metabolism was closely correlated with suppression of the auxin-induced cell wall expansion, suggesting a causal relationship between xyloglucan degradation and the cell wall expansion. In growing plant stems, the epidermis is under a tension imposed by the inner tissues and thereby controls extension of the whole stem. Auxin was shown to induce stem elongation by modifying the epidermal cell wall

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(Tanimoto and Masuda, 1971). In dark-grown squash (Curbita maxima Duch) hypocotyls (Wakabayashi et al., 1991) and pea epicotyls (Bret-Harte and Talbott, 1993), auxin substantially accelerated depolymerization or degradation of xyloglucan in the epidermis but not in inner tissues. In pea epicotyls, which had been placed horizontally to cause a gravitropic response, xyloglucan was specifically degraded in the lower side (the growing side) of the epicotyls (Talbott and Pickard, 1994). These observations were interpreted as strong evidence for the “cleavage model” of cell wall loosening, which states that splitting of the load-bearing xyloglucan molecules by hydrolase is the key step controlling architectural changes that lead to cell wall extension and, hence, cell expansion growth (Cosgrove, 1989; Fry, 1989; Hayashi, 1989; Hoson, 1993). Two endoglucanases (EC 3.2.1.4) capable of cleaving p-1,4-glucosidic linkages in plant cell walls were isolated from auxin-treated pea epicotyls (Wong et al., 1977). Subsequently, similar glucanases were found in several plant species. Studies of genes encoding endo-p-l,4-glucanases showed them to be involved in cell wall degradation in abscission zones of bean (Tucker and Milligan, 1991) and tomato (Lashbrook et al., 1994) as well as ripening fruit of avocado (Cass er al., 1990) and tomato (Lashbrook et al., 1994). Recently, a gene coding for a similar endo-P-1,4-glucanase was isolated from poplar (Populus d b a ) suspension-cultured cells (Nakamura et al., 1995) and pea epicotyls (Wu et al., 1996). Although these glacanases exhibit hydrolytic activity toward p-1,4-glucosyl linkages in carboxymethyl cellulose or lichenan, they did not act efficiently toward xyloglucans (Hayashi and Ohsumi, 1994; Ohmiya et al., 1995). Thus, these glucanases most likely act on cellulose but not on xyloglucan cross-links. O n the other hand, xyloglucan-specific p-1,4-glucanase was isolated from germinating cotyledons of nasturtium (Edwards et al., 1985), and degraded efficiently and specifically storage xyloglucans in the cell wall. As will be described in following sections, this enzyme is a member of the EXGT-related gene family.

b. Molecular Grafting Model Suppose that only hydrolytic cleavage of load-bearing linkages in a cellulose-xyloglucan framework is involved in the cell wall modification responsible for wall expansion. This means that the M , of xyloglucan will gradually decrease as wall expansion proceeds, and the framework will finally break down. Furthermore, it is difficult to use the cleavage model to explain the process through which nascent cellulose microfibrils spun out at the terminal complex are integrated into a preexisting cellulose-xyloglucan framework, a process essential for cell wall deposition. As a matter of fact, in growing plant cells, the matrix polymer, as well as cellulose microfibrils, is continuously metabolized so as to compensate for the wall thinning due to wall extension, and the M, of xyloglucan is

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precisely controlled during the cell extension process (Nishitani and Masuda, 1980, 1982a; Wakabayashi et al., 1993; Talbott and Ray, 1992b). Experiments using wall synthesis inhibitors, such as DCB, monensin, and galactose, have shown that the synthesis of matrix polysaccharides is essential for cell wall extension and suggest an essential role of integration of new wall material into the preexisting wall framework (Inouhe and Yamamoto, 1991; Hoson and Masuda, 1992). This situation constitutes a puzzle that could not be solved simply by studies of hydrolases and strongly suggests the presence of an alternative mechanism by which the cell wall framework is continuously reorganized by enzymes capable of mediating reconnection between preexisting xyloglucan cross-links and integration of newly secreted cellulose and xyloglucans into the preexisting framework structure. This puzzle was partially resolved by the discovery of endoxyloglucan transferases.

111. Endoxyloglucan Transferese

A. Enzyme Reaction of EXGT 1. Identification and Purification of EXGT Endoxyloglucan transferase activity was discovered early in the 1990s through three independent lines of research, each being carried out toward a different goal. The first indication of the existence of an endo-type transglycosylase activity in plants was noticed by Fry’s group during their investigation aimed at elucidating the mechanisms by which concentrations of biologically active xyloglucan oligosaccharides are regulated in vivo. They fed growing suspension-cultured cells of spinach (Spinach oleracia) 3Hlabeled xyloglucan nonasaccharide and traced its fate during several hours of incubation. They found that a proportion of the 3H-labeled oligosaccharide was incorporated into a buffer-soluble, extracellular polymer. This polymer was stable in concentrated solutions of acetic acid and sodium hydroxide but was degraded by a Trichoderma viride /3-1,4-glucanase preparation (Baydoun and Fry, 1989). They speculated that the labeled xyloglucan nonasaccharides became attached to soluble extracellular xyloglucans by transglycosylation activity present in the extracellular space. This enzymatic activity was designated xyloglucan endotransglycosylase activity (XET) (Smith and Fry, 1991). Subsequently, similar enzyme activities were found in dilute buffer-soluble fractions of tissue homogenate derived from various plant species and were considered to be involved in cutting and rejoining cross-links between xyloglucan chains. These findings led Fry and

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colleagues (1992) to propose that the XET activity is responsible for the wall loosening required for plant cell expansion. Independently, we obtained evidence for transglycosylation activity between xyloglucan molecules during research undertaken to explore enzymes involved in structural modification of the cell wall, a process responsible for cell expansion growth (Nishitani and Tominaga, 1991). We prepared an apoplastic fluid of azuki bean epicotyls by a low-speed centrifugation procedure without degrading tissue integrity and incubated it with xyloglucans of a defined mass average M , (420 kDa). The reaction caused the generation of xyloglucans with higher M , (820 kDa) and lower M , (149 kDa) components than that of the initial substrate. Clearly, the data indicated the presence, in the apoplast or cell wall space of azuki bean epicotyls, of polymerization activity as well as degradaton activity toward xyloglucans. These results were explained as being from the action of an endo-type transglycosylase that mediated transfer of a large segment of a xyloglucan molecule to another xyloglucan polymer. This hypothesis was confirmed by measuring segment-transfer activity from high M , xyloglucan to a fluorescently labeled xyloglucan oligosaccharide (Nishitani, 1992). The enzyme was purified and designated endoxyloglucan transferase (EXT) based on its unique mode of action (Nishitani and Tominaga, 1992). Purification of the enzyme was followed by molecular cloning of the cDNA encoding EXT from several plant species, showing that the EXT protein is ubiquitous at least among angiosperms (Okazawa efal.,1993). Currently, the abbreviation EXT is replaced with EXGT in order to avoid confusion with extensin (Ext) genes. The third line of evidence came from Reids’s group, which isolated a xyloglucan-specific p- 1,4-glucanase involved in degradation of storage xyloglucans in cotyledons of nasturtium (Tropaeolummajus) during germination (Edwards et al., 1985, 1986). Further examination of the mode of action of this hydrolase showed that the xyloglucan-specific j3-1,4-glucanase exhibited, under certain specific conditions, a transglycosylase activity and thereby mediated transfer of an oligomeric unit of xyloglucan to another xyloglucan oligosaccharide (Farkas et al., 1992; Fanutti er al., 1993). The nasturtium xyloglucan-specific p-glucanase was renamed XET. The purified nasturtium enzyme exhibited a different catalytic activity from that of the purified azuki bean EXGT. The major difference in the mode of action between the two enzymes is that under ordinary conditions the former exhibited hydrolytic activity, whereas the latter did not. The implication of the difference in enzymatic actions on their biological functions will be discussed later. 2. Mode of Action of Azuki Bean (V. angularis) EXGT a. Enzymafic Reaction When a mixture of low M , labeled xyloglucan and high M , nonlabeled xyloglucan was incubated with purified azuki bean

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EXGT, medium-sized labeled xyloglucans were generated, indicating that a portion of nonlabeled high M , xyloglucan (donor substrate) was transferred to the low M , xyloglucan (acceptor substrate). The average molecular size of xyloglucan segments transferred from the donor to the acceptor was calculated by subtracting the M , of the acceptor from that of the transglycosylation product, which was represented by the average M, of labeled xyloglucan generated by the enzyme reaction. The ratio of the molecular size of the transferred segment to that of the donor molecule ranged between 0.49 and 0.65, irrespective of the donor size. This means that individual donor substrates were cleaved, on average, around their midpoints and transferred to the acceptor molecule, thereby generating products with an average M , equal to nearly half the size of the donor substrate. This is convincing evidence that the enzyme is an endo-type transferase (Nishitani and Tominaga, 1992). On the other hand, when a xyloglucan preparation with a defined M , distribution was incubated with the azuki bean EXGT, the disproportioning of the M , distribution without any detectable change in its average M, was detected. This means that a xyloglucan fragment split from a donor substrate is always transferred to an acceptor xyloglucan, but not to a water molecule, thereby maintaining an average M,. Furthermore, during this reaction, little or no oligomer or monomer is generated. Thus, this transferase exhibits no glycanase or glycosidase activity. Masking of the reducing terminus of donor xyloglucan substrate by reduction with sodium borohydride did not affect the EXGT-catalyzed enzyme reaction. This result unambiguously excludes the possibility of preexisting reducing termini of xyloglucans being involved in the transglycosylation reaction and indicates that the newly generated reducing end of the split fragment is linked to the nonreducing terminus of an acceptor xyloglucan molecule. 'H NMR analyses showed that the ratio of individual anomeric protons in xyloglucans did not change during the enzyme reaction, confirming the view that a single type of glycosidic linkage, namely P-1,4-glucosidic linkage, is involved in both the splitting and reconnection reactions. These findings indicate that the EXGT-catalyzed reaction consists of (i) recognition and splitting of P-l,4-linked glucosyl residues on the xyloglucan main chain in an endo-type fashion, and (ii) reconnection of the newly generated glucosyl residue at the reducing terminus of the split xyloglucan to the 4 - 0 position of the glucosyl residue at the nonreducing terminus of other xyloglucan molecules (Nishitani and Tominaga, 1992) (Fig. 4).

b. Substrate Speci$city

Pyridylamino oligosaccharides with different side chains were used to characterize the acceptor substrate specificity of the purified azuki bean EXGT. No significant difference in the acceptor substrate activity was observed among the three pyridylamino xyloglucan oligo-

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FIG. 4 Diagrammatic presentation of EXGT-mediated molecular grafting between xyloglucan molecules. EXGT catalyzes both endo-type cleavage of ~-1,4-glucosyllinkage at an unsubstituted glucosyl residue and linking of a newly generated reducing terminus to the nonreducing terminus of another xyloglucan at the 4 - 0 position, thereby mediating transfer of a large segment of xyloglucan t o another xyloglucan.

saccharides. On the other hand, deletion of a single xylosyl residue at the nonreducing terminus of the pyridylamino xyloglucan heptasaccharide resulted in the loss of the acceptor activity (date not shown). Pyridylamino

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cellohexaose or laminarihexaose did not act as the acceptor substrate. Clearly, a nonreducing terminal /3-1,4-glucosyl residue with an a-1,6xylosyl side chain is required for the acceptor substrate activity. These results suggest that azuki bean EXGT recognizes the acceptor substrate in an exo-type fashion (Nishitani and Tominaga, 1992). An XET preparation derived from pea epicotyl exhibited a similar acceptor substrate specificity (Fry et al., 1992). The donor substrate specificity of EXGT was analyzed using various polysaccharides with different glycosidic linkages. EXGT acted equally efficient toward fucosylated and nonfucosylated xyloglucans used as donor substrates, indicating that fucosyl side chains are not required for the EXGT-mediated transglycosylation or do not interfere with it. In contrast, the EXGT did not act toward carboxymethylcellulose or /3-1,3-1,4-mixed glucan (Nishitani and Tominaga, 1992). Thus, the enzyme exhibits and strict donor substrate specificity for xyloglucans with its basic structural features. Higher reaction rates were achieved when xyloglucans with higher M , were used as the donor substrate. Xyloglucans with a M , of less than 10 kDa showed little or no donor substrate activity. On the other hand, the acceptor substrate activity did not depend on the M , of acceptor molecules. This means that EXGT preferentially splits higher M , xyloglucans in the cell wall space and transfers the split end to any xyloglucan polymer or its oligosaccharides, thereby helping to maintain the uniformity of xyloglucans with respect to M , distribution. The M , dependency of the donor substrate specificity of the EXGT makes possible the incorporation of free xyloglucan oligomers into the immobilized xyloglucans in the cell wall. As a result, the concentrations of xyloglucan oligosaccharides in the cell wall might be maintained at a low level. The mechanism by which the azuki bean EXGT preferentially acts on a higher M , xyloglucan donor substrate is thus of great importance but remains to be resolved. c. p H Dependency The azuki bean EXGT exhibited maximum transferase activity at p H 5.8 (Nishitani and Tominaga, 1992). A crude apoplastic solution derived from azuki bean epicotyls (Nishitani and Tominaga, 1991) and a crude extract from pea epicotyls (Fry et al., 1992) showed optimal pH at p H 5.4 and 5.5, respectively. These enzyme activities declined steeply as the pH value increased. The pea crude enzyme preparation was less than half as active at p H 7.0. Azuki bean apoplastic enzyme solution exhibited little or no activity at pH higher than 6.2. According to the acid growth theory, auxin causes acidification of the cell walls by stimulating hydrogen ion secretion into the cell walls and thereby induces the cell wall modification that leads to cell expansion (Cleland, 1971). This hypothesis has been challenged by evidence that the effects of auxin and acidic pH on cell expansion are additive, and it is

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currently accepted that they act via separate mechanisms (Kutschera, 1994). The pH of apoplastic solutions derived from epicotyls of azuki bean ranged from 6.2 to 6.6 (Nishitani and Tominaga, 1991). Auxin decreases the p H value by 1 unit in several plant tissues (Jacobs and Ray, 1976). Because the azuki bean EXGT exhibits its optimal transferase activity at p H 5.7, auxin can upregulate the activity in the apoplastic space via its acidification process. This view is consistent with the additive action of auxin and acidic pH on the cell expansion growth. Hormonal regulation of EXGT activity will be discussed under Section V,B in the context of expression of genes encoding EXGTs and related proteins.

3. Detection Procedures To detect the transferase, three different procedures are currently available. Figure S illustrates the principles of these procedures: (Fig. 5A) measurement of the transfer of a portion of the donor substrate to a radioactive acceptor, (Fig. 5B) measurement of the transfer of a portion the donor substrate to a fluorescently labeled acceptor, and (Fig. SC) measurement of changes in the M , distribution profile.

a, Radioactive Acceptor Procedurefor XET Measurement of XET activity can be carried out using radioactive xyloglucan oligosaccaharides and polymeric xyloglucans as acceptor and donor substrates, respectively. The radioactive oligosaccharides are synthesized by labeling H at position 1 of the reducing terminal glucosyl unit of a xyloglucan nonasaccharide by exchanging the 'H with 'H in the presence of 3H2 to obtain [reducing terminus-1-'H (RT-l-3H)] xyloglucans (sp act ca. 12 T B q h o l ) (Smith and Fry, 1991). The reaction mixture contains 80 pg of xyloglucan polymer, 1.4 kBq of 3H nonasaccharide, and enzyme extract in a total volume of 40 pl. After the reaction is terminated by addition of 20% formic acid, the mixture is applied to a piece of Whatmann 3MM filter paper followed by drying and washing in running tap water to remove free 3H nonasaccharides. The transglycosylase activity is estimated by measuring the radioactivity adsorbed to the piece of filter paper by a scintillation counter (Fry et a/., 1992). b. Fluorescently Labeled Acceptor Procedure for EXGT Fluorescently labeled xyloglucan oligomers are prepared by tagging the reducing terminus of xyloglucan polymer or oligosaccharide with 2-aminopyridine by reductive amination (Hase et af., 1979) to obtain pyridylamino xyloglucan polymer or oligosaccharide. The reaction mixture contains 20 pmol of 500-kDa nonlabeled xyloglucan (10 pg), 200 pmol of the pyridylamino xyloglucan oligosaccharide or polymer (ca. 10 kDa), and the enzyme preparation in

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FIG. 5 Diagrammatic representation of three detection procedures for endo-type xyloglucan transferase activity. (A) Procedure used to measure XET activity. High M , xyloglucans (open bars) and radioactive xyloglucan oligomers (solid bars), in which H at position 1 of its reducing terminus is replaced with jH, are used as the donor and acceptor substrates, respectively (Smith and Fry, 1991). Transfer of a high M , fragment of the donor xyloglucan to the acceptor substrate produces polymeric-labeled xyloglucans, which can be absorbed to a piece of Whatmann 3MM filter paper. The transglycosylation activity is measured by counting the radioactivity absorbed to the filter paper by a scintillation counter. (B) Procedure used for demonstration and characterization of EXGT. In this procedure, a fluorescently labeled xyloglucan oligomers (solid bars), which was prepared by tagging the reducing terminus of xyloglucan with 2aminopyridine (PA) by reductive amination (Nishitani and Tominaga, 1992), was used as the acceptor substrate. The transglycosylation reaction produces fluorescent xyloglucans with increased M,. The M,distribution profile, as well as the amount of the fluorescently labeled xyloglucans generated during the reaction, is analyzed by gel permeation chromatography using a fluorescence detector. This method makes possible direct characterization of the product in terms of M , changes. (C) Xyloglucan-disproportioningreaction. Procedure used to demonstrate endo-type transglycosylation between xyloglucan polymers. Xyloglucans with defined M , distribution profiles (open and solid bars) are employed for both the acceptor and donor substrates. Because transfer of a large segment of xyloglucan from one xyloglucan (donor) to another xyloglucan (acceptor) produces higher and lower M,xyloglucan molecules than substrate, the enzymatic activity can be detected by measuring the peak broadening by use of GPC.

10-20 pl of 0.2 M sodium acetate buffer at p H 5.8. After incubation at 25°C for 1 h, the reaction is terminated by addition of 0.1 M sodium hydroxide solution. The reaction product is resolved by gel permeation

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chromatography using glass-packed columns of TSKgel G3000PW (8 x 300 mm) and G5000PW (8 X 300 mm) connected in series, and the content of pyridylamino xyloglucans in the eluent is monitored by a fluorescence detector with the excitation wavelength at 320 nm and the emission wavelength at 400 nm. The transfer of xyloglucan from the high M , nonlabeled xyloglucan to the low M , pyridylamino xyloglucan is assessed by measuring the area of the peak for high M , pyridylamino xyloglucan on the chromatogram (Nishitani, 1992). c. Xyloglucan Disproportioning Reaction In this procedure, xyloglucans with defined M , distribution profile are employed for both the acceptor and donor substrates. Xyloglucan (10 pg) is incubated with enzyme preparation in 10 p1 of 0.2 M sodium acetate buffer at pH 5.8 at 25°C. After the reaction. the M , distribution profile of xyloglucans in the reaction product is resolved by gel permeation chromatography using columns of TSKgel G3000PW and G5000PW connected in series, and the polysaccharide content in the eluate is monitored by a pulsed amperometric detector, which responds specifically to carbohydrates. The enzyme reaction causes broadening of the xyloglucan peak without any change in the elution volume for the peak top position. This peak broadening indicates that both higher and lower M , species for xyloglucan molecules are generated by the transglycosylation reaction (Nishitani, 1992).

B. In Muro Functions 1. EXGT-Mediated Molecular Grafting in Muro To assess the molecular-grafting activity of EXGT in the cell wall space, a purified azuki bean EXGT was incubated with a mixture of fluorescently labeled xyloglucan oligomers and isolated cell wall that had been prepared by autoclaving ethanol-boiled cell walls derived from epicotyls of azuki bean. If the EXGT can mediate the molecular grafting reaction between the labeled xyloglucan oligomers (acceptor) and immobilized xyloglucan molecules in the isolated cell wall preparation (donor), then the fluorescently labeled acceptor substrate will be incorporated into the cellulosexyloglucan complex and immobilized in the wall material. Thus, the enzyme action can be detected by measuring the amount of the fluorescently labeled oligosaccharide moiety incorporated into the insoluble wall fraction. After the enzyme reaction, the insoluble wall fraction of the reaction products was separated'from the water-soluble fraction by a filtration procedure and was digested with purified T. viride endo-j3-1,4,-r>-glucanase(EC 3.2.1.4), a cellulase that can specifically hydrolyze p-1,4-linkages on the unsubsti-

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tuted glucosyl residues to liberate structural units of xyloglucan. Highperformance liquid chromatography analysis of the xyloglucan fragments liberated from the water-insoluble fraction of the reaction product indicated that the EXGT actually functions in the cell wall matrix and recognizes wall-bound xyloglucans as donor substrates (Fig. 6). This finding indicates potential action of EXGT in integration of newly secreted cellulosexyloglucan complex into the preexisting cell wall framework, thereby producing a cell wall with rearranged framework structure (Fig. 7).

0 0 C Q 0 u)

?!

0

a

G

I I I 1

0

I

I

20 40 Elution volume, ml

FIG. 6 EXGT-mediated molecular grafting in muro. Three micrograms of native or denatured EXGT purified from the apoplastic space of epicotyls of azuki bean was incubated with a mixture of 600 pg of the isolated cell wall and 0.6 pg of pyridylamino xyloglucan heptasaccharide in 40 pI of 0.2 M sodium acetate buffer at pH 5.8. After the reaction at 25°C for 12 h, the insoluble component or cell wall fraction was recovered by washing with the acetate buffer using a Ultrafree C3HV filter (Milipore) and subjected to enzymatic degradation with 30 pg of purified Trichoderma viride endo-1,4-~-o-g~ucanase (EC 3.2.1.4). which specifically hydrolyzed unsubstituted (1-4)-~-~-glucosyl residues in xyloglucan main chains. A portion of the solubilized fraction was resolved by an HPLC system equipped with a fluorescence spectrofluorometer (Shimadzu SPD 6A) set at an excitation wavelength of 310 nm,emission wavelength of 390 nm, and a column of TSKgel Amide 80 (4.6 X 250 mm). The column was eluted with 40 ml of 0.1 M sodium acetate buffer containing a linear gradient of 4 5 6 5 % acetonitril at a flow rate of 1 ml/min. A peak of fluorescently labeled xyloglucan oligosaccharide, which eluted at 15.3 ml, was detected when incubated with the native enzyme but not with denatured enzyme, indicating the occurrence of EXGT-mediated molecular grafting in muro.

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FIG. 7 Hypothetical representation of EXGT-mediated integration of newly synthesized cellulose-xyloglucan (C-X) complex into preexisting cellulose-xyloglucan framework. A hypothetical cellulose-xyloglucan complex generated in the vicinity of a cellulose crystallization site can be integrated into the preexisting cell wall by repeated reactions of molecular grafting between xyloglucan cross-links. CMF, cellulose microfibrils; XG. xyloglucan.

2. Localization The azuki bean EXGT protein was isolated from the apoplastic solution, which was obtained by low-speed centrifugation of 1-cm sections of the azuki bean epicotyls mounted on filtered funnel following infiltration in 50 mM magnesium chloride solution. EXGT proteins in tobacco BY-2 cells were also extracted by 50 mM magnesium chloride solution but were not found in the culture medium. The fact that 50 mM magnesium chloride facilitates liberation of the enzyme from the cell wall seems to suggest involvement of the pectic framework in the immobilization of azuki bean EXGT in the cell wall. Immunohistochemical localization showed that nasturtium xyloglucanspecific p-1,4-glucanase was exclusively localized in the cell wall of germinated nasturtium seedlings (de Silva et af., 1993). A crude preparation containing XET activity was also obtained by simple homogenization of plant tissues with a dilute buffer solution (Fry et al., 1992). These results

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are consistent with the view that EXGT and its related proteins are localized extracellularly and are loosely bound to the cell wall. This view was confirmed by the presence of a signal polypeptide composed of 20 amino acid residues upstream of the amino terminus of the mature azuki bean EXGT protein. Because this sequence contained a high content of hydrophobic residues, such as Ser, Lue, and Ala, particularly at its central part, it is quite probable that these sequences serve as a signal destined for final transfer to the extracellular space via the endoplasmic reticulum (Okazawa et al., 1993). Similar signal sequences with characteristic hydrophobicity were found in all EXGT-related proteins derived from various plant species. The presence of two pairs of cysteine residues in carboxyl-terminal regions of all EXGTs suggest the possibility of dimeric or oligomeric forms in the cell wall space. Although no direct evidence has yet been obtained to support this view, such a hypothetical interaction would play a role in regulating EXGT activities in muro.

IV. XRPs A. XRP Gene Family Structural studies of xyloglucan-specific /3-1,4-glucanase (NXGI; also termed XET) isolated from nasturtium (T. rnajus) (de Silva et al., 1993, 1994) and EXGTs derived from several plant species, including Arabidopsis (Arabidopsis thaliana) and azuki bean (Okazawa et al., 1993), revealed a structural similarity between the two functionally related proteins. These structural analyses also disclosed the presence of another structurally related Arabidopsis gene termed meri-5 (Medford et al., 1991). Meri-5 had been identified as a meristematic tissue-specific gene by differential screening. Expression profiles of a meri-5 promoter-GUS fusion gene in transgenic tobacco and Arabidopsis plants suggested specific functions of the protein in the meristematic dome and branching points in the shoot and root, although the function of the gene product per se was unknown. The sequences for the meri-5 cDNAs derived from two ecotypes of Arabidopsis [Landsberg errecta (Arrowsmith and de Silva, 1995) and Columbia (Xu et al., 1996)] were recently reexamined, and the original sequence was revised. The newly deduced amino acid sequence for the meri-5 protein shows much more similarity to EXGTs and the NXGI. Over the past few years, genes encoding proteins structurally related to meri-5, NXG1, and EXGT have been isolated in rapid succession from various plant species. The phylogenetic tree for these genes is shown in

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Fig. 8. The soybean BRUI was isolated as a brassinosteroid-regulated gene (Zurek and Clouse, 1994; Zurek et al., 1994). The maize wus1100.5 was isolated as a gene inducible by oxygen deprivation (Peschke and Sachs, 1994; Saab and Sachs, 1995), whereas Arabidoposis TCH4 was isolated as a touch-induced gene (Braam, 1992; Zurek et al., 1994). tXET-BZ and tXET-B2 were isolated from a tomato fruit cDNA library (de Silva et al., 1994). Nasturtium X E T l ( T m X E T ) was isolated from an epicotyl cDNA library of nasturtium seedlings as a homolog of nasturtium N X G l (Rose et al., 1996). Recently, Braam’s group (Xu et al., 1995) has reported an

FIG. 8 Phylogram of deduced amino acid sequences of XRP. The possible evolutionary relationship among the members of the XRP family were estimated using a “malign” program prepared by DNA Data Bank of Japan (DDBJ news letter, No. 15, pp 51-57. 1995). This family can be classified into three subfamilies, based on both divergence in the primary structure and enzyme reaction. Members of subfamilies I and I I exclusively exhibit transferase activity, whereas a member of subfamily 111 show hydrolytic activity with transferase activity. References: a, Arrowsmith and de Silva (1995): b, Xu et al. (1995); c, Xu et ( I / . (1996); d. Medford e t a / . (1991), Arrowsmith and de Silva (1995); e. Saab and Sachs (1995); f. Zurek and Cluse (1994); g, S. Okamoto etal. unpublished results; h, Okazawa era/. (1993); i, Atkinson and Redgwell (1995; Accession No. L46792); j, Rose et a/. (1996); k, Nishitani ef nl. (1996. Accession No. D86730); and I, de Silva et 01. (1993).

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additional five members of this gene family, termed XTR2, -3, -4, -6, and -7, from Arabidopsis. We have also isolated two additional XRP members (EXGT-A3 and -A4) from Arabidopsis (Okamoto et al., unpublished data). Thus, the XRPs constitute a fairly large multigene family, which consists of at least 10 members from Arabidopsis. Recombinant proteins of soybean BRUI and Arabidopsis TCH4 (Xu et al., 1995) as well as tomato tXET-BI (de Silva et al., 1994) exhibited XET activity. On the other hand, the purified protein of nasturtium NXGI exhibited not only transferase activity but also hydrolytic activity toward xyloglucans. Although the protein function of other members of this family is not known, their structural relation to these proteins strongly implies that they have enzymatic activities toward xyloglucans. Thus, this gene family was termed XRP (Nishitani, 1995). Each member of this family contains a potential N-terminal signal sequence with high levels of hydrophobic amino acid residues and is likely to be secreted into the cell wall space. This is consistent with the view that members of XRP possess enzymatic activity toward xyloglucans in the cell wall framework and play roles in rearrangement of the cellulose-xyloglucan framework in the cell wall space. With respect to their deduced amino acid sequences as well as enzymatic functions, the XRP family can be classified into three subfamilies. Subfamily I consists of nasturtium XETl and several EXGTs from various plants, whereas subfamily I1 contains meri-5, BRUI, tXET-BI and -2, TCH4, wus11005, and XTR3, -6, and -7. Subfamily I11 includes N X G I , XTR2 and -3, and EXCT-A3. A similar classification for the XRP family has been proposed by Xu et al. (1996). It is noteworthy that each subfamily contains two or more XRP members from Arabidopsis. B. Catalytic Site

The conserved amino acid sequence D-E-I-D-I/F-E-F-L-G (Fig. 9, box a) found in XRPs is also conserved in several bacterial endo-P-1,3-1,4glucanases (Borriss et al., 1990). The bacterial endo-~-1,3-1,4-glucanases cleave /I-1,-4-glycosyl linkages on 3-0-substituted glucopyranose units in P-13-1P-mixed glucan. These bacterial endo-P-l,3-1,4-glucanasesdo not show sequence similarity to either bacterial endo-P-1,4-glucanase or to barley P-1,3-1,4-glucanase (Haj and Fincher, 1995). Amino acid residues essential for the bacterial /3-1,3-1,4-glucanase activities have been investigated by site-directed mutagenesis. A recombinant Bacillus licheniformis endo P-l,3-1,4-glucanase produced by the E134Q mutant, in which the Glu 134 was replaced by Gln, showed catalytic activity of less than 0.3% of the wild-type activity (Planas et al., 1992). In B. licheniformis, the activi-

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FIG. 9 Sequence alignment of putative catalytic site amino acid residues in XRP members from plants and bacterial /3-1,3-1.4-glucanases. Box a indicates the putative catalytic center for plant XRPs. Note I or F residues indicated in this box and the flanking regions indicated by bars b, c, and d.

ties of the E134Q and D136N recombinant proteins were reduced to 0.5% of the wild-type, whereas E138Q mutation yielded a completely inactive recombinant enzyme (Juncosa et al., 1994). These findings show that Glu 138 (i.e., the second E in Box a) is the most likely candidate for the acid catalyst and that other surrounding residues, Glu 134 and Asp 136, may affect the catalytic activity. Thus, it is quite likely that the conserved sequence D-E-I-D-I/F-E-F-L-Gin plant XRP also serves as a catalytic center for the enzymatic splitting of p-1,4-glycosyl linkages in the xyloglucan main chains in the course of the transglycosylation reaction. All the XRPs, except for N X G l and XTR2, contain a consensus sequence of D-E-I-D-F-E-F-L-G-N,In NXGl and XTR2, which belong to subfamily 111, the first phenylalanine residue in the consensus sequence is replaced with an isoleucine residue (D-E-I-D-I-E-F-L-G-N).In NXGl, the first isoleucine is further replaced with leucine (D-E-L-D-I-E-F-L-G-N). According to the three-dimensional structure of a hybrid Bacillus /3-1,3-1,4-glucanase (EC 3.2.1.73) analyzed by X-ray crystallography at a resolution of 2.9 the sequence of D-E-I-D-I-E residues is located along the bottom of the active site, and their side chains protrude into the active site cleft (Keitel et al., 1993). Thus, replacement of amino acid residues in these sequences would cause a significant change in the three-dimensional structure of the active site cleft and, hence, in the enzymatic functions, including substrate specificity and reaction specificity.

A,

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Braam’s group (Xu et al., 1996) noticed additional structural differences in the flanking regions of the D-E-I-D-IF-E-F-L-G sequence between subfamily I11 and subfamilies I and 11. The members of subfamily I11 possess three additional amino acid residues on the N-terminal side of the conserved sequence (Fig. 9, bar c), whereas those of subfamilies I and I1 lack these residues. Each member of subfamilies I and I1 contains a potential site for N-linked glycosylation (N-X-SIT) on the C-terminal side of the conserved sequence (Fig. 9, bar b). In members of subfamily 111, such as XTR2 and XTR4, the potential site for N-glycosylation is not located next to the consensus sequence but is displaced toward the C-terminal side by 15 residues (Fig. 9, bar d). N X G l lacks the consensus sequence for Nglycosylation site. Nasturtium N X C l (a member of subfamily 111) literally hydrolyzes xyloglucans, whereas azuki bean EXGT (subfamily I) (Nishitani and Tominaga, 1992) and tomato tXET-Bl (subfamily 11) do not exhibit hydrolytic activity (de Silva et al., 1994). Rose et al. (1996) compared the substrate specificity of nasturtium N X G l (subfamily 111) with nasturtium XETl (subfamily I) using a crude homogenate prepared from epicotyls or cotyledons of nasturtium seedlings, respectively. They showed that NXGl exhibited higher XET activity toward nonfucosylated xyloglucans, whereas XETI acted on nonfucosylated and fucosylated xyloglucans with equal facility. Such differences in substrate specificity between these subfamilies are likely to be due to their structural difference around the putative catalytic cleft. Although significant differences in enzymatic activity between subfamilies I and I1 have not yet been studied in detail in terms of enzymology, the divergent amino acid sequences among the two subfamilies potentially imply different enzymatic activities. This means there is a possibility that XRPs with divergent catalytic activities cooperate in enzymatic functions required for construction, rearrangement, and degradation of the cellulosexyloglucan framework structure, a complex process composed of several different types of reactions.

V. Regulation of XRP Gene Expression A. Spatial and Temporal Regulation of XRP Expression

XET and EXGT activities were observed in various tissues including leaf, stem, root, peduncle, pestil, and fruit. In epicotyls of 6-day-old azuki bean seedlings, higher levels of apoplastic EXGT activity were found in the upper growing regions (Nishitani and Tominaga, 1991). A similar distribution pattern of extractable XET activity was observed along the epicotyls

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of 7-day-old etiolated pea seedlings, in which higher XET activities were observed in the third and second internodes rather than the first internode on a fresh weight basis (Fry et al., 1992). In maize root. XET activity expressed on a fresh-weight basis of the root tissue showed a close correlation with the relative growth rate profile along the root (Pritchard et a/., 1993). These results seem to indicate a positive correlation between the total activity of the endo-type transferase as evaluated by either XET o r EXGT activity and cell wall deposition activity in individual organs. In Arabidopsis, the meri 5 mRNA is expressed preferentially in meristernatic tissues (Medford eta/., 1991), whereas TCH4 is expressed in trichome, lateral root primordia, vascular bundles, and leaves (Xu etal., 1995). Thus, XRP members within a single subfamily from Arabidopsis show differential gene expression profiles. In nasturtium seedlings, XETI mRNA is expressed in all vegetative tissues except for germinating cotyledons, whereas NXCJ mRNA is exclusively expressed in cotyledons (Rose et af., 1996). In this plant, the two divergent members of the XRP family show mutually opposite patterns of gene expression. Azuki bean EXGT-VI (formerly Azuki bean EXT) is predominantly expressed in the growing stem of azuki bean seedlings. Within a single epicotyl, the highest levels of EXGT-VJ mRNA expression were observed in tissues in which cell elongation had just finished but there was still a high activity of cell wall deposition. Thus, a profile of high expression of EXGT-Vl mRNA along the epicotyl coincides well with that of the cell wall deposition activity, but not simply with the cell elongation rate (E. Tomita et al., unpublished data). Immunohistochemical localization as well as RNA gel blot analysis showed that much higher levels of EXGT-VJ mRNA and protein are expressed preferentially in epidermal cell walls than in the inner cells of azuki bean epicotyls (E. Tomita et af., unpublished data). Epidermal cell walls in plants are much thicker than those in inner tissues and exhibit higher deposition rates. Physiological studies have shown an important role of the epidermal cell wall in the regulation of stem growth in several plant species (Tanimoto and Masuda, 1971; Kutschera, 1994). These results are consistent with the idea that EXGT-V1 has a part in secondary wall deposition as well as primary wall construction. In tomato fruit, tXET-BZ is expressed during their maturation. This gene is also expressed in stems. The recombinant protein derived from tomato tXET-B2 cDNA was shown to have XET activity with no detectable hydrolytic activity, a similar enzymatic activity of purified azuki bean EXGT. Levels of rXET-BI transcript as evaluated by ribonuclease protection assay increased as the maturation proceeded, peaking at the pink stage (Arrowsmith and de Silva, 1995). This result is also consistent with the idea that the transferase is involved in construction of fruit cell wall rather than its

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degradation. On the other hand, in persimmon fruit, the highest level of XET activity was observed in the growth stage when the fruit had reached its maximum size and the average M,of xyloglucans was drastically decreasing (Cutillas-Iturralde et al., 1994). This result implies the possible involvement of XET activity in the xyloglucan degradation. Consideration of all these findings suggests the likelihood of different members of XRP being preferentially expressed in different tissues at different growth stages, although the complete picture of the XRPs in terms of expression profiles has not yet been elucidated, even for a single species. Temporally and spatially regulated expression patterns of individual XRP members imply differences in their physiological roles in cell wall construction, including those associated with cell plate formation in meristematic cells, the cell wall modification in expanding tissues, thickening of secondary walls in nongrowing tissues, cell wall degradation during fruit ripening and abscission, as well as simple degradation of storage xyloglucans in cotyledons and endosperm.

6 . Hormonal Regulation

1. Auxin Auxin plays a crucial role in regulating the cell wall modification that leads to cell expansion (Masuda, 1990). This hormone is also involved in the regulation of cell division and cell differentiation, including development of tracheary elements and adventitous root formation. To explain its pleiotropic effects, the existence of multiple receptors for auxin has been assumed, and several genes involved in signal transduction pathways from auxin perception to early gene expression have been discovered (Abel and Theologis, 1996; Nagata et al., 1994). However, little is known about the genes responsible for the cell wall modification as governed by auxin, a series of biochemical processes that are mediated chiefly by several types of carbohydrate-related enzymes. Endo-P-1 ,Cglucanase has long been marked as a promising candidate for the auxin-regulated enzyme capable of modifying cell wall structure (Verma et al., 1975). However, because endo-P-1,4-glucanase does not exhibit enough activity toward xyloglucans, its role in auxin-induced modification of xyloglucan cross-links in the cell wall architecture is still unclear (Ohmiya et al., 1995; Hayashi and Ohsumi, 1994). In addition, the construction and reorganization of the cellulose-xyloglucan framework in complicated lamellae structure could not be accomplished by simple hydrolytic cleavage of xyloglucan cross-links.

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Recent findings of cDNAs encoding a series of XRPs have opened another path for exploring auxin-regulated cell wall enzymes and have furnished new clues for resolving the molecular mechanisms for the wall organization. In Arabidopsis seedlings that had been grown in liquid culture on a rotary shaker under continuous light, externally applied IAA at 1 pM increased transcript levels of TCH4, EXGT-A1 (formerly Arabidopsis EXT), and XTR3 within 30 min after the hormone treatment (Xu et al., 1996). Auxin also upregulated mRNA levels of EXGT-VZ (formerly azuki bean EXT) in epicotyl sections of azuki bean as measured by RNA blot (E. Tomita et al., unpublished data). When we consider auxin-induced cell wall modification, we need to take into account that auxin does not always regulate enzymatic action via either de novo synthesis or activation of cell wall enzymes. Some of its action might be exerted through modification of the molecular environment for cell wall enzymes, such as pH, the pore size of the cell wall matrix, and weak interactions between enzymes and wall components such as those between lectins and polysaccharides, which are modulators capable of affecting enzymatic actions (Hoson, 1993). A steep pH dependency of EXGT activity suggests that the enzymatic activity may be regulated indirectly by auxin through acidification in the cell wall caused by auxin. According to porosity measurements using several different procedures, globular proteins smaller than 25 kDa can diffuse relatively freely through the primary cell wall, whereas those larger than 75 kDa are hindered and diffuse slowly (Read and Bacic, 1996). Therefore, the mobility of each XRP, which ranges between ca. 30-34 kDa, will be affected by a subtle change in the wall porosity. There is evidence that the pectic framework forms a wall of the finest mesh size in the cell wall architecture and plays a central role in determining the pore size (Baron-Epel ef al., 1988). This means that alteration of the three-dimensional structure of the pectin framework may directly affect the activity of XRP. Because auxin increases the wall porosity in some plant tissues (Yamamoto, 1995), the mobility of XRP in the cell wall space might be facilitated indirectly by auxin action. O n the other hand, any factors that can cause association of EXGT molecules to form oligomers would induce reduction of enzyme mobility in the cell wall space and hence reduce activity in muro. 2. Brassinosteroids

Brassinolide is an endogenous plant growth regulator isolated from pollen extracts of Brassica napus (Grove et al., 1979). Several other structurally related steroid compounds with similar biological activities have also been identified from a wide variety of plant species and are generically termed Brassinosteroids (Mandava, 1988).External application of brassinosteroids

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at 10-100 nM profoundly elicits shoot growth promotion, which is among its versatile growth effects on different organs in various plant species including azuki bean, pea, soybean, and maize. Arabidopsis cpd gene encodes a cytochrome P450, which is a putative enzyme essential for biosynthesis of brassinosteroids (Szekeres et al., 1996). The cpd mutant impaired in the brassinolide synthesis displays dwarfism, and the phenotype can be completely restored to the wild type by application of brassinosteroids. Arabidopsis dwarf mutants, cbbl and cbb3, are also impaired in brassinolide biosynthesis. In these mutants, expression levels of meri-5and TCH4 genes are significantly low compared to those in the wild type (Kauschmann et al., 1996). In epicotyl sections of azuki bean, brassinolide enhances longitudinal cell elongation. This growth stimulation is correlated with the increased percentage of transversely oriented cortical microtubules in epidermal cells. This result indicates that brassinolide enhances the longitudinal cell expansion by organizing cortical microtubules transversely to the cell axis, thereby causing the deposition of transversely oriented cellulose microfibrils (Mayumi and Shibaoka, 1995). In etiolated squash hypocotyl segments, a brassinosteroid causes cell wall changes, as can be seen from the mechanical properties of tissue sections particularly in the inner tissue (Tominaga et al., 1994). This suggests the possible involvement of this hormone in the regulation of the cell wall organization. In soybean epicotyls, application of brassinosteroids causes elongation growth of the sections within 2 h after the hormone application. Ribonuclease protection assays showed that the brassinosteroid-enhanced stem growth was accompanied by an increase in the mRNA specific for BRUI, a member of XRP, within 2 h after the hormone application (Zurek et al., 1994). Exposure of Arabidopsis seedlings to 1 pM solution of 2,4-epibrassinolide, a kind of brassinosteroid, resulted in elevation of TCH4 gene expression with the mRNA accumulation peaking at 2 h after treatment (Xu et al., 1995, 1996). The TCH4 gene expression is rapidly upregulated in response to physical stimuli, such as watering and touch (Braam, 1992). Because the kinetics of induction by brassinosteroids is slower that those of physical induction, it seems unlikely that the thigmomorphogenesis caused by mechanical stimuli is directly mediated by brassinosteroids. Other XRP members from Arabidopsis did not significantly respond to brassinosteroids (Xu et al., 1996). Thus, each XRP member shows a differential responsiveness to phytohormones. Although our understanding of the relationship between the brassinosteroid upregulated expression of some XRP members and the hormone-induced morphological changes during the entire life of a plant is primitive, the molecular approach using these XRP probes offers a

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method for exploring the mechanism by which brassinosteroids promote growth in plants.

3. Other Hormones In addition to auxin and brassinosteroids, gibberellic acid also causes conspicuous morphological changes upon external application to various plants. In epicotyls of a gibberellic acid-responsive dwarf variety of pea (Pisurn sativum L. var Feltham Firs), the extracted XET activity is roughly correlated with elongation growth of epicotyls induced by external application of gibberellic acid (Potter and Fry, 1993). Gibberellic acid promoted elongation of lettuce hypocotyls and increased the extractable XET activity per unit fresh weight of the hypocotyl tissue (Potter and Fry, 1994). In cucumber seedlings, gibberellic acid evoked prolonged promotion of elongation over a few days, but evoked a small increase in XET activity on a fresh weight basis. In azuki bean epicotyl sections, mRNA levels of EXGT-Vl (formerly azuki bean EXT) as estimated by RNA gel blot analyses were slightly upregulated by gibberellic acid as well as by auxin (E. Tomita et al., unpublished data). These two hormones were shown to stimulate cell wall deposition in these sections (Hogetsu et al., 1974). In azuki bean epicotyls, gibberellic acid stimulates cell wall synthesis not only in young growing tissues but also in older regions of epicotyls where secondary walls are actively deposited (Nishitani and Masuda, 1982b). These lines of circumstantial evidence imply a rough correlation between gibberellic acid-stimulated cell wall synthesis and the expression of some members of XRP, including EXG T-V1.

C. Environmental Signals A member of the XRP family (wus11005)was isolated from maize as a flooding-induced gene (Peschke and Sachs, 1994; Saab and Sachs, 1995). The level of mRNA hybridizing to this clone increased in shoots of maize seedlings subjected to hypoxic stress. Increase of the mRNA level began within 6 h and continued until 72 h. Other abiological stresses, such as heat shock at 40°C and watering with 0.05 N hydrochloric acid or 5 M sodium chloride, did not increase the mRNA level. In Arabidopsis plants, mechanical stimuli, such as touch, water spray, and wind, caused the development of shorter petioles and bolts. These growth responses are known as thigmomorphogenesis and are considered to be mediated, at least partly, by a calcium-mediated transduction of signals from the mechanical stimuli (Braam and Davis, 1990). TCH4 gene was isolated as an Arabidopsis gene that responds rapidly to touch and other

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physical stimuli (Braam, 1992). Because external application of calcium or heat shock also causes a significant and rapid increase in its mRNA level in the seedlings, this gene expression is considered to be regulated via a calcium-mediated signal transduction pathway (Braam, 1992). Some growth supression caused by hypoxic conditions and mechanical stresses are closely associated with a shift in the cell wall metabolism from a primary wall-directed one to the secondary wall deposition type of wall construction. The touch-induced TCH4 and the flooding-induced wus12005 might be involved in the wall rearrangement required for such a secondary wall thickening. Although higher levels of mRNAs for TCH4 and meri-5 were observed in dark-grown Arabidopsis seedlings than in light-brown ones, there is no evidence that light directly regulates expression of XRP members (Xu et al., 1995, 1996).

VI. Overview of Cell Wall Construction during Plant Growth and Development: A Hypothetical Scheme Figure 10 visualizes the hypothetical unit processes involved in rearrangement of the cellulose-xyloglucan framework structure during morphological changes in plant cell walls, including those processes leading to construction and extension of the primary walls, deposition of secondary walls, and wall degradation.

A. Cleavage of Load-Bearing Cross-Links Simple cleavage of load-bearing xyloglucan cross-links will lead to increased mobility of microfibrils within the cell wall. This cleavage might be mediated by either hydrolase or endoxyloglucan transferase. Xyloglucan-specific hydrolase, such as endo-P-1,4-glucanases and some members of XRP in subfamily 111, can split xyloglucan cross-links by simple hydrolysis (Fig. 10, A). On the other hand, XRP members in subfamilies I and I1 can cleave xyloglucan cross-links by transferring a split end of the xyloglucan crosslink to a free xyloglucan oligomer (Fig. 10, A). Complete cleavage of cross-links between cellulose microfibrils will result in disintegration of the cell wall framework, a process that seldom occurs in the lifetime of a plant except for special cell wall degradation during fruit ripening, abscission, and the storage degradation in seed germination. When auxin causes cell expansion of tissue sections floating in incubation medium without any substrate for wall synthesis, the average M,of xyloglu-

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c2

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FIG. 10 Unit processes required for rearrangement of the cellulose-xyloglucan framework. (A) Hydrolase-mediated simple cleavage of xyloglucan cross-links by action of endo 0-1.4endoglucanase or XRP subfamily I11 (hydrolase). (B) EXGT-mediated cleavage of xyloglucan cross-links by transglycosylation between a free xyloglucan molecule and a cross-linking xyloglucan molecule. This reaction can be catalyzed by XRP members in subfamilies I and 11. (C1 and C2) EXGT-mediated molecular grafting. The interchange o r molecular grafting between xyloglucan cross-links is catalyzed by the action of XRP members in subfamilies I and 11. This reaction can mediate wall synthesis as well as rearrangement of the cell wall framework. which is required for cell wall deposition in both the primary and secondary wall depositions. (D) Hydrogen-bonding-mediated loosening of the cellulose-xyloglucan framework. A hypothetical process that leads to repeated disruption and reformation of hydrogen bonding between xyloglucans and cellulose will result in displacement of the spatial arrangement of cellulose microtibrils. CMF, cellulose microtibrils: XG, xyloglucan; circles at the end of xyloglucans indicate nonreducing termini.

can decreases, indicating partial splitting of xyloglucan cross-links during cell wall expansion (Nishitani and Masuda, 1981, 1982a, 1983). The transferase-mediated cleavage of xyloglucans (Fig. 10, A, bottom) can be stimulated by increasing concentrations of soluble xyloglucan polymers or oligomers, which serve as acceptor molecules for the transglycosylation. Farkas and Maclachlan (1988; Farkas et al., 1992) observed that xyloglucan nonasaccharide at 0.2 mM apparently enhanced by several fold the hydrolytic activity of the xyloglucan-specific endo-/3-1,4-glucanase preparation derived from pea and nasturtium. In these reactions, xyloglucan oligosaccharides serve as acceptor molecules. The endo-type transglycosylation produces xyloglucan molecules with M, = (M, of donor xyloglucan + M , of acceptor xyloglucan)/2. As described previously, auxin increases the concentrations of water-soluble xyloglucan oligomers in the free space of

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pea epicotyls (Terry and Jones, 1981). The auxin-induced liberation of xyloglucan oligomers in the cell wall space would lead to acceleration of the XRP-mediated degradation of xyloglucans. Thus, hydrolase and transferase acting toward xyloglucans can interact synergistically to accelerate xyloglucan degradation. Although cleavage of load-bearing xyloglucans in the cell wall framework can increase the mobility of cellulose microfibrils to allow cell wall expansion due to turgor pressure, their action alone cannot cause rearrangement of the wall architecture leading to cell wall synthesis. As a matter of fact, in intact stem tissues, in which cell wall materials are continuously synthesized, no decrease in M , of xyloglucan is observed during stem growth (Nishitani and Masuda, 1980; Wakabayashi et al., 1993).

B. EXGT-Mediated Molecular Grafting Both splitting and reconnection of the interconnections between cellulose microfibrils must be required for spatial rearrangement of cellulose microfibrils, which occurs continuously during cell wall deposition irrespective of the cell type (Fig. 10, C). The interchange or molecular grafting between load-bearing xyloglucan cross-links could only be achieved by the action of XRP members in subfamilies I and 11. Although our understanding of the mechanism by which cellulose microfibrils are polymerized and crystallized is still poor, a common assumption is that the microfibrils are crystallized at the terminal complex located on the plasma membrane, whereas xyloglucans are polymerized in the Golgi apparatus and are secreted into the cell wall space (White et al., 1993; Brummell et al., 1990). Presumably, at the surface of the plasma membrane, xyloglucans are adsorbed to and intercalated into the cellulose microfibrils by means of still unknown mechanisms to form a cellulose-xyloglucan complex. Conceptually, the XRPmediated molecular grafting reaction makes possible the integration of the hypothetical cellulose-xyloglucan complex into the preexisting framework structure (Fig. 10, C). The empirical demonstration and characterization of this hypothetical process in terms of molecular interactions will be one of the most important steps for a full understanding of the cell wall construction process and, hence, of cell growth and differentiation. In Arabidopsis, there exists at least 10 XRPs with potentially different enzymatic functions and different expression profiles with respect to expression tissues specificity and responsiveness to hormones and other signals. Meri-5 is preferentially expressed in meristematic tissues in which primary wall construction and wall expansion predominate. O n the other hand, EXGT-A1 (formerly Arabidopsis E X T ) and TCH4 are predominantly expressed in tissues with massive deposition of secondary walls as well as primary walls. These facts strongly suggest different physiological roles

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for each tissue. However, little is known about the differential enzymatic functions of XRP members in terms of catalytic activity. Further investigation using purified enzyme as well as mutants with impaired activity of a single XRP member is needed to understand the divergent roles of individual XRP members in the cell wall construction.

C. Disruption of Interactions between Xyloglucans and Cellulose Microfibrils A new family of cell wall proteins, termed expansins, with the potential for altering the mechanical properties of the cellulose-xyloglucan framework was identified by Cosgrove (1989) in the early 1990s through investigation of an enzyme activity capable of inducing wall creep or long-term extension of frozen-thawed wall specimens. Expansins purified from cucumber (Cucumis sativum) hypocotyls catalyze the extension, in v i m , of isolated cell wall specimens under tension at acid pH (McQueen-Mason et af., 1992). The highly purified expansin fractions do not exhibit either XET activity or hydrolase activity toward cell wall components (McQueen-Mason et al., 1993). Rheological analyses show that these proteins reduced the mechanical strength of filter paper, which is essentially composed of pure cellulose, but did not exhibit any detectable cellulase activity. Because the mechanical strength of paper is chiefly due to hydrogen bondings between cellulose microfibrils, this suggests that expansin action involves the disruption of hydrogen bonding between cellulose microfibrils. Furthermore, expansinmediated wall extension was increased by concentrated urea solution, which weakens hydrogen bonding between wall polymers. On the other hand, the expansin action was reduced in solution in which water was replaced with deuterated water, which strengthens hydrogen bonds (McQueenMason and Cosgrove, 1994). This line of evidence strongly suggests that in growing plant cells, expansins catalyze the disruption of hydrogen bonding between cellulose microfibrils and other matrix polysaccharides and thereby mediate slippage between these macromolecules. Expansins have been shown to bind weakly to crystalline cellulose, with the binding being greatly increased by unknown component of cell wall matrix polymers. Clostridium celulovorans produces cellulose-binding protein A (Shoseyov et al., 1992). The cellulose-binding domain (CBD) of this protein has strong affinity to cellulose (Goldstein et al., 1993). Shoseyov (1995) has shown that CBD drastically affects growth of pollen tubes and root hairs. Although such a cellulose-binding protein has not yet been isolated from plants, these findings imply the presence of modulators involved in regulation of the cellulose-xyloglucan interaction and, hence, an alternative mo-

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lecular process by which cellulose microfibrils are rearranged via displacement of xyloglucans along the cellulose microfibrils.

VII. Concluding Remarks The wall expansion process is apparently achieved by rearrangement of the cell wall architecture, which includes integration of new wall material into the preexisting framework structure. Because only hydrolase was known to be present in the cell wall, the process responsible for the splitting and rejoining of the cross-links between microfibrils constituted an intricate puzzle until the discovery of endo-type xyloglucan transferases (EXGT and XET) in the cell wall space. Currently, the rearrangement of the xyloglucan cross-links in the cell wall framework can be explained by the action of two categories of enzymes: (i) xyloglucan-specific hydrolase capable of cleaving cross-links and (ii) endoxyloglucan transferase capable of mediating both splitting and reconnection of cross-links among cellulose microfibrils. The discovery of XRPs disclosed that both types of enzymes belong to a single multigene family, a finding with profound implications for the evolutionary traits of the cellulose-xyloglucan framework in plants. In Arabidopsis at least 10members of XRP have so far been identified, indicating a fairly large size of this gene family. Characterization of the mode of enzymatic actions of individual XRP members and their implications for the physiological roles in specific plant tissues is also a prerequisite for a complete understanding of the mechanism of the cell wall expansion in plants. The mechanism by which each organism constructs its shape through a series of complicated but specific developmental processes has long been a basic theme in biology. Remarkable progress during this decade in elucidating genes for receptors and transfactors that govern plant morphogenesis has shed light on the molecular mechanism underlying the fundamental process for plant development. On the other hand, the final steps in morphogenesis in plants are expressed via the construction processes of the cell wall, which determines the shape of the plant. However, there still is a gap or missing link in the transduction pathways between the upstream genes and the final steps that are directly responsible for the construction processes during plant growth and morphogenesis. There is considerable evidence that members of the XRP family serve as key enzymes in a wide spectrum of cell wall construction processes and thereby play roles in the final steps of morphogenesis in plants. It is noteworthy that gene expression of some members of this gene family is upregulated by auxin and/or brassinosteroids, two major classes of phyto-

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hormones regulating the cell wall expansion process. Furthermore, expression of some members of this family is controlled by environmental signals, such as mechanical stress and light conditions, which affect patterns of morphogenesis. Arabidopsis mutants deficient in the brassinosteroid biosynthetic pathway werc recently identified. These mutants include cbbl and cbb3 (Kauschmann et al., 1996), cpd (Szekeres et al., 1996), and det 2 (Li et al., 1996). All these mutants display reduced growth such as dwarfism. Several transgenic plants and mutants with altered auxin levels and aberrant sensitivity to auxin have long been isolated and characterized (Hobbie and Estelle, 1994; Reid and Ross, 1993). This means that searching the signal transduction pathway involved in the signal-mediated XRP gene expression may enable us to elucidate the hidden molecular processes that intervene between the signal perception and the plant response expressed as cell wall modification. Isolation of mutants impaired in individual XRP members is necessary for a complete understanding of all the physiological functions of the XRP family in plants.

Acknowledgments 1 am grateful to A. B. Bennett. G . P. Bolwell, N. C. Carpita. A. G. Darvill, S. C. Fry, T. Hoson. J . M. Lahavitch. G. A. Maclachlan. K. Roherts. L. D. Talbott, and 0. Shoseyov for providing reprints and preprints. Thanks are due to my colleagues Shigehisa Okamoto and Etuko Tomita, Kagoshima University, for their invaluahle discussions. This work was supported by Grants-in-Aid for Scientific Research on Priority Areas (No. 05276103) and (B) (No. 07454220) from the Ministry of Education. Science. Sports and Culture, Japan, and JSPSRFTF96L00403 from The Japan Society for the Promotion of Science.

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Microtubule-Microfilament Synergy in the Cytoskeleton R. H. Gavin Department of Biology, Brooklyn College of the City University of New York, Brooklyn, New York 11210

This review describes examples of structural and functional synergy of the microtubule and actin filament cytoskeleton. An analysis of basal body (centriole)-associatedfibrillar networks includes studies of ciliated epithelium, neurosensory epithelium, centrosomes, and ciliated protozoa. Microtubule and actin filament interactions in cell division and development are illustrated by centrosome motility, cleavage furrow positioning, centriole migration, nuclear migration, dynamics in the phragmoplast, growth cone motility, syncytial organization, and ring canals. Model systems currently used for studies on organelle transport are described in relation to mitochondria1transport in axons and vesicular transport in polarized epithelium. Evidence that both anterograde and retrograde motors are associated with one organelle is also discussed. The final section reviews proteins that bind both microtubules and actin filaments and are possible regulators of microtubule-microfilament interactions. Regulatory roles for posttranslational modifications,microtubule and microfilament dynamics, and multisubunit complexes are considered. KEY WORDS: Actin microfilament, Centriole, Basal body, Growth cones, MAPS, Microtubules, Molecular motors.

1. Introduction

During the past two decades, refinements in both transmission and scanning electron microscopy techniques and advances in video-enhanced optical microscopy have contributed to our understanding of the cytoskeleton as a highly cross-linked network in which actin filaments form cross-links with microtubules (MTs) and intermediate filaments to integrate cytoskeletal Inremarional Review of Cytology. Vol. 173 0074-7696/97 $ 2 S . N

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organization. Numerous studies (e.g., Schliwa and van Blerkom, 1981;Hartwig et al., 1980) were instrumental in defining the cytoskeleton as an interlocking network. There is convincing evidence (Verkhovsky and Borisy, 1993) that myosin filaments are also integral components of the cytoskeleton. The structural and functional interaction of these various fibrillar systems is the focus of intense investigation in cell biology. This review describes a diverse group of selected examples that illustrate structural and functional synergy of the MT and actin filament cytoskeleton. The review does not comprehensively treat the literature on MTs, actin filaments, and their respective motors. Several excellent reviews on these topics have been published in recent years and have been cited in the text. Three criteria were used to evaluate examples for inclusion in this review: (i) physical closeness of the two cytoskeletal elements to one another within a structure or a group of structures as judged by electron microscopy or by video-enhanced optical microscopy, (ii) interdependence of the two networks in a structure or function relationship as demonstrated by pertubation of the systems with pharmacological agents, antibodies, or other sitespecific agents, and (iii) existence of accessory binding proteins that could function as mediators of MT-microfilament interactions. Because studies on MT-microfilament synergy are in their infancy, many of the examples cited here meet only the first criterion.

II. Basal Body-AssociatedFibrillar Networks Basal bodies and centrioles form structural associations with various cytoskeletal fibrillar complexes and are therefore excellent models for the investigation of interactions between MTs and microfilaments. Examples of centriole- or basal body-associated fibrillar complexes include the pericentriolar material, a major MT organizing center for cytoskeletal microtubules (Brinkley, 1985);the basal foot, a basal body-associated fibrillar complex that organizes both MT and microfilament networks (Reed et al., 1984);and the cage, a fibrillar chamber that encloses the basal body cylinder in the ciliate cytoskeleton (Williams, 1986; Hoey and Gavin, 1992). This chapter describes basal body-associated fibrillar networks in ciliated epithelium, neurosensory epithelium, and ciliated protozoa.

A. Basal Foot in Ciliated Epithelium The apical cortex of ciliated epithelial cells contains numerous ciliated basal bodies interconnected by associated fibrillar attachments. Basal bodies and

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associated fibrillar systems in these cells have been the focus of several studies including a detailed ultrastructural analysis of ciliated epithelial cells in freshwater mussel gill (Reed et al., 1984). In gill epithelium, each basal body possesses a cilium at its distal end. A dense tapering structure, called the basal foot, projects from the side of the basal body at its proximal end and is attached to a network of MTs and microfilaments. Serial sections showed that MTs and microfilaments originate at the basal foot, which could function as a nucleating site for both MTs and microfilaments (Reed et al., 1984). Immunofluorescence microscopy and immunogold electron microscopy were used to localize myosin to the apical pole of ciliated epithelial cells in culture (Klotz et al., 1986). This ultrastructural analysis showed that anti-myosin antibodies labeled a fibrillar complex that is attached to the basal foot. The interlocking network of MTs and microfilaments in the apical cortex of epithelial cells could provide positional information and integrate the response of the cell surface to physiological stimuli (Reed et al., 1984). The demonstration of myosin in the apical cortex raises the possibility of myosin-powered transport or contractile events in the apical region. Microinjection of antibodies could be used to further analyze the role of actin microfilaments and myosin in the apical cortex.

6.Neurosensory Epithelium 1. Photoreceptor Cells The mammalian retina consists of several cellular layers surrounded by a layer of pigmented epithelium. The inner side of the epithelial layer contains the light-sensitive processes of photoreceptor cells. The nomenclature for these cells is derived from their shape. Rods are slender, narrow projections, whereas cones are broad and tapering. Photoreceptor cells are differentiated into an inner segment, which contains mitochondria and other metabolic machinery, and an outer segment consisting of a series of flattened membranous disks formed from the plasma membrane. The outer segment is connected to the inner segment by a nonmotile cilium with a 9+0 array of MTs anchored to a basal body. Within the cilium, actin filaments project between MT doublets with their minus ends near the lumen of the axoneme and their plus ends at the plasma membrane (Arikawa and Williams, 1989). Anti-myosin antibodies localized to the actin domains within the connecting cilium (Williams et al., 1992). New disk membranes are formed at the connecting cilium by evagination of the ciliary membrane (Arikawa et al., 1992). Williams et al. (1992) suggested that the cytoskeleton of the connecting cilium plays a role in disk morphogenesis. These authors proposed that actin and myosin constitute a contractile sys-

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tem within the ciliary cytoskeleton and force generated by this system could be exerted on the ciliary plasma membrane and result in membrane protrusions.

2. Cochlea Hair Cells

The organ of Corti is a spirally arranged band of epithelial cells in the mammalian cochlea. These sensory epithelial cells, known as hair cells, contain many long microvilli that consist of actin filament bundles. In the early phases of cochlea development, a single cilium is associated with hair cells. Later in the developmental process, the cilium disappears, although the basal body remains. Unconventional myosins have been localized to hair cells and proposed as modulators of ion channels in neurosensory epithelium (Gillespie et al., 1993; Assad and Corey, 1992; Hudspeth and Gillespie, 1994; Solc et al., 1994). For a recent review of these myosins, the reader is referred to Bahler (1996).

3. Microtubule-Microfilament Interactions Weil et al. (1995) proposed that myosin VII mediates MT-microfilament interactions in neurosensory epithelial cells. The focal point for these interactions would be the basal bodylcilium in photorecetor cells and in the cochlea. Several observations are consistent with this proposal. Defects in myosin genes have been correlated with abnormalities in the mammalian retina and inner ear. In humans, Usher syndrome type I B is characterized by hearing loss, vestibular dysfunction, and retinitis pigmentosa. Usher syndrome patients exhibit abnormalities in the organization of the nonmotile ciliary axoneme in photoreceptor cells and myosin VII gene defects that include deletions, missense mutations, and premature stop codons (Weil et al., 1995). Many individuals with genetic deafness show abnormalities of the inner ear neurosensory epithelium and a degeneration of the organ of Corti (Weil et al., 1995). Hearing impaired mouse mutants show similar defects in the neurosensory epithelium. One of the mouse mutants known as shaker exhibits head tossing and circling due to vestibular dysfunction and cochlear defects. The shaker gene encodes a myosin VII, and in shaker mutants defects in the head domain of myosin VII have been identified (Gibson et al., 1995). Future studies with these two genetic systems will undoubtedly define the interactive roles of MTs actin filaments, and myosins in neurosensory mechanisms.

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C. Ciliated Protozoa 1. Proximal Structures of the Basal Body The ciliate cytoskeleton contains hundreds of ciliated basal bodies interconnected by fibrillar networks that make it ideally suited for investigations that focus on the synergic action of MTs and microfilaments. In ciliates such as Tetrahymena or Paramecium, cortical basal bodies are arranged in longitudinal rows and in interconnected groups that form an anterior feeding complex known as the oral apparatus. The organization of basal bodyassociated fibrillar complexes in the Tetrahymena cytoskeleton has been extensively investigated. Basal bodies within the oral apparatus are interconnected by a network of MTs and microfilaments that are attached to a dense fibrous structure at the proximal end of each basal body (Gavin, 1977;Fig. 1A). The fibrillar nature of the dense proximal structure indicates that it could be a nucleating site for both MTs and microfilaments. The localization of centrosomal antigens to proximal fibrillar structures of basal bodies in the ciliate cytoskeleton is a further indication that these structures could have nucleating activity. In Terruhymenu, immunofluorescence microscopy revealed the localization of y-tubulin and pericentrin to basal bodies of the oral apparatus (Stearns and Kirschner, 1994), and in Paramecium immunogold electron microscopy was used to localize human centrosoma1 antigens to a fibrillar network at the proximal end of oral apparatus basal bodies (Keryer et al., 1990).

2. Basal Body Cage: A Microtubule-Microfilament Complex Each basal body within the Tetrahymena oral apparatus is contained within a separate, filamentous cage that is connected to basal body MTs by a meshwork of microfilaments as illustrated in Fig. 1A. Immunofluorescence microscopy and immunogold electron microscopy revealed the localization of actin to the network of filaments that connect basal body triplet MTs with the filamentous cage wall (Hoey and Gavin, 1992). Because actin and myosin can interact to form a contractile system, the presence of one of these components in a subcellular location invariably leads to speculation about the presence of the other. Recently, Garces and Gavin (1995) provided the first identification of myosins in Tetrahymena by employing biochemical, immunochemical, and polymerase chain reaction (PCR) approaches. A 180-kDa Tetrahymena cytoskeletal polypeptide (p180) was identified as a myosin heavy chain based on reactivity with an anti-myosin antibody, ATPase activity, and ATP-dependent binding to actin filaments. The p180 has been colocalized with actin to cage filaments that connect basal body MTs with the cage wall (GarcCs e f al., 1995).

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FIG. 1 Electron micrographs of basal bodies. (A) A longitudinal section through a row of basal bodies in the oral region cortical cytoskeleton of Tetruhymena. Each basal body is contained within a separate filamentous cage that is connected to basal body microtubules by a meshwork of acin microfilaments (arrow). Note the dense filamentous base of the cage and the associated accessory microtubules observed in transverse section at the bottom left side of the micrograph. Bar-0.23 pm. (B)Immunogold staining with an affinity-purified, antimyosin heavy chain antibody. The secondary antibody was anti-IgG linked to 15 nm colloidal gold particles. The left arrow identifies a region where most of the basal body is not in the plane of section. However, a cluster of intracage filaments that connect cage wall with the basal body microtubules is clearly visible and heavily labeled with colloidal gold particles. The right arrow locates another region where the basal body wall is not in the plane of section. However, the cage wall and its base are clearly visible and labeled with colloidal gold particles. The micrograph in A is of a comparable section stained with preadsorbed anti-myosin heavy chain antibody followed by IgG secondary antibody conjugated to 15 nm colloidal gold particles.

PCR was used to search for gene sequences that code for myosins in Tetrahymena (GarcCs and Gavin, 1995). Conserved amino acid motifs in the N-terminus head domain in all known myosins were used to design slightly degenerate PCR primers for the amplification of Tetrahyrnena genomic DNA. Sequencing of a 765-bp PCR product revealed extensive predicted amino acid sequence homology with unconventional myosins VII and VIII. Based on this analysis, it was concluded that the 765-bp PCR

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product is a fragment of the first myosin gene to be discovered in ciliated protozoa, and the name TETMYO-1 is proposed for this new myosin (J. GarcCs and R. Gavin, manuscript in preparation). Studies that will identify the protein encoded by TETMYO-1 and the relationship between TETMYO-1 and p180 are in progress (J. GarcCs and R. Gavin, personal communication). Gene fragment-mediated genomic knockout could be used to determine whether TETMYO-1 is an essential or nonessential gene (Gaertig and Gorovsky, 1995).

3. Ciliary Motility Interactions between basal bodies and microfilament complexes could play an important role in controlling the direction of ciliary motility as suggested more than three decades ago by Gibbons (1961) and subsequently by Dirksen and Satir (1972). Changes in direction of ciliary movement are evident from observations of ciliated protozoa that reveal that the organisms are not limited to movement in only one direction. Although much of the current research focuses on the central pair MTs as a possible regulator of the ciliary bend (Smith and Sale, 1994), the localization of actin and myosin to basal body-associated fibrillar complexes provides the basis for at least two models that define a possible role for the basal body in the regulation of ciliary motility. In a model proposed by GarcCs et al. (1995), changes in the direction of ciliary movement could be induced by myosin-powered contraction of basal body-associated actin filaments that could alter the spatial positioning of the basal body. Although there is no direct evidence for basal body reorientation in ciliates, studies on an algal cell have demonstrated that contraction of basal body-associated centrin fibers (Salisbury, 1995) reorients basal bodies and flagella during a photophobic response (McFadden er al., 1987). Another model is based on a proposed role for myosin in modulating ion channels (Bahler, 1996). Myosin-mediated shifts within a basal body-associated fibrillar complex, for example, the cage, could modulate ciliary membrane ion channels and induce changes in ciliary motion. Although these proposed functions for actin and myosin are hypothetical at this point, the existence of these two contractile proteins in Tetruhymena provides a genetic model for dissecting their function through the use of gene knockouts (Gaertig and Gorovsky, 1995). Basal bodyassociated fibrillar complexes as regulators of ciliary motility define a potentially pivotal role for the basal body and for MT-microfilament interactions in cell motility. The implications go well beyond motility in ciliated protozoa. Ciliated basal bodies with associated fibrillar complexes are present in many vertebrate tissues, e.g., tracheal epithelium, and ciliary motion is integral to their physiology.

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D. Centrosomes An actin homolog, centractin, has been cloned, sequenced, and localized to the centrosome, an interphase MT organizing center (MTOC) in most eukaryotic cells (Clark and Meyer, 1992). To further explore the relationship between centractin and MTs, fibroblasts were treated with colcemid, which depolymerized cytoskeletal MTs but left intact the centriolar cylinders. In colcemid-treated cells, anti-centractin antibody labeled the centrosomes, an indication that maintenance of the MT cytoskeleton is not required for the localization of centractin to centrosomes (Clark and Meyer, 1992). It is unclear whether centractin localizes to the pericentriolar material or is associated with the centriole cylinders. Centractin in the centrosome complex, through its affinity for actin-binding proteins, could link the actin cytoskeleton with the MT cytoskeleton.

111. Microtubule-Microfilament Interactions in Cell Division and Development

A. Centrosome Movement Centrosome movement is an example of the dynamic interplay between MT and actin filament networks. A role for the MT and actin cytoskeleton in centrosome motility was demonstrated by treating human leucocytes with a tumor-promoter drug 12-0-tetradecanoylphorbol-13-acetate(TPA) (Euteneur and Schliwa, 1985). In drug-treated cells, the two centrosomal centrioles, each with surrounding astral MTs, separated by a distance of several micrometers. Centrosome splitting was inhibited when cells were treated with nocodazole prior to treatment with TPA, an indication that intact MTs are required for the drug-induced centrosome splitting and that the force required for splitting is probably exerted on the MTs. Similarly, disruption of the actin cytoskeleton with cytochalasin inhibited TPAinduced centrosome splitting. The influence of actin organization on centrosome migration is demonstrated by meiosis in Drosophila. Centrosomes migrate to the nuclear membrane where they nucleate astral MTs at the onset of the first meiotic division of spermatocytes. Subsequently, the two asters separate and migrate to positions opposite one another on either side of the nucleus close to the nuclear membrane. Mutations in twinstar, a Drosophila gene that encodes a homolog of the actin filament-severing protein cofilin, resulted in defective centrosome migration (Gunsalus et al., 1995). In mutant pheno-

215 types, the two asters failed to associate with the nuclear membrane and were delayed in their migration to opposite poles (Gunsalus et al., 1995). MICROTUBULE-MICROFILAMENT SYNERGY

B. Cortical Movements in Development In several motility and developmental systems, there is continual and directed flow of material adjacent to the plasma membrane. This movement of material is referred to as cortical flow and is important for the correct positioning of morphogenetic determinants during development. Direction of cortical flow reflects the distribution of actin microfilaments within the cytoskeleton. In the first cell cycle of Caenorhabdiris, a transient cleavage furrow forms. During this interval, known as pseudocleavage, actindependent contractions in the anterior region of the cortex result in cortical flow toward the posterior end of the egg (Hird and White, 1993). Experiments with nocodazole suggest that spindle orientation affects the distribution of the actin cytoskeleton in Caenorhabditis embryos. Nocodazole-induced pertubation of spindle location resulted in changes in cortical actin distribution and corresponding changes in the polarity of cortical flow (Hird and White, 1993). The spindle interzone, the region of the anaphase spindle between the separating chromosomes, is important for mediating the interaction between spindle and cortex. In studies of mitosis in a rat cell line, the midanaphase spindle interzone was distinguished by its overlapping MTs that formed dense “stem bodies” (Katsumoto et al., 1993). Microtubules from the stem bodies were in close association with actin filaments in the equatorial region of the cell at the initiation site of the cleavage furrow. These MTs remained located at the cell equator as the contractile ring filaments accumulated in a manner that suggested that MTs might act to trigger actin filament accumulation. In many dividing cells, movement of surface receptors is coupled to the mobility of the actin cortex. These receptors can be labeled and tracked with fluorescent particles or latex beads during cell division (Wang et al., 1994). Organized movement of surface-bound beads was not detected until the onset of anaphase and the formation of the spindle interzone where movement was most active in contrast to the poles where movement was random. Surface receptor movement was inhibited by cytochalasin, an indication that the cortical activity is actin-based. Interactive positioning of the actin and MT cytoskeleton plays a role in the maintenance of syncytial organization during Drosophila embryogenesis. In the early development of the Drosophila embryo, a syncytial monolayer of cortical nuclei undergoes several rounds of synchronous division. Fidelity of nuclear divisions within the monolayer is maintained by actin-based,

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transient membrane invaginations that separate adjacent spindles (Postner et al., 1992). Myosin 95F, a class VI myosin, powers the redistribution of cortical particles to the actin-based membrane invaginations (Mermall et al., 1994). Inhibition of myosin 95F activity by microinjection of anti-myosin 95F antibodies resulted in the failure of the cortical particles to associate with the transient furrows, which did not form properly (Mermall and Miller, 1995). The membrane invaginations in antibody-injected cells did not extend to the depth of the mitotic spindle and, consequently, MTs from one spindle encroached upon neighboring spindle domains, which resulted in fusion of nuclei and eventual disorganization of the blastoderm. C. Spindle Orientation Determines Cleavage Furrow Position

That cleavage furrow position is determined by spindle orientation is a long-established tenet of developmental biology (Strome, 1993).The cleavage plane is always established between the spindle poles, and pertubation of spindle location results in an altered location of the cleavage furrow. In Caenorhabditis embryos, rotation of the centrosome alters spindle orientation and creates a new division plane (Hyman and White,1987; Hyman,1989). Studies of Xenopus development suggest a role for the spindle interzone in determining the location of cleavage furrows. Progression of the cleavage furrow is known to coincide with a wave of high calcium concentration. Injection of calcium buffers into Xenopus eggs demonstrated that a high intracellular calcium concentration is required for cleavage furrow induction, maintenance, and extension (Miller et al., 1993). Injection of calcium buffers induced eccentric furrows located along a meridian through the animal pole in a manner suggesting that the signal for furrow induction derives from an expanding plate or disc such as the extension of the metaphase plate known as the diastema (Miller et al., 1993). The diastema region appears to be comparable to the “stem bodies” described by Katsumoto et al. (1993). Thus, cleavage furrow position was related to the spindle interzone rather than to the asters. D. Centriole Migration

Centriole (basal body) migration and macrocilia formation have been studied in the ctenophore Beroe^ (Tamm and Tamm, 1988). Macrocilia are compound ciliary organelles that contain several hundred axonemes enclosed within a common membrane. Basal bodies of the macrocilia arise in close association with dense fibrogranular bodies and develop a long

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striated rootlet at one end. The basal body-rootlet complex, while attached to actin filaments, migrates to the cell surface. Actin filaments were observed behind the migrating basal bodies but not ahead of them. Tamm and Tamm (1988) proposed that basal bodies are propelled toward the cell surface by the oriented assembly of actin filaments. In order to be consistent with the polymerization-driven model, the attached actin filaments would have uniform polarity with their plus ends attached to the centriole complex. It is unclear how polymerization could be accomplished while the filaments are still attached to the centriolar complex. A role for actin polymerization in driving the forward motility of structures has been extensively discussed by Mitchison and Cramer (1996). During ciliogenesis in epithelial cells, newly formed basal bodies migrate to the cell surface where cilia elongation occurs. Immunofluorescence microscopy and immunogold electron microscopy have been used to localize myosin to the fibrillar complexes that connect to basal bodies (Klotz et aL, 1986; Lemullois et af., 1987) and provide tentative support for a contractile system that could power the migration of basal bodies to the cell surface.

E. Spindle Positioning and Cytokinesis in Plant Cells During interphase, MTs are arranged in cortical networks linked to the plasma membrane. At the onset of M-phase, major cytoskeletal reorganizations occur. An array of MTs, microfilaments, and their associated proteins emerges at a cortical site that predicts the future division plane. This MTmicrofilament complex, known as the preprophase band (PPB), is initially a broad band of MTs oriented transversely to the long axis of the cell (Liu and Palevitz, 1992). The PPB is formed from a rearrangement of existing cortical MTs (Eleftheriou and Palevitz, 1992) or by polymerization of new MTs (Cleary et al., 1992). Actin microfilaments, which are either recruited from the cortical microfilament array or polymerized de novo, colocalize with MTs in the PPB (Cleary et af., 1992). As PPB MTs become more densely packed, the PPB becomes narrower. Interaction of the MT and microfilament arrays within the PPB is illustrated by experiments that showed that PPB MTs did not become densely packed in the presence of cytochalasin (Eleftheriou and Palevitz, 1992), and disruption of PPB MTs with colchicine resulted in the loss of cortical microfilaments (Mineyuki and Palevitz, 1990). The transient PPB disintegrates prior to nuclear envelope breakdown, and the cortical interzone once occupied by the PPB is subsequently “recognized” by an expanding cell plate that fuses with the plasma membrane to form a new cell wall. The cell plate develops within the interzone from a structure known as the phragmoplast that consists of vesicles for cell wall

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formation, MTs, and microfilaments (Staiger and Lloyd, 1991; Staehlin and Hepler, 1996). Development of the phragmoplast was studied by injecting fluorescein-labeled tubulin and rhodamine-labeled phalloidin into stamen hair cells of Tradescantia in order to monitor MT and microfilament dynamics with confocal imaging (Zhang et al., 1993). During phragmoplast development, interzone MTs associate laterally to form a bundle of MTs that align parallel to the long axis of the cell with their plus ends overlapping each other in the interzone (Zhang et a!., 1993; Staehlin and Hepler, 1996). Phragmoplast microfilaments arise de novo in late anaphase and are oriented parallel to the phragmoplast MTs on either side of the developing nuclei but are not present in the interzone (Zhang et al., 1993). HMM decoration of isolated phragmoplasts showed that most of the microfilaments are of uniform polarity with their plus ends oriented toward the cell plate (Kakimoto and Shibaoka, 1988 ) as would be expected if the filaments are tracks for the translocation of myosin coated vesicles toward the cell plate. As development proceeds, MT depolymerization results in a decrease in the length of the initial phragmoplast cluster of MTs, but continued addition of new MTs expands the girth of the cluster so that it reaches the cell perimeter (Zhang et al., 1993). Within the expanded phragmoplast, MTs in the central interzone depolymerize, whereas phragmoplast MTs at the perimeter remain tightly packed until complete depolymerization of the phragmoplast occurs at cytokinesis (Zhang et al., 1993). Microfilament dynamics parallels MT dynamics, suggestive of synergistic action. As the phragmoplast cluster of MTs expands toward the periphery, so do the microfilaments, as if both cytoskeletal elements are under coordinate control (Zhang et al., 1993).

F. Interdependence of Microtubule and Actin Filament Arrays Studies of meiotic cells have been useful in defining an interdependence between MT and microfilament arrays. Rhodamine-labeled phalloidin was used to localize actin filaments within and around the meiotic spindle in eggplant cells (Traas et al., 1989). Cytochalasin fragmented preexisting actin fiilament bundles and prevented spindle formation in dividing eggplant cells, which suggests a role for microfilamentsin the organization of spindle MTs. The organization of the MT array in the transition from prophase to metaphase has been studied in maize (Staiger and Cande, 1990, 1991). In wild-type cells, the metaphase spindle converged to form characteristic focused poles. Rhodamine phalloidin staining of actin microfilaments was focused in a small spot at the spindle poles. In mutant cells, the metaphase

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spindle remained divergent and the polar staining of actin microfilaments was broad, indicating a more diverse arrangement of the microfilaments. Other studies illustrate the interdependence of microfilament and MT arrays. In the late stages of cytokinesis in Spirogyra, disruption of the MT array with oryzalin prevented the reorganization of the microfilament array that was normally associated with the completion of the cell wall (Sawitzky and Grolig, 1995). The ban mutants in fission yeast appear to affect both the MT and the actin cytoskeleton (Verde et al., 1995). The MT defects in these mutants include shorter interphase MTs, abnormal bundling of interphase MTs to one side of the cell, and abnormally short mitotic spindles. These mutants also display a smaller number of cortical actin patches, which appear larger in comparison with cortical patches in wild-type cells. Although the ban gene product has not been identified, it appears to have a role in the regulation of both the actin and MT cytoskeleton. Drosophilu bristles are surface projections that provide the organism with tactile and chemosensory information. These structures have been the focus of a recent reexamination (Tilney et al., 1995). The bristle shaft contains a central core of MTs and membrane-associated, cross-linked microfilament bundles. Ultrastructural examination of early postpuparium development revealed tiny bristles that contained MTs but no actin filaments, an indication that the early stages of bristle elongation from the surface can procede without actin filaments. Does the MT cytoskeleton within the bristle shaft orchestrate the positioning of microfilament bundles? The authors promised future studies that would further explore the interaction between MTs and microfilaments in this interesting complex.

G. Nuclear Migration Nuclear migration in plant cells is coordinated with preferential growth at the cell apex (Nagai, 1993; Willamson, 1993). In protonemal cells of the fern Adiantum, MTs and microfilaments connect the nucleus to both the apical and basal cortex. During cell growth, the nucleus maintains a constant distance from the apex. Nuclear-cytoskeletal interactions in these cells were investigated by using rhodamine-phalloidin, anti-tubulin antibodies, colchicine, and cytochalasin B in conjunction with confocal laser microscopy (Kadota and Wada, 1995). Depolymerization of microfilaments by cytochalasin B resulted in the cessation of both apical growth and nuclear movement. Depolymerization of MTs by colchicine partially inhibited apical growth. Nuclear movement in colchicine-treated cells continued but in a basal direction. Simultaneous application of both cytochalasin B and colchicine resulted in the cessation of both tip growth and nuclear movement. The experiments by Kadota and Wada further showed that centrifu-

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gation of protonemal cells resulted in the dislocation of the nucleus to a basal position without disruption of the MT and microfilament connections from nucleus to cell apex. The centrifuged cells recovered, and the nucleus returned to its original apical position. Cytochalasin alone or colchicine alone had no effect on apical-directed movement of the nucleus in centrifuged cells. However, apical-directed nuclear movement in centrifuged cells was inhibited by the simultaneous application of cytochalasin and colchicine. These various experiments show that both MTs and microfilaments are involved in apical-directed movement of the nucleus in fern protonemal cells and that in the presence of agents that disrupt one of these cytoskeletal elements, apical-directed movements continued on the other cytoskeletal element. There is a further indication from these studies that microfilaments are involved in basal-directed nuclear movements. Molecular motors could power these movements. The bidirectionality of the microfilamentassociated movement would require a myosin motor on actin filaments of mixed polarity. The MT-associated nuclear movement suggests a nucleusassociated dynein or kinesin motor and polarized MTs that connect nucleus to cell apex.

H. Cytoplasmic Streaming in Ring Canals In Drosophilu, four incomplete cell divisions of a germline stem cell produce a cluster of 16 cells interconnected by a series of cytoplasmic bridges known as ring canals. One of these cells with four ring canals differentiates into the oocyte, whereas the other 15 cells become nurse cells. Nurse cells and the developing oocyte, interconnected by ring canals and surrounded by follicular epithelium, constitute the Drosophilu egg chamber. The margins of the ring canals contain conspicuous actin bundles (Warn et al., 1985; Riparbelli and Callaini, 1995). Microtubules originate from the MTOC in the prooocyte and extend through ring canals into nurse cells so that a single MT cytoskeleton is formed (Theurkauf et al., 1992, 1993). Cytoplasmic components such as specific mRNAs are translocated through the ring canals during the early stages of oogenesis. A role for egg chamber MTs in the transport of specificmorphogenetic determinants has beeen described (Theurkauf et al., 1992) and is strenghtened by the finding that a Drosophilu gene encoding a cytoplasmic dynein is preferentially transcribed in the nurse cell complex, and the encoded dynein is preferentially accumulated in the oocyte (Li et ul., 1994). In the early stages of egg chamber development, the cortical cytoplasm of the oocyte contains a dense actin network, and microfilament bundles are evident in the nurse cells as revealed by laser scanning confocal microscopy

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(Riparbelli and Callaini, 1995). Late in the vitellogenesis process, there is bulk transfer of cytoplasm from nurse cells to oocyte and subsequent streaming of the ooplasm. Transfer of nurse cell cytoplasm is a cytochalasinsensitive process and thus requires actin filaments (Gutzeit, 1986). Inhibitors of MT assembly prevent ooplasm streaming (Theurkauf et al., 1993). The beginning of bulk cytoplasmic flow from nurse cells to oocyte coincides with drammatic changes in the actin and MT cytoskeleton in both the nurse cells and the oocyte. In nurse cells, actin bundles radiate from the plasma membrane and form a cage around the nucleus (Riparbelli and Callaini, 1995). In the oocyte, the cortical network of actin filaments becomes less dense, and a parallel array of subcortical MTs is formed in a manner suggestive of coordinate control for the two cytoskeletal reorganizations (Riparbelli and Callaini, 1995; Theurkauf, 1994).

I. Growth Cones Mobility of nerve cells is confined to the growth cone, a region at the tips of axons and dendrites. Neurons grow through the extension of axons and dendrites under guidance from the growth cone, which contains a wide variety of signal receptor molecules (Dodd and Schuchardt, 1995; Tanaka and Sabry, 1995). Growth cones move and sense their environment through surface protrusions known as filipodia and lamellipodia. Filipodia have a spike-like morphology and contain cross-linked bundles of actin filaments in contrast to the web-like lamellipodia, which are filled with a meshwork of 40- to 100-nm-wide actin filament bundles and branching actin filaments (Lewis and Bridgman, 1992). Although most of the actin filaments have their plus ends toward the leading edge of the growth cone, some filaments have their minus ends toward the leading edge (Lewis and Bridgman, 1992). Growth cone MTs have their plus ends oriented toward the leading edge of the growth cone (Heidemann et al., 1981) and are largely confined to the central region of the structure (Lin and Forscher, 1993). As growth cones explore the extracellular environment, lamellipodia extend and contract with extensive changes in both the actin and MT cytoskeletons. Actin polymerization occurs at the lamellar leading edge while actin filament disassembly takes place at the center of the growth cone. A characteristic feature of lamellar extensions is the movement of actin filaments from the distal growth cone margin toward the growth cone center (Forscher and Smith, 1988). Treatment of neurons with cytochalasin resulted in the immediate cessation of actin filament assembly but did not block retrograde translocation of actin filaments, an indication that translocation is not driven by distal actin polymerization and is perhaps a motor-driven process (Forscher and Smith, 1988).

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When growth cones encounter a positive environmental signal, a stable attachment of the lamellipodium to the substrate ensues with further changes in both the actin and MT cytoskeletons. In a study by Lin and Forscher (1993), neurons were positioned so that one growth cone would make contact with another growth cone. Subsequent to cell-to-cell contact, the rate of retrograde actin flow decreased (Lin and Forscher, 1995). However, actin filament assembly continued and resulted in the accumulation of actin filaments at the contact site (Lin and Forscher, 1993). The observed retardation in retrograde flow is proposed to involve actin-binding proteins that retard retrograde flow while permitting the actin-based motors to continue cell movements in the direction of the lamella (Lin and Forscher, 1995). A decrease in retrograde flow of actin filaments is accompanied by the forward protrusion of MTs into the contact site at the region of actin accumulation. The dynamic nature of MTs (Mitchison and Kirschner, 1984) enables them to extend into and retract from the lamellar protrusions as the lamellipodia spread and contract during exploration of the environment. Is a myosin motor responsible for retrograde flow of actin filaments? Both myosin I and I1 have been localized to growth cones (Bridgman and Dailey, 1989; Lewis and Bridgman, 1996; Miller et al., 1992; Wagner er al., 1992; Rochlin et al., 1995). A study by Lewis and Bridgman (1996) provides evidence for a membrane-bound myosin that could function as a motor enzyme for retrograde translocation of actin filaments in growth cones. Antibodies raised against the head region of a mammalian myosin I localized to the innermost surface of the plasma membrane of growth cones and were often associated with growth cone actin filaments (Lewis and Bridgman, 1996). Disruption of growth cone actin filaments with cytochalasin B did not alter the immunogold labeling of the plasma membrane, an indication that the myosin is stably associated with the plasma membrane (Lewis and Bridgman, 1996).

IV. Organelle Transport on Microtubule and Microfilament Tracks A. Model Systems for Organelle Transport

There is extensive experimental evidence for organelle transport as a MTbased process employing kinesin and dynein motors and a microfilamentbased process employing myosin motors (Fath and Burgess, 1994; Goldstein, 1993; Langford, 1995; Skoufias and Scholey, 1993). These were regarded as disparate motility systems until Kuznetsov and co-workers (1992) reported the movement of axoplasmic membranous organelles on

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both MT and microfilament tracks and proposed that a single organelle could display both a MT-based and a microfilament-based motor protein. Consequently, there is now much interest in the synergistic action of MTs and microfilaments in organelle transport. Much of the research on organelle transport has utilized three model systems: the nonpolarized cell in culture, the neuron, and the polarized epithelial cell. Each possesses a different arrangement of cytoskeletal MTs. In the nonpolarized cell at interphase, MTs are radially arranged with their minus ends at the centrosome and their plus ends toward the plasma membrane. The endoplasmic reticulum in nonpolarized cells is distributed near the plus ends of MTs whereas the Golgi complex is located near the minus ends of MTs. The neuron is a highly polarized cell with a single long axon, which conducts nerve impulses away from the cell body, and several shorter dendrites, which form synaptic junctions with axons from other neurons. Within the cell body, the MTOC, nucleus, and Golgi are in close proximity to one another. In the axon, MTs are polarized with their minus ends toward the cell body and their plus ends distal to the cell body (Heidemann et al., 1981), whereas in dendrites MTs are of mixed polarity (Baas, et al., 1988). The axon is also rich in actin filaments (Fath and Lasek, 1988), although they are not known to be of uniform polarity. In contrast to the organization in axons, the minus ends of cytoskeletal MTs in the polarized epithelial cell are near the centrioles in the apical cytoplasm, and the plus ends of MTs are located near the Golgi at the basal end of the cell (Achler et al., 1989). This section focuses on mitochondria1 transport in axons, vesicular transport in polarized epithelial cells, and evidence that both anterograde and retrograde motors are associated with one organelle.

E. Mitochondria1 Transport in Axons Because axons lack the machinery for protein synthesis, molecules synthesized in the cell body must be transported to the nerve terminal. Suggestions that molecular motors power transport within the axon date back more than two decades (Ochs, 1972). The work of Hirokawa (1982) was among the first to describe MT-microfilament links to membranous organelles and to suggest a mechanism by which these organelles move on MT tracks powered by ATPase activity. The nature of this transport system is a major focus in cell biology today. Axonal transport has been extensively investigated, and the reader is referred to a recent review (Langford, 1995). However, there are relatively few studies that have explored a possible synergistic role for MTs and microfilaments in organelle transport. In one of these studies (Morris and

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Hollenbeck, 1995), video-enhanced microscopy of chick neurons was used to study axonal transport of mitochondria in control cells and in cells cultured in the presence of cytochalasin or vinblastine, which produced neurites that lacked microfilaments or MTs, respectively. In control cells, bidirectional movement of mitochondria was observed, although mitochondria were stationary most of the time. Of the time spent in movement, more was devoted to anterograde rather than retrograde motion. Therefore, net movement of mitochondria in control cells was anterograde, toward the nerve terminal. In cytochalasin-treated cells, which were devoid of a microfilament array but contained normal MT and neurofilament systems, bidirectional axonal transport of mitochondria was observed with net anterograde movement. In vinblastine-treated cells that were devoid of MTs but that contained normal microfilament and neurofilament arrays, bidirectional mitochondrial transport was observed, although at a slower rate compared to control or cytochalasin-treated cells, with net retrograde movement. Depolymerization of both MT and microfilament arrays resulted in a neurofilament network that did not support mitochondrial movement. Quantitative analysis of axonal mitochondrial transport suggests that MT-based transport and microfilament-based transport are not completely independent systems but are possibly under coordinate control (Morris and Hollenbeck, 1995). In cytochalasin-treated neurons in which transport of mitochondria was on MTs alone, anterograde movements were reduced by about one-third compared with control cells, whereas retrograde movements did not change significantly and net movement remained anterograde. In vinblastine-treated cells mitochondrial transport was on microfilaments alone, and there was a reduction in anterograde movements in addition to a threefold increase in retrograde movements that resulted in net retrograde movement. Although the study by Morris and Hollenbeck (1995) did not identify motor molecules involved in axonal mitochondrial transport, the bidirectional nature of the transport on axonal MTs of uniform polarity indicates that both plus end-directed and minus end-directed motors are associated with mitochondria. Kinesin and dynein are the most likely candidates for these motors. The kinesin superfamily contains both plus end-directed and minus end-directed motor proteins (Goldstein, 1993). Therefore, two different motors from the kinesin superfamily could possibly account for the bidirectional nature of the axonal transport. Kinesin-mitochondrion associationshave been demonstrated. A novel member of the kinesin superfamily in mammalian brain has been cloned and sequenced (Nangaku er al., 1994). This protein, designated KIFlB, is a monomer, N-terminal type motor that translocated mitochondria on MTs in a plus end-directed manner in vitro (Nangaku et al., 1994). In another study, antibodies against the head region of a Drosophila kinesin cross-reacted with a 116-kDa kinesin

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heavy chain in a purified mitochondria fraction from rat brain (Jellai et al., 1994). Movement of mitochondria on actin filaments (Morris and Hollenbeck, 1995) suggests that myosin is associated with mitochondria. Because all known myosins are plus end-directed motors (Cheney et al., 1993;Mooseker and Cheney, 1995), bidirectional movement of mitochondria on microfilaments indicates the axon contains microfilaments of mixed polarity. A myosin-mitochondrion association has also been demonstrated. In photoreceptor cells of arthropods, light stimulation causes aggregation of mitochondria (Sturmer et al., 1995). Microfilament bundles labeled with phalloidin and/or antiactin were shown to be aligned in close association with mitochondria along the path of mitochondria translocation. More direct evidence for an association of myosin with mitochondria comes from studies on yeast. Isolated yeast mitochondria exhibited an ATP-sensitive, F-actin binding activity that indicates the presence of a myosin motor (Lazzarino et al., 1994). Degradation of the outer mitochondria1 membrane resvlted in the loss of F-actin binding activity, an indication that the putative myosin is located on the surface of the mitochondrion. Rhodamine phalloidinlabeled yeast actin filaments and isolated yeast mitochondria were used in a filament sliding assay for motility. Translocation of the actin filaments on the yeast mitochondria was observed in an ATP-dependent manner (Simon er al., 1995).

C. Vesicular Transport in Polarized Epithelial Cells Immunoblotting and immunofluorescence microscopy were used to demonstrate that both myosin I and cytoplasmic dynein were present in a Golgi fraction isolated from intestinal epithelial cells (Fath et al., 1994). Both motor proteins were extractable with high salt, which suggests that they are vesicle peripheral membrane proteins. In highly polarized cells such as intestinal epithelia, the MT array is not extensive in the apical microvillus region. This observation led Fath and Burgess (1993) to suggest that Golgi vesicles possess both MT-based and actin filament-based motors that could provide transport from the trans-Golgi to the apical plasma membrane. In the model proposed by these authors, dynein would translocate vesicles from the Golgi to the apical cytoplasm, and myosin would complete the translocation of vesicles through the terminal web to the apical plasma membrane. D. Different Motors on One Organelle?

Are different motors displayed on one organelle? The studies described in the previous section indicate that a population of membranous organelles

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may contain more than one motor protein and therefore raise the possiblity that one organelle could display a spectrum of anterograde and retrograde motors. The investigations of Kuznetsov and co-workers (1992) suggest that a single organelle possesses both a MT-based motor and an actin filament-based motor and can travel on both MT and actin filament tracks. There is convincing evidence that a subset of Golgi membranes displays both dynein and myosin I (Fath et al., 1994). Bidirectional axonal transport of mitochondria indicates that mitochondria contain MT-based and actin filament-based motors (Morris and Hollebeck, 1995), although it is not known whether a single mitochondrion displays both types of motors. If there are multiple motors on an organelle, what factors determine which motor is deployed? What are the molecules that regulate the interaction between motor and track and enable the motor to load its cargo and engage the appropriate track? A regulator of organelle deployment must have the ability to create cross-communication among MTs, actin filaments, MT motors, and actin filament motors. The regulation of motor-track interaction will be discussed under Section V.

V. Regulation of Microtubule-Microfilament Interactions A. Tubulin- and Actin-Binding Proteins Tubulin- and actin-binding (TAB) proteins are good candidates for regulators of MT-microfilament interactions. Several proteins exhibit this dual binding capacity and are discussed in the following sections.

1. MAP-2 and T Microtubule-associated proteins are possible regulators of MTmicrofilament interactions. The acidic C-termini of 6 and P-tubulin contain binding sites for MAPs (Paschal et al., 1989). The microtubulebinding domain of MAP-2 (Lewis et al., 1988) and T (Himmler et al., 1989) consists of conserved C-terminal 18-amino acid repeats that are positively charged. The first studies to demonstrate an in vitro effect of MAPs on MT-microfilament interactions suggested that MAPs mediate the formation of MT-microfilament gels (Griffith and Pollard, 1978, 1982). In these studies, low shear viscometry and electron microscopy were used to study the interaction between purified actin filaments and MTs. Mixtures of actin filaments with MTs and their heterogeneous associated proteins (MAPs) had higher viscosities than the separate

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polymers. Actin filaments with MAP-free MTs exhibited low viscosity, and MAPs in the absence of MTs increased the viscosity of actin polymers. Electron microscopy of these high viscosity, MT-microfilament mixtures revealed the close association between the two fibrillar structures. Sattilaro and collaborators (1981) studied the effect of MAPs on actin filament organization. A heat-stable MAP-2 fraction induced an ATPdependent bundling of actin filaments in vitro. Quantitative analysis of the bundled filaments revealed -1 MAP-2 molecule for every 28 actin filaments. Formation of MAP-actin bundles was inhibited by ATP. The activity of MAP-2 and T on actin filament gelation and filament bundling was investigated by Kotani and co-workers (1985). Their study indicated that for actin filaments, MAP-2 is a gelation factor, whereas T is a bundling factor. MAP-induced gelation of actin filaments is not contradictory to MAP-2-induced bundling of actin filaments because, as pointed out by Kotani et af. (1985), high concentrations of gelation factors, such as filamin, can induce actin bundling. Lopez and Sheetz (1994) investigated the effect of MAP-2 on kinesin and cytoplasmic dynein activity. These investigators used a MT gliding assay with MAP-2-coated tubulin and either kinesin or cytoplasmic dynein and showed that a MAP-2 concentration as low as one MAP-2 per 69 tubulin dimers inhibited MT motility by about 75%. Their study further showed that the basis for the inhibition did not appear to be the C-terminal MT-binding domain in MAP-2 because T protein, which contains the conserved C-terminal amino acid repeat that is present in MAP-2, did not inhibit gliding motility. Furthermore, MAP-2 chymotryptic fragments containing the MT-binding domain did not inhibit gliding motility. The authors proposed that the side arm of MAP-2 is responsible for MAP-2 inhibition of MT gliding. They further suggested that the MAP-2 side arm could interfere with the interaction of the motor with MTs and increase the rate of MT release from the motors. MAP-2 inhibition of motor activity has important implications for the regulation of organelle transport. For example, selective binding of MAP-2 to MT tracks within an axon could inactivate MT-dependent transport on a subset of the MT tracks. Consequently, organelle transport might be diverted to other MT tracks or to microfilament tracks. Antibodies have been used to study the interaction of MAPs and T protein with actin filaments. A monoclonal antibody was produced by immunization with a synthetic peptide that contained a portion of the sequence from the MAP-2-binding domain in P-tubulin (Rivas et af., 1988). This antibody was characterized as an auto anti-idiotypic antibody that recognized the tubulin binding domain on MAP-2 and T. On immunoblots of cytoskeletal proteins from cultured cells, the anti-idiotypic antibody recognized T proteins but did not detect MAP-2 (Cross et af., 1993). In

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a double-labeling immunofluorescence experiment with the anti-idiotypic antibody and rhodamine phalloidin, the antibody colocalized with the phalloidin staining of stress fibers, an indication that the antibody detected T epitopes associated with actin filaments (Cross et al., 1993). In another double-labeling experiment, the MT staining pattern with an anti-6tubulin antibody was distinct from the staining pattern with the anti-idiotypic antibody, further indicating that the T protein detected by the anti-idiotypic antibody was associated with actin stress fibers and was not associated with MTs (Cross et al., 1993). These data suggest that T can mediate interactions between MTs and actin stress fibers in cultured cells. 2. MAP-1

MAP-1A belongs to a group of three high-molecular-weight MAPs that includes MAP-1B and MAP-1C (cytoplasmic dynein). Solid-phase immunoassays and cosedimentation assays were used to investigate the interaction of purified MAP-1A with actin (Pedrotti et al., 1994a,b). These assays showed that MAP-1A exhibited binding affinity for both G- and F-actin and induced the gelation and cross-linking of actin filaments. The actin binding site on both MAP-1A and MAP-2 was studied by coincubation of actin with both MAPs. Incubation of F-actin with either MAP-1A or MAP2 resulted in the binding of the respective MAP to the actin filaments. However, the inclusion of both MAP-1A and MAP-2 in the incubation assay with F-actin resulted in the preferential binding of MAP-2 to the actin filaments, which suggests that MAP-2 and MAP-1A share a common actin-binding domain. Further evidence for the interaction of MAP-1 with actin filaments is the decoration of actin stress fibers by a monoclonal antibody against MAP-1 (Asai et af., 1985). 3. Calmodulin, Caldesmon, and Synapsin A study by Kotani et af. (1985) indicates a role for calmodulin in the regulation of MT-microfilament interactions. Calmodulin, in a calciumdependent and reversible manner, increased the critical concentration of MAPs required for actin filament gelation. These authors suggested that the binding of calmodulin to MAP-2 inactivated the MAP-2 actin crosslinking activity. The ability of actin-binding proteins to modulate MT function is illustrated by studies of caldesmon (Ishikawa et af., 1992a,b). Both muscle and nonmuscle caldesmon bind to brain MTs in vitro (Ishikawa et af., 1992b). Binding of caldesmon to MTs was inhibited by the presence of Cacalmodulin, and purified caldesmon decreased the critical concentration of tubulin for in vitro polymerization (Ishikawa et al., 1992a). Limited proteo-

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lysis was used to map the MT binding site of caldesmon to a 34-kDa Cterminal domain that is near the actin binding site and the calmodulin binding site in the caldesmon protein (Ishikawa et al., 1992a). A dimeric form of caldesmon cross-linked actin filaments (Bretscher, 1984) and bundled MT in v i m (Ishikawa, 1992a). In a motility assay, caldesmon inhibited dynein-powered sliding of MTs (Ishikawa, 1992a). Synapsin I is another protein with binding affinities for both MTs and actin filaments ( Petrucci and Morrow, 1987). I n vitro, this phosphoprotein bundled and cross-linked actin filaments. Phosphorylation of synapsin with a calcium and calmodulin-dependent kinase I1 reduced the actin-bundling and actin-binding activity of synapsin. However, phosphorylation of synapsin I enhanced its MT-binding activity. Synapsin could be a link between vesicles and actin-based motility systems.

4. Ezrin and Fodrin Characterization of an 80-kDa, ezrin-like protein from chicken erythrocytes revealed properties of both MT- and microfilament-associated proteins (Birgbauer and Solomon, 1989). The 80-kDa protein co-assembled with brain tubulin in virro and localized to marginal band MT by immunofluorescence microscopy. It also colocalized with the phalloidin staining in erythrocytes and was stably associated with cytoskeletons prepared by detergent extraction of erythrocytes. The ezrin-like protein might be involved in promoting interactions between MTs and microfilaments in the growth cone (Goslin et al., 1989 ). The protein was closely aligned with actin filaments in the growth cone as determined by rhodamine- phalloidin staining. However, its localization was distinct from that of MTs. When neurons were treated with nocodazole, depolymerization of MTs resulted in the rapid disruption of the ezrin protein staining pattern. The ezrin protein was redistributed proximally along the axon, but it always remained distal to the receding edge of the MTs, which suggests that the location of the protein relies on positional information provided by intact MTs (Goslin et af., 1989). In the presence of prolonged nocodazole treatment and the absence of MTs, the ezrin protein was not detected by immunofluorescence. Microtubules reassembled when nocodazole was removed, and the original pattern of staining associated with the ezrin protein was achieved after the completion of MT assembly, which is a further indication that MTs might provide positional information for the localization of proteins in the growth cone. Studies with antisense oligonucleotides in growth cones suggest that localization of ezrin might be dependent on the presence of T and provide further evidence for r as a mediator of MT-microfilament interactions. An antisense oligonucleotide was used to suppress r in growth cones (Caceres

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and Kosik, 1990;Caceres et af.,1991,1992;Ditella et al., 1994 ). Suppression of T resulted in the disappearance of the ezrin protein from the growth cone, although the protein was present in the neuronal cell body. T reappeared when the antisense oligonucleotide was removed from the culture medium. Suppression of T also affected the actin cytoskeleton in growth cones. In control neurons, phalloidin staining revealed actin filaments as radial striations in the lamellipodia, whereas in muppressed neurons, phalloidin staining was diffuse and not organized as radial striations. Future studies might focus on the disruption of ezrin function by microinjection of antibodies, which could enable one to study the role of the protein in growth cone motility and axon formation. Disruption of ezrin function might also be achieved by overexpression of mutated ezrin genes in neurons or by microinjection of antisensense RNA to ezrin gene sequences. Fodrin is another protein that interacts with both MTs and actin microfilaments. This high-molecular-weight (260-kDa) spectrin-like protein has been purified from neuronal tissue. In vitro, purified fodrin cross-linked actin filaments, bound calmodulin in a Ca-dependent manner, (Bennett et af., 1982) and bundled MTs (Ishikawa et al., 1983).

6.Posttranslational Modifications Posttranslational modifications such as phosphorylation may also play important roles in the regulation of motor-track interactions. Griffith and Pollard (1978) and Satillaro et al., (1981) noted the inhibitory effect of ATP on MAP-actin interactions. This nucleotide inhibition was investigated further by Selden and Pollard (1983), who observed a 70% increase in phosphate content when heat-stable MAPs (MAP-2) were incubated with ATP. Incubation of MAPs without nucleotides resulted in a 28% decrease in phosphate content. The effect of MAP phosphorylation on MAP-actin interaction was also investigated. Filament cross-linking activity was measured by low shear viscosity, and a reversible, inverse relationship between actin filament cross-linking and phosphate content of MAPs was observed. MAPs with the highest phosphate content (i.e., most highly phosphorylated) had the lowest actin filament cross-linking activity. MAP cross-linking activity could be reversed by treatment with phosphatases. In a recent study, the effect of kinases and phosphatase inhibitors on MT-dependent vesicle transport in cultured cells was investigated (Hamm-Alvarez et al., 1993). Various pharmacological agents stimulated vesicle transport. Of particular interest is okadaic acid, which inhibited serinekhreonine protein phosphatases and increased the frequency of MT-dependent vesicle movement by more than sixfold (Hamm-Alvarez et al., 1993).

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C. A Role for Microtubule and Actin Filament Dynamics Several studies on neuronal cells have focused on MT and microfilament dynamics as complementary interactive forces within cells that may play a role in regulating MT-microfilament interactions. In growth cones, as discussed previously, the forward intrusion of MTs into the lamellipodium is correlated with changes in the growth cone actin cytoskeleton. What factors control MT extension into growth cone lamellipodia? Forscher and Smith (1988) proposed that the alignment and forward protrusion of MTs is modulated by changes in actin dynamics. These authors suggested that depletion of actin in regions adjacent to the growth cone contact site allows MTs to enter the lamellar leading edge. Their suggestion is supported by experiments with cytochalasin. In cytochalasin-treated neurons, actin filaments were capped and further actin assembly was prevented. This resulted in a depletion of the actin network and the invasion of MTs deep into the lamellar periphery (Forscher and Smith, 1988). In neurons in which growth cones had engaged a contact site, the MT extension process became random in the presence of cytochalasin, an indication that MTs become aligned for forward protrusion by interaction with actin filaments (Lin and Forscher, 1993). Microtubule dynamics may also play a role in the interaction of growth cone MTs and microfilaments. Pertubation of MT dynamics with vinblastine, at concentrations that inhibited dynamics without an effect on polymerization, inhibited forward MT extension. However, in a significant minority of the drug-treated cells, forward extension of MTs was not inhibited, although the treated growth cones exhibited a wandering rather than a persistent forward motion (Tanaka et af., 1995). Microtubules within PC 12 neurites have been described as under compression, supporting tension within the actin cytoskeletal network (Joshi et al., 1985). Incubation of neurite-bearing cells with MT depolymerization reagents resulted in the retraction of neurites and reversion of the cell to a round morphology due to the force of released tension created by MT depolymerization (Solomon and Magendantz, 1981;Joshi et al., 1985). Actin filaments have a role in neurite retraction as demonstrated by experiments in which disruption of actin filament networks with cytochalasin prevented neurite retraction (Solomon and Magendantz, 1981; Joshi et af., 1985). Is neurite retraction a result of MT-mediated contraction of the actin cytoskeleta1 network? Recent studies suggest an association between MT depolymerization and contraction of the cytoskeletal network. In cultured cells, such as chicken embryo fibroblasts, contraction of the actin-myosin cytoskeletal network exerts tension that can be measured when the cells are cultured in a collagen matrix (Kolodney and Elson, 1993; Kolodney and Wysolmerski, 1992). Contraction of the cytoskeletal network can be induced by MT depolymerization (Dennerll et af.,1988). The possibility that contrac-

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tion is regulated through an effect of MT depolymerization on phosphorylation of the myosin light chain (LC,,) was investigated by using nocodazole, a MT depolymerizing agent, and paclitaxel (taxol), a MT stabilizing agent (Kolodney and Elson, 1995). In that study, nocodazole-induced contraction in fibroblasts was inhibited and reversed by paclitaxel, and the amount of phosphorylated LC20in nocodazole-treated cells was 40-70% higher than the level of phosphorylated LCzoin untreated control cells, an effect that was also inhibited and reversed by paclitaxel. An important implication of these studies is that coupling of MT dynamics with myosin light chain phosphorylation could regulate actomyosin activity, for example, the activation of an actin filament-based motor system for intracellular transport.

D. A Microtubule-Based Motor Compensates for an Actin-Based Motor Myo2p is a class V myosin in yeast (Johnston et al., 1991). In the mutant my05 cells at the restrictive temperature fail to produce buds although they continue to increase in size (Johnston ef al., 1991). The phenotype of the my02 mutant is similar to the phenotype of actin mutants (Adams and Pringle, 1984) that affect polarized secretion. The temperature sensitivity of the my02 mutant can be suppressed by overexpression of the gene S M Y l , which encodes a member of the kinesin superfamily (Lillie and Brown, 1992). Immunofluorescence microscopy showed that Smylp and My02p colocalized to regions of active growth in the cell. It is unclear how a kinesin-like protein, which is a MT-based motor, can compensate for the loss of a myosin motor activity. Although kinesin and myosin do not share amino acid sequence identity, analysis of their crystal structures revealed that secondary structural elements of the two proteins overlap one another in the catalytic P-loop domain and therefore raise the possibility that these two proteins evolved from a common ancestor and share similiar forcegenerating mechanisms (Kull et al., 1996). Can a kinesin interact with actin filaments? A large body of experimental evidence demonstrates the motor capabilities of kinesins and myosins on MTs and actin microfilaments, respectively. In order to consider the possibilty that kinesins interact with actin filaments ,we cannot be limited by previously established parameters and should consider novel reaction conditions in order to experimentally test this idea.

E. Multisubunit Complexes 1. Dynactin Because of its potential for interaction with both MTs and microfilaments, the dynactin complex may be involved in the regulation of dual, or perhaps

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multiple, motors on an organelle. The dynactin complex is an activator of dynein-mediated vesicle movement on MTs (Gill, et al., 1991; Schroer and Sheetz, 1991). It consists of at least nine different polypeptides, which include p150 (dynactin), the homolog of Drosophila glued150;actin-related protein Arpl; conventional actin, and several other polypeptides. A recent ultrastructural analysis of the dynactin complex revealed that it contains a short filament composed of actin-related protein Arpl and probably conventional actin (Schafer, et al., 1994). The actin-like filament in the dynactin complex provides a possible binding site for myosin. The N-terminal region of p150gluedcontains a conserved MT binding site (WatermanStorer et al., 1995). Therefore, the dynactin complex has the potential for interaction with both actin filament-based and MT-based motor systems. The p150 could be a link between the dynactin complex and a dynein motor as indicated by the recent demonstration that p150 binds the dynein intermediate chain (Vaughan and Vallee, 1995). Other components within the dynactin complex could have affinities for motor polypeptides, for example, the role of the 50-kDa component of the dynactin complex in targeting dynein to the kinetochore (Echeverri et al., 1996). Much additional work must be done in order to establish a regulatory function for the dynactin complex in organelle transport. 2. Dynein Regulatory Complex

The dynein regulatory complex (DRC) is another potential regulator of cross-communication among actin filaments, MTs, and dynein. Generation of axonemal bends requires the action of multiple dynein molecules arranged as a series of inner and outer arms along the axoneme. In Charnydornonas, six axonemal proteins, including actin and caltractinkentrin, form a complex referred to as dynein regulatory complex (Piperno et al., 1992). Suppressor mutants that are missing DRC components have a reduced amount of inner dynein arms 12 and 13, which suggests that the DRC is in close proximity to inner arm dynein. Although the function of DRC is unknown, the presence of caltractin, a calcium-binding protein, suggests a regulatory function related to axonemal motility.

F. Regulation of Motor-Track Interactions This section has provided possible clues to the identification of molecules that regulate the interaction between motor and track. As discussed under Section 111, a regulator of motor deployment must have the ability to create cross-communication among MTs, actin filaments, MT motors, and actin filament motors. An organelle with both a MT-based and an actin filament-

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w Microtubule Track

Microfilament Actin-based Motor

FIG. 2 Schematic diagrams of the regulation of motor-track interactions by a tubulin- and actin-binding (TAB) protein. The cargo in these diagrams is equipped with a microtubulebased motor and an actin filament-based motor. The TAB protein is depicted as a wedgeshaped object that, when bound to the microtubule, interferes with the movement of the motor on a microtubule track. TABs could be permanently or transiently bound to the microtubule and are proposed to exist in a phosphorylated or dephosphorylated state. Phosphorylated TABs would inhibit the interaction between microtubules and actin filaments,

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based motor could be switched from a MT track to microfilament track by the action of a regulatory molecule that interacts with both MTs and actin filaments. MAP-2, through its ability to cross-link MTs and actin filaments, could mediate the switching of cargo from a MT track to a microfilament track as illustrated in Fig. 2. The switching mechanism could be regulated by MAP-2 phosphorylation or by calmodulin, both of which inhibit MAP interactions with actin filaments. MAP-2 could further regulate motor selection through a direct inhibition of dynein or kinesin motor activity. Caldesmon, because of its ability to bind MTs, actin filaments, and calmodulin and to directly inhibit dynein motor activity, is another good candidate for mediating cross-communication among motors and tracks. Binding of caldesmon to MT tracks could suppress dynein-powered transport on MTs, whereas the actin binding site on caldesmon would allow a microfilamentbased transport to continue.

VI. Concluding Remarks This review has sought to identify diverse examples of structural and functional synergy of the MT and actin filament cytoskeleton. As more detailed knowledge of cytoskeletal proteins becomes available, future studies will employ greater use of disruptive agents and techniques that target specific components of interacting cytoskeletal systems in order to dissect structurefunctional relationships. Examples of highly selective pertubation agents and techniques include site-specific monoclonal antibodies, antisense RNA, and site-specific genomic knockouts. Future review articles will undoubtedly focus on the role of myosin filaments in the synergy of MTs and actin filaments. The next decade should be an exciting one for advances in the explorartion of synergy in the cytoskeleton. Acknowledgments The author gratefully acknowledges the able assistance and invaluable criticism of Dr. Jorge GarcCs and grant support from the National Science Foundation.

whereas dephosphorylated TABS would promote the cross-linking of microtubules and actin filaments. (A) The cargo is engaged on a microtubule track and is translocated on any part of that track until a phosphorylated TAB (depicted as a black. wedge-shaped object) derails the cargo as indicated by the downward arrow. (B)The cargo is deflected from the microtubule track by a dephosphorylated TAB that has promoted the cross-linking of an actin filament to the microtubule. The actin filament-based motor on the deflected cargo engages the actin filament and the cargo is translocated along the actin filament.

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Insulin Internalization and Other Signaling Pathways in the Pleiotropic Effects of Insulin Robert M. Smith, Shuko Harada, and Leonard Jarett Department of Pathology and Laboratory Medicine, University of Pennsylvania Medical Center, 633 Gates Building, Philadelphia, Pennsylvania 19104

Insulin is the major anabolic hormone in humans and affects multiple cellular processes. Insulin rapidly regulates short-term effects on carbohydrate, lipid, and protein metabolism and is also a potent growth factor controlling cell proliferation and differentiation. The metabolic and growth-related effects require insulin binding to its receptor and receptor phosphorylation. Evidence suggests these events result in subsequent substrate phosphorylation and activation of multiple signaling pathways involving Src homology domain-containing proteins and the internalizationof the insulin:receptor complex. The role of insulin internalization in insulin action is largely speculative. For more than two decades, extensive investigation has been carried out by numerous laboratories of the mechanisms by which insulin causes its pleiotropic responses and the cellular processing of insulin receptors. This chapter reviews our current knowledge of the phosphorylation signaling pathways activated by insulin and presents evidence that substrates other than insulin receptor substrate-1 are involved in insulin’s regulation of immediate-early gene expression. We also review the mechanisms involved in insulin internalizationand present evidence that internalization may play a key role in insulin action through both signal transduction processes and translocation of insulin to the cell cytoplasm and nucleus. KEY WORDS: Insulin action, Insulin receptor, Signal transduction, Endocytosis, Endosome, Caveolae, Immediate-early gene expression.

1. Introduction Insulin is the major anabolic hormone in humans and has well-known biological effects that fall into two major categories. Insulin rapidly regulates I,trc~rnnrronol K c r i m , of C v r r ~ k ~ g V ~d ~. .17.3

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ROBERT M. SMITH ETAL.

short-term effects on carbohydrate, lipid, and protein metabolism. Insulin is also a potent growth factor (Taub et al., 1987; Randazzo et al., 1990) and controls cell proliferation and differentiation through rapidly induced and long-term effects. Both the metabolic and growth-related effects require insulin binding to its plasma membrane receptor and subsequent activation of intracellular effector or signaling mechanisms. For more than two decades, extensive investigation has been carried out by numerous laboratories of the mechanisms by which insulin causes its pleiotropic responses. Several reviews on the consensus phosphotyrosine cascade signal transduction pathway have been published. The purpose of this chapter is to summarize work from many laboratories, including our own, that suggest that insulin internalization and multiple signaling pathways play physiologically important roles in insulin’s actions.

A. Insulin Signal Transduction Network As illustrated in Fig. 1,the simple “black box” used to depict the signaling mechanism 20 years ago has been augmented by a complex and everexpanding network of interacting, overlapping, and bypassing pathways that Cheatham and Kahn (1995) appropriately referred to as a “signaling network.” The proximal and presumably essential elements involved in the signal transduction network are the binding of insulin to the receptor a subunit and the autophosphorylation and activation of the intrinsic tyrosine kinase on the receptor’s @ subunit. These elements are common to a large family of tyrosine kinase receptors. The unique node in the insulin network is the insulin receptor substrateldocking protein, IRS-1, with its 22 tyrosine phosphorylation sites and amino acid residues at these sites which confer specificity for the binding of proteins containing Src homology (SH2) domains. Most tyrosine kinase receptors, including the insulin receptor (see below), have some SH2 docking domains in the receptor itself. The interaction with IRS-1 and activation of a variety of SH2 domain-containing enzymes, e.g., p85 a subunit and p85 @ subunit of PI 3-kinase, GRB-2, Syp, and Nck, provides one branching point that could account for many of insulin’s pleiotropic effects. Although IRS-1 is a key element in insulin’s signaling, we now know it is not the only intracellular insulin substrate. IRS-l-deficient transgenic mice compensate using a substrate termed IRS-2 (Araki et al., 1995). Another circuit in the insulin signaling network, which is shared by other tyrosine kinase receptors, is the insulin-induced tyrosine phosphorylation of Shc, which binds GRB-26OS (Skolnik et al., 1993). Some investigators have suggested that Shc/GRB-2/SOS complex may be the predominant mechanism by which insulin and cytokines activate the ~21‘”’ pathway

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245

Insulin Receptor p-subunit Autophosphorylation and Kinase Activation

. DNA synthesis and cell proliferation FIG. 1 Schematic representation of insulin signal transduction pathways.

(Pruett et al., 1995). As discussed later in this chapter, we have demonstrated that Shc may be the primary insulin-sensitive tyrosine phosphorylated substrate responsible for insulin's effects on immediate-early gene expression in 32D myeloid precursor cells that lack IRS-1. Another insulin-sensitive circuit involves the association and activation of the p85 subunit of PI 3kinase with the tyrosine phosphorylated COOH terminus of the insulin receptor (Levy-Toledano et al., 1994; Van Horn et al., 1994), which may require some unidentified cytoplasmic factors (Liu and Livingston, 1994). p85 may then link the insulin receptor to p62 GAP-associated protein and GTPase activating protein (GAP), which could then activate the Ras pathway independently of the IRS-1 or Shc circuits (Sung et al., 1994). Staubs et af. (1994) have shown that, in addition to p85, Syp binds to Tyr'"' in the COOH terminus of the insulin receptor and GAP associates with Tyr"O in the juxtamembrane region. These direct interactions of SH2

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domain-containing proteins with the insulin receptor provide a mechanism that may bypass the IRS-1 docking protein to activate distal molecules in the insulin signal transduction network. Despite the overwhelming evidence that tyrosine phosphorylation of IRS-1 plays a central role in insulin signaling (Rose et al., 1994; Sun et al., 1992), the sheer number of potential signaling circuits that are competing for limited, and frequently cell-specific, concentrations of substrates (Yamauchi and Pessin, 1994) suggests that other mechanisms of signal transduction have yet to be resolved. In fact, current schemes depicting the insulin signaling network retain the black box labeled as alternative or other substrates, e.g., pp15, pp42, pp85, pp120 (Roth et al., 1992), pp60 (Hosomi et al., 1994), CytPTK (Shisheva and Shechter, 1992), and PHAS-I (Hu et al., 1994) as well as the numerous serinehhreonine kinases (Czech et al., 1988) that are rapidly phosphorylated by insulin and/or other peptide growth factors. Holgado-Madruga et al. (1996) demonstrated GRB-2-associated binder-1 (Gab-1), which has homology to IRS-1, binds to GRB-2 through a SH3 domain and is phosphorylated by insulin and EGF, providing another signaling molecule in insulin’s signal transduction network. It has been suggested that phosphorylation of cytoplasmic substrates, in the absence of receptor autophosphorylation, may be sufficient to elicit insulin responses (Yamamoto-Honda et al., 1993). Mutational analyses of the insulin receptor have demonstrated that some responses are not dependent on intrinsic receptor kinase activity (Gottschalk, 1991; Moller et al., 1991). Studies using anti-receptor antibodies have resulted in similar conclusions (Sung, 1992). We have shown that insulin stimulates immediate-early gene egr-1, but not c-fos, expression to the same extent in Chinese hamster ovary (CHO) cells overexpressing wild-type or kinase-deficient human insulin receptors or only low levels of endogenous receptors (Harada et al., 1995a). In contrast, insulin increased PI 3-kinase activity and IRS-1 phosphorylation to detectable levels only in CHO cells overexpressing wildtype insulin receptors. These observations suggested that insulin-induced egr-1 expression in CHO cells was regulated by insulin-sensitive signaling mechanisms not necessarily controlled by IRS-1 phosphorylation and PI 3-kinase activation. Alternative tyrosine phosphorylated substrates of -120 kDa are currently being investigated, as discussed later in this chapter. A few studies have suggested that the major proximal element in the network, the insulin receptor, may not be essential for some insulin responses. For example, Roth ef al. (1981) constructed an insulin-ricin B hybrid that bound to rat HTC hepatoma cell ricin receptors and stimulated amino acid uptake to a significantly greater extent than insulin or ricin B alone. Hofmann et al. (1983) also used an insulin-ricin B hybrid and demonstrated increased glucose incorporation into glycogen in MDCK cells that had no detectable insulin receptors and no ricin-B or insulin effect.

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247

Because ricin is one of many molecules that is translocated to the cytoplasm, the effects observed in these studies may have resulted from the translocation of insulin to the cytoplasm. Miller (1988) reported that microinjection of insulin into the cytoplasm of Xenopus laevis oocytes resulted in increased RNA and protein synthesis. We showed that trypsin treatment of H35 hepatoma cells, which resulted in undetectable insulin binding to plasma membrane receptors, did not significantly affect insulin’s stimulation of immediate-early gene transcription (Lin et al., 1992). Because fluid-phase endocytosis (see below) and insulin’s accumulation in the nucleus at high insulin concentrations were not affected by trypsin treatment (Harada et al., 1992), these results also suggested that the translocation of insulin to the cytoplasm or nucleus may play a physiologically important role in insulin action. These studies, although in conflict with the basic tenets of insulin signal transduction, suggest that insulin might be able to activate alternative pathways that are not directly related to its binding to plasma membrane receptors if an alternative method of cellular uptake, e.g., ricin receptors or fluid-phase endocytosis, results in the translocation of intact insulin to the cytoplasm or nucleus. Whether or not insulin internalization plays a role in insulin action is still unproved and controversial. Several studies have suggested that endosomeassociated insulin receptors may play an important signal transduction role (Smith and Jarett, 1983; Khan et al., 1986; Klein et al., 1987; Bevan et al., 1995, 1996). Other studies have demonstrated specific insulin binding to intracellular organelles including the nucleus (Goldfine and Smith, 1976; Horvat, 1978; Goidl, 1979) and association of insulin with cytoplasmic proteins (Hari et al., 1987; Harada et al., 1995b). These results suggest, and this chapter intends to demonstrate, that insulin internalization may be something more than a circumstantial event and could be physiologically relevant.

6.Signaling Mechanisms Used by Other Internalized Hormones and Growth Factors

Although there are some clear distinctions between the actions of insulin and other polypeptide hormones and growth factors, significant similarities also exist. An understanding of the mechanisms used by these other agents may provide insights into potential pathways used by insulin. Cytoplasmic translocation and nuclear accumulation of hormones and growth factors is common. Epidermal growth factor (EGF) (Raper et al., 1987), acidic fibroblastic growth factor-1 (FGF-1) (Wiedtocha et al., 1994; Zhan et al., 1992; Prudosky et al., 1996)’ basic FGF (FGF-2) (Amalric et al., 1994; Hawker and Granger, 1992, 1994), interleukin-1 (Weitzmann and Savage,

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1992),prolactin (Clevenger et af., 1991), angiogenin (Moroianu and Riodan, 1994), nerve growth factor (NGF) (Eveleth and Bradshaw, 1992), growth hormone (Lobie et al., 1994) and insulin-like growth factor-l(1GF-1) (Blazer-Yost et af., 1992;Soler et al., 1990) all accumulate in nuclei (Burwen and Jones, 1987; Hopkins, 1994; Jans, 1994; Morel, 1994; Levine and Prystowsky, 1995). These hormones also have plasma membrane receptors and signal transduction systems in the cytoplasm that are believed to be activated by the ligand binding to the plasma membrane receptor. Several laboratories report that nuclear accumulation of ligands, irrespective of membrane signaling events, is required for full biological response. For example, FGF-1 exogenously added to cells activates the tyrosine kinase activity of the membrane receptor and stimulates DNA synthesis, but the effect on DNA synthesis requires nuclear translocation (Wiedtocha et al., 1994). FGF-2 utilizes a heparin sulfate proteoglycan to internalize and translocate to the nucleus (Amalric et al., 1994), where it activates DNA transcription and increases casein kinase I1 activity. Prolactin occupancy of the prolactin receptor results in a phosphorylation cascade implicated in the prolactin-induced activation of immediate-early genes, whereas cell proliferation was dependent on nuclear accumulation of the hormone (Prystowsky and Clevenger, 1994).Similar findings of obligatory nuclear accumulation have also been reported for angiogenin (Moroianu and Riodan, 1994). The ability, and frequently the requirement, of these hormones to enter the cytoplasm and nucleus demonstrates that cellular mechanisms exist to affect the translocation of polypeptide hormones into the cytoplasm and nucleus and provide support for the hypothesis that cytoplasmic and nuclear translocation of insulin may play an important role in its actions.

II. Mechanisms of Insulin Internalization Our laboratory and others have investigated insulin internalization in a variety of normal insulin target cell types, e.g., adipocytes, hepatocytes, fibroblasts, etc., and in cultured cells used as models of insulin target tissues, e.g., 3T3-Ll adipocytes, H35 hepatoma cells, and cells transfected with the cDNA of wild-type or mutated human insulin receptors. These studies have used biochemical techniques, i.e., measuring '251-insulininternalization (Backer et af., 1990, 1991, 1992; Paccaud et al., 1992; Rajagopalan et af., 1995;Reynet et af.,1994;Yamada et af.,1995) and ultrastructural techniques (Carpentier and McClain, 1995; Carpentier, 1992; Smith et al., 1991a, 1993, 1996; Shah et af., 1995).

INSULIN INTERNALIZATION AND OTHER PATHWAYS

A. Fluid-Phase, Constitutive, and Ligand-Induced Endocytosis of Insulin and Insulin Receptors Insulin is internalized by two mechanisms: fluid-phase and receptormediated endocytosis (Harada et al., 1992; Moss and Ward, 1991). Receptor-mediated endocytosis is further subdivided into constitutive and ligand-induced processes as discussed below. Because of the high affinity of the insulin receptor, the receptor-mediated pathway accounts for the majority of intracellular insulin at physiological insulin concentrations ( 4 nM). Fluid-phase endocytosis of insulin and other molecules is proportional to their extracellular concentration. The absolute rates of fluid-phase endocytosis can vary greatly among different cell types. Fluid-phase endocytosis becomes a major component of insulin internalization at concentrations greater than 10-50 nM (Smith and Jarett, 1990; Moss and Ward, 1991), depending on cell type. However, physiological concentrations of insulin increase fluid-phase endocytosis of extracellular molecules (Gibbs et al., 1986; Miyata et al., 1988), presumably including insulin. The mechanism by which insulin stimulates fluid-phase internalization is not completely understood, although it probably involves a signal transduction cascade resulting from the autophosphorylation of the insulin receptor (Kotani et al., 1995). It is therefore likely that some mutations in the insulin receptor affecting receptor phosphorylation, or differences in receptor phosphorylation caused by intracellular kinases or phosphatases, will decrease insulin’s effects on fluid-phase endocytosis. The magnitude of fluidphase endocytosis can be determined and appropriate corrections made to eliminate its contribution to ligand internalization. If these determinations are not made, or if conditions that favor fluid-phase endocytosis are not avoided, the internalization of insulin will not be indicative of the internalization of the insulin receptor. Another complication in assessing the mechanisms involved in insulin receptor internalization results from the fact that insulin receptors are internalized by two processes: insulin-stimulated and constitutive receptor internalization. Insulin-stimulated receptor internalization results from the redistribution and aggregation of dispersed occupied insulin receptors into endocytic structures. Ultrastructural studies using cultured cells have generally revealed that, at the earliest time points, the majority of the labeled insulin receptors are dispersed on microvilli (Carpentier and McClain, 1995; Carpentier et al., 1993; Smith et al., 1991a, 1993, 1996) (also see below). After several minutes, the labeling of the nonvillous plasma membrane is increased and the occupied receptors are aggregated compared to the initial distribution. This observation has been interpreted as evidence of an insulininduced aggregation and migration of insulin receptors from the microvilli

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to the plasma membrane. Occupied insulin receptors are later visualized in endocytic structures on the membrane and in endosomes within the cytoplasm. There is some disagreement whether insulin receptors are internalized exclusively by coated pits (Carpentier and McClain, 1995) or in both coated and noncoated invaginations or caveolae (Backer et al., 1991; Goldberg et al., 1988; Smith et al., 1991a, 1993). Some of this disagreement may be due to cell-specific differences (Smith and Jarett, 1988). In the absence of insulin, unoccupied receptors are constitutively internalized and recycled to the plasma membrane at a relatively constant, but probably cell-specific, rate with a TIE of -7 min (Smith and Jarett, 1983, 1987). Insulin affects the internalization, recycling and recruitment of numerous membrane proteins (Corvera et al., 1989; Smith et al., 1991b) as well as fluid-phase endocytosis (Gibbs et al., 1986; Miyata et al., 1988), which utilizes both coated and noncoated caveolae structures that contain the insulin receptors. It is likely that insulin affects the constitutive internalization of unoccupied insulin receptors via a signaling cascade resulting from occupied receptors. However, if the cell expresses mutated receptors, those signaling cascades may be impaired. Insulin binding to constitutively internalizing insulin receptors inevitably complicates the assessment of insulin-stimulated receptor internalization.

B. Insulin Receptor Mutations and Their Effects on Insulin Internalization Ligand-induced insulin receptor internalization has been presumed to require tyrosine phosphorylation of the insulin receptor /3 subunit. Previous ultrastructural analyses by Carpentier and colleagues (1993, Carpentier and McClain, 1995) and our laboratory (Smith el al., 1991a, 1993) have demonstrated that the majority of insulin initially bound to receptors on the microvilli of cultured cells and, after insulin binds, kinase-competent receptors rapidly migrated to the nonvillous surface and aggregated. In contrast, mobility of kinase-deficient receptors was severely restricted. Carpentier and McClain (1995) suggested that insulin binding and activation of the receptor kinase releases a constraint anchoring the receptor to the microvilli. Two observations in a recently completed study (Smith et al., 1996) in CHO cells expressing mutant insulin receptors suggest there may be an alternative explanation. The CHO cells used in this study were transfected with the wild-type insulin receptor expression plasmid pCVSHVIRc as well as expression plasmids encoding mutant human insulin receptors in which alanine replaced LYS'"~(CHOAIO~S), phenylalanine or alanine replaced Tyrgm(CHOmw or CHOAgm), or there was a deletion of (CHOAgm). Receptor mobility was assessed ultrastructurally Ala954-Asp965

INSULIN INTERNALIZATION AND OTHER PATHWAYS

251

using colloidal-gold insulin (Au-Ins) (Smith et al., 1991a, 1993). First, the cells failed to migrate kinase-competent receptor expressed in CHOAghO from the microvilli compared to the wild-type receptors as shown in Table I. These results demonstrated that activation of the p subunit kinase activity was not sufficient to release an anchoring constraint associated with the receptor on the microvilli. Second, in prefixed CHOAIOIS cells used to demonstrate the distribution of unoccupied insulin receptors, the percentage of kinase-deficient receptors on the nonvillous membrane was significantly reduced compared to the wild-type receptor. This observation is consistent with the hypothesis that the kinase-deficient insulin receptor has an impaired ability to interact with the cellular machinery involved in the constitutive movements of the receptor to and from the microvilli. Although it is not clear how the substitution of Ala"Ix for Lys'o'x might have this effect, if this hypothesis is correct the decreased mobility of insulin-occupied kinase-deficient receptors may not be directly related to insulin's inability to activate the receptor kinase. In addition to the migration of occupied receptors from the microvilli, insulin-induced receptor internalization is distinguished from constitutive receptor internalization by the concentration or aggregation of the occupied receptors in endocytic structures (Carpentier, 1992). Coated pits are a specialized endocytic mechanism responsible for the concentrative endocy-

TABLE I Redistribution of Au-Ins-Occupied Insulin Receptors to the Nonvillous Plasma Membrane of CHO Cell Clones % of total extracellular particles

Cell type

CHOtIIRc CHOAwi CHOFY~,I) CHOAY~,II CHOAIIIIX

Prefixed

5 Min (A)

18.1 2 3.2 12.3 5 2.8 14.3 t 2.3 15.5 2 2.8 7.7 % 2.6*

41.6 2 5.8 (23.5) 29.5 2 3.8* (17.2) 39.0 2 3.1 (24.7) 16.7 ir 1.8* ( 1 . 1 ) 8.5 t 4.1%(0.8)

15 Min (A) 44.8 2 38.0 2 44.5 ir 26.5 5 19.4 2

3.8 (26.7) 4.2 (25.7) 2.9 (30.2) 3.2* (11.0) 3.0* (11.7)

____~

Nore. The initial distribution of occupied insulin receptors was determined on cells prefixed with 4% paraformaldehydc in PBS buffer for 30 min at 4°C and washed with 50 mM Tris-HCI in PBS to neutralize reactive amino groups. The prefixed cells were resupended in KRM buffer and incubated in 17 nM Au-Ins for 60 min at 4°C. Unfixed CHO cells were incubated with 17 nM Au-Ins at 37°C for the indicated times. After incubations. cells were diluted 10fold in 2%glutaraldehyde in PBS buffer at 4°C and processed for electron microscopic analysis (Smith e t a / . . 1988). The location of Au-Ins particles on a minimum of 100 randomly selected cells was determined for each incubation condition in three experiments. Au-Ins particles were analyzed for the location, e.g., microvilli and nonvillous plasma membrane (Smith et a/., 1991). Results presented are the mean 2 SD of the observations in three experiments. * Significantly different from CHOl1IRc cells at same condition at p < 0.005.

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tosis of most, if not all, ligand-receptor complexes (Smythe and Warren, 1991; Anderson, 1992). The NPEY960and GPLY953motifs in the insulin receptor are similar to the NPVYW and YZoXFWsequences that are required for internalization of low-density lipoprotein and transferrin receptors, respectively, by coated invaginations. Analysis of 12SI-insulininternalization in CHO cells expressing receptors with alanine (CHOA960)or phenylalanine (CHOF960)substitutions or a deletion of Ala954-Asp965 (CH0A960) was used in an attempt to deduce the importance of the tyrosine phosphorylation in these motifs in insulin internalization and action (Backer et al., 1990, 1991). At low insulin concentrations, internalization was not affected in CHOF960but was decreased by -40 or -70% in CHOAgmand CHOA960cells, respectively. Because this motif was supposed to be involved in the coated invagination-mediated internalization of insulin receptors, Backer et al. (1992) concluded that tyrosine phosphorylation was not required for the receptor to be internalized by coated pits but the alanine substitution and deletion mutations affected the conformation of the coated pit docking site. Recent studies (Smith et al., 1996) confirmed that phosphorylation of Tyr 960 was not required for the aggregation of receptor in coated pits. However, the alanine substitution, which decreased insulin internalization and which Backer et al. (1992) proposed would cause a destabilization of the /3 turn in the juxtamembrane region of the receptor and prevent the normal association of the IRA960in the coated invagination, did not affect the ability of the receptor to aggregate in coated invaginations. The alanine substitution affected the movement of the receptor from the microvilli to the cell surface as shown in Table I. The deletion mutation, which was presumed to cause a severe conformational change and “bad fit” to the coated invagination, did have that effect, but the more significant cause of the diminished internalization was the inability of the receptors to aggregate and move from the microvilli as shown in Table 11. There may be numerous problems associated with using cells expressing mutated insulin receptors to study the binding and internalization of insulin receptors and the biological effects presumably related to insulin receptor structure. One of the problems may be small but crucial changes in the conformation of the receptor fl subunit that prevent normal association of the receptor with intracellular molecules. Another problem with these cells are potential artifacts resulting from the overexpression of proteins. Our ultrastructural observations of various transfected cells have shown that many have significant morphological differences from parental or siblingtype cells. The effects that these differences may have on insulin processing and responses are often unknown.

253

INSULIN INTERNALIZATION AND OTHER PATHWAYS TABLE II Aggregation of Au-Ins-Occupied Insulin Receptors on CHO Cell Clones % of total particles

Cell type

Prefixed

CHOHIRC CHOA%o CHOCHOAW CHOAlols

6.2 4.4 6.8 6.0 7.0

? 1.2 ? 0.8

2 1.2

t 1.0 ? 1.4

5 Min (A) 18.7 ? 2.4 (12.5) 10.6 ? 1.8* (6.2) 15.0 2 2.1 (8.2) 5.7 t 0.8* (-0.3) 7.4 t 2.1* (0.4)

Note. Prefixed and unfixed CHO cells were incubated with 17 nM Au-Ins and prepared for electron microscopic analysis (Smith et al., 1988). The extent of receptor aggregation, i.e., the clustering of AuIns particles, on the microvilli, nonvillous plasma membrane, caveolae, and coated pits were determined as previously described (Smith et al., 1991). Results presented are the mean ? SD of the observations in three experiments. * Significantly different from CHOHIRc cells at same condition at p < 0.005.

C. Potential Role of Endosome-Associated Insulin in Biological Effects Endosomes isolated by cell fractionation techniques are sometimes characterized on a time continuum, i.e., early to late, that correlates with differences in the processing of the internalized ligand. It is debatable whether these endosomes are structurally and functionally discrete vesicles that use carrier vesicles to transport ligands between them (Griffiths and Gruenberg, 1991) or the same vesicles that mature and acquire different functions during this time frame (Stoorvogel ef al., 1991). Another alternative is that the in vivo structures are not really vesicles but rather an intestine-like continuous tubular system with discrete functional sections (Hopkins et al., 1990; Tooze and Hollingshead, 1991). Whatever the structure of the endosomal apparatus may be, until recently the primary function ascribed to this system was that of ligand degradation and receptor processing and recycling (Burgess et al., 1992a). More than a decade ago, it was suggested that the internalization of insulin provided a potential signal transduction mechanism. The original role of kinase-activated receptors in endosomes was theorized to be to move through the cytoplasm activating IRS-1, which

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was then considered to be the exclusive signal transduction molecule, which in turn would carry out its signaling functions through the binding and activation of numerous SH2 domain-containing signaling molecules. This theory has been supported by studies by Khan et al. (1986) and Klein et al. (1987) demonstrating increased autophosphorylation and tyrosine kinase activity of the insulin receptor associated with endosomes. Studies by Kublaoui et al. (1995) failed to detect IRS-1 associated with the plasma membrane in insulin-treated adipocytes; however, endosomal fractions contained 20% of the cellular IRS-1. These results suggested that endosomal insulin receptors may play a major role in signal transduction through IRS-1. Recently, tyrosine-phosphorylated IRS-1 has been detected in the intracellular "low-density microsome" fraction of insulin-treated 3T3-Ll adipocytes by Heller-Harrison et al. (1995). However, because the association and phosphorylation of IRS-1 in the microsomes occurred at 4"C, they suggested it may not be related to insulin receptor internalization, which is not supposed to occur at 4°C. Studies with the E G F receptor (Di Guglielmo et al., 1994) demonstrated that SH2 domain-containing proteins, i.e., Shc and GRB-2, associated with the internalized, tyrosine-phosphorylated receptor in the endosome. Despite the demonstration that SH2 domain-containing molecules associate with isolated tyrosine phosphorylated insulin receptors (Staubs et al., 1994), no association with insulin receptors occurred in hepatic endosomes as a result of insulin treatment (Di Guglielmo et al., 1994), perhaps because of the rapid dephosphorylation of endosomal insulin receptors (Burgess et al., 1992b). In our continuing attempt to determine the potential role of insulin internalization in its effects on cell processes we developed a cell fractionation scheme using differential and iodixanol gradient (Ford et al., 1994) centrifugation to isolate membrane fractions from H35 hepatoma cells that showed time-dependent association of 0.7 nM '251-insulinas described elsewhere (R. Smith et al., manuscript in preparation). Electron microscopic examination revealed that the three major fractions isolated contained vesicles of various sizes, consistent with the different densities in the gradient. Coomassie blue staining of fractions subjected to SDS-PAGE suggested that the fractions consisted of vesicles composed of different proteins (data not shown). Western blot analysis identified the three fractions as caveolae, endosomes, and plasma membranes. As shown in Fig. 2, the association of '251-insulin with the caveolae, endosomes, and plasma membranes was specific, i.e., displaced by 4 pM unlabeled insulin. Significant amounts of '251-insulinwere associated with the caveolae at 4°C and incubation at 37°C for 5 or 15 min increased caveolae-associated insulin by about 50%. Association of '251-insulin with the endosome fraction was both time and temperature dependent; there

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INSULIN INTERNALIZATION AND OTHER PATHWAYS

a

r

3 -

1.5

1.0

3

u)

C

E

0.5 0.0

Caveolae

Endosomes

Plasma Membranes

Membrane Fraction

..

FIG. 2 Time- and temperature-dependent insulin association to subcellular membrane fractions. H35 hepatoma cells were incubated with 0.7 nM '251-insulinin the presence of 4.2 p M unlabeled insulin for 15 min at 37°C or in the absence of unlabeled insulin at 4°C for 15 min at 37°C for 5 or 15 min The cells were washed at 4°C and membrane fractions isolated on a Iodixanol gradient (R. Smith et al., manuscript in preparation). '251-insulin associated with each subcellular fraction was determined in a gamma counter and the results were expressed as fmol insulin per milligram protein. Depicted are the mean results of three experiments: the SD was too small to illustrate.

was no specific accumulation of insulin in endosomes at 4°C. Insulin binding to the plasma membrane was not significantly different under the time and temperature conditions used in these studies. We analyzed the effects of insulin on the protein tyrosine phosphorylation in the cytosol and three membrane fractions as shown in Fig. 3. H35 hepatoma cells were incubated at 4 or 37°C for 0, 5, or 15 min with 8.5 nM insulin. The cells were subjected to subcellular fractionation and equal protein concentrations of cytosol, caveolae, endosomes, and plasma membranes were solubilized and subjected to SDS-PAGE and Western blot analysis with anti-phosphotyrosine antibody. At 37°C insulin caused a 15 fold increase in phosphorylation of IRS-1 within 5 min in the caveolae fraction. This increase was substantially larger than the 3- to 6-fold increases seen in the cytosol, endosomes, or plasma membrane fractions. Interestingly, insulin-induced tyrosine phosphorylation of the insulin receptor fl subunit was approximately equal in all three membrane fractions. We also observed insulin-induced phosphorylation of an unidentified 72-kDa protein in the caveolae and to a lesser extent in the endosomes. This substrate was not apparent in the plasma membrane fraction. Insulin incubation for 15 min caused a 2-fold increase in the tyrosine phosphorylation of the 46-kDa isoform of Shc in caveolae, which was not detected in the endosomes or plasma membrane fraction. The phosphorylated 52-kDa

256

ROBERT M. SMITH E r AL. Caveolae

Cytosol

Endosomes

Plasma Membranes

5

5

IRS-I -D

Minutes at 3 7°C

0

5 1 5

0

5 1 5 0

1 5 0

15

FIG. 3 Effect of insulin on protein tyrosine phosphorylationin cytosol and membrane subcellular fractions of H35 hepatoma cells. H35 cells were incubated with 8.5 nM insulin for the indicated times at 37°C. Isolated subcellularfractionswere prepared (R. Smith et al., manuscript in preparation),solubilized, and subjected to SDS-PAGE and Western blot analysis (Harada ef al., 1995a).

Shc isoform was found in all three membrane fractions. When the cells were incubated at 4°C (data not shown), we observed virtually identical insulin-induced tyrosine phosphorylation of IRS-1 in all three membrane fractions, confirming the report of Heller-Harrison et a/. (1995). We also detected increased tyrosine phosphorylation of the insulin receptors in the endosomes. Because there was no insulin internalization into the endosomes at 4"C, as shown in Fig. 2, these data suggest that, even at 4"C, insulinsensitive phosphotyrosine kinases and some signaling mechanisms are able to cause the phosphorylation of endosome-associated IRS-1 and, interestingly, the insulin receptor. Figure 4 illustrates the concentration of various membrane proteins and signaling molecules in the three fractions as determined by Western blot analysis. Caveolin, a marker of membrane caveolae, was associated almost exclusively with the fraction we call caveolae. Vesicle-associated membrane protein-2 was enriched in the endosome fraction and found to lesser extents in both the caveolae and plasma membranes. Insulin receptors were found at approximately the same concentration in all fractions and at all time points except the 0-min incubated endosomes. The small insulin-induced increase in endosome-associated insulin receptor may represent receptor internalization. However, the absence of an increase in insulin receptors in the caveolae suggests that the total number of insulin receptors in those structures is not significantly affected. This contrasts with observations in other studies that suggest insulin-occupied receptors aggregate in endocytotic structures (Carpentier, 1992; Smith et af., 1991a). One explanation for the difference between the Western blot and ultrastructural analyses is that the latter only observed occupied receptors, which are only a fraction of the

Caveolae

Endosomes

Plasma Membranes

insulin Receptor

IRS-I Shc

Gab-I Minutes at 31°C

0 5 1 5

0

5 1 5

0 5 1 5

FIG. 4 Effect of insulin on protein distribution in subcellular membrane fractions isolated from H35 hepatoma cells. H35 cells were incubated with 8.5 nM insulin for the indicated times at 37°C. Isolated subcellular fractions were prepared as described elsewhere (R. Smith et al., manuscript in preparation), solubilized, and subjected to SDS-PAGE and Western blot analysis (Harada er al., 1995a).

total receptor concentration. Because only a small fraction of the occupied receptors aggregate in endocytotic structures, the effect on the total concentration of receptors in the internalization pathway may be, as suggested by the Western blot analysis, undetectable. At Time 0, the concentrations of IRS-1, Shc, and Gab-1 were greatest in the caveolae fraction and 5-10 times higher than those in the endosomes or plasma membranes (Fig. 4) or cytosol (data not shown). The concentration of IRS-1 in both the caveolae and endosomes was increased about 1.5-fold by 5 and 15-min insulin incubation at 3TC, probably as a result of IRS-1 translocation from the cytosol as suggested by Heller-Harrrison et al. (1995). There was no change in IRS-1 concentration in any of the fractions when cells were incubated at 4°C (data not shown). Insulin treatment at 37°C had no effect on the concentrations of Shc and Gab-1 associated with the membrane fractions. These results suggest the distribution of these signaling molecules, in contrast to IRS-1, among the membrane fractions does not appear to be regulated by insulin receptor tyrosine phosphorylation. The relatively high concentration of IRS-1, Gab-1, Shc, and pp72 in the structures involved in internalization of insulin provides additional support for the hypothesis that insulin internalization is physiologically relevant and linked to pleiotropic effects of insulin.

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ROBERT M. SMITH E T A .

111. Translocation of Insulin t o the Cytoplasm and Nucleus Results from several laboratories provide evidence that insulin specifically binds to isolated intracellular structures, particularly the nucleus. Goldfine and Smith (1976) were the first to demonstrate insulin binding to receptors in the nucleus and nuclear membrane and suggested that such an interaction could play an important role in insulin action. During the same era, other studies confirmed those observations (Goidl, 1979; Goldfine et al., 1985; Horvat, 1978). Others (Hari et al., 1987; Shii and Roth, 1986) showed that insulin interacted with and was degraded by insulin-degrading enzyme (IDE) in the cytoplasm of intact cells. These studies support the hypothesis that insulin is translocated from endosomes to the cytoplasm after it is internalized. In 1987,our ultrastructural studies observed covalently labeled monomeric ferritin-insulin complex in nuclei of 3T3-Ll adipocytes (Smith and Jarett, 1987). Subsequently, nuclear insulin has also been demonstrated in a variety of proliferating cultured cell types using immunoelectron microscopic techniques (Blazer-Yost et al., 1992; Heyner et al., 1989; Smith and Jarett, 1993; Soler et al., 1989; Thompson et al., 1989). Because insulin is known to affect nuclear processes, such as the uptake of macromolecules (Jiang and Schindler, 1988; Soler et al., 1992) and gene transcription (O’Brien and Granner, 1991), our observations led to the hypothesis that insulin internalization and translocation to cytoplasm and nucleus could be physiologically relevant (Lin et al., 1992).Biochemical studies characterized part of the nuclear uptake process (Smith and Jarett, 1990), including the demonstration that nuclear accumulation of insulin resulted from both receptor-mediated and fluid-phase endocytosis (Harada et al., 1992). The insulin in the nucleus was associated with the nuclear matrix and was intact or was bound to a high molecular weight complex (Thompson et al., 1989). Nuclear accumulation of intact insulin was increased more than fivefold when IDE activity was inhibited by 1,lO-phenanthroline (Harada et al., 1993). Because IDE is primarily a cytoplasmic enzyme (Akiyama et al., 1988), our results provided additional evidence that internalized insulin left the endosome and entered the cytoplasm, prior to accumulating in nuclei, as suggested by the in vivo cross-linking of 1251-insulinto IDE reported by Hari et al. (1987). Although our early studies using covalently linked ferritin-insulin (Smith and Jarett, 1982) showed a significant number of ferritin particles in nuclei (Smith and Jarett, 1987),endogenous cytoplasmic ferritin was indistinguishable from that labeling the insulin (Blackard et al., 1986; Smith and Jarett, 1988). Experiments using a noncovalently linked colloidal gold-insulin complex (Smith et al., 1988) were not successful in demonstrating cyto-

INSULIN INTERNALIZATION AND OTHER PATHWAYS

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plasmic or nuclear insulin at least in part because the insulin dissociated from the gold particle in intracellular acidic organelles (Smith and Jarett, 1993). Studies using electron microscopic autoradiography reported 1251insulin grains in the cytoplasm and nucleus (Bergeron et al., 1979; Carpentier et af., 1979; Fan et af., 1983). However, autoradiography did not provide sufficient spatial resolution to determine whether the insulin was actually in the cytoplasm or nucleus or associated with nearby plasma membrane or endosomes, as was usually assumed. To circumvent many of the problems in earlier studies, we prepared a covalently linked nanogold-insulin complex (nG-I) (Shah et al., 1995). The complex was easily identified on or within cells after preembedding silver intensification, it competed with '251-insulinfor binding to the insulin receptor, and it was biologically active. In addition, nG-I appeared to be a substrate for IDE because it inhibited the degradation of '251-insulin in intact cells and isolated cytosol. nG-I was observed bound to insulin receptors on microvilli and plasma membrane on the cell surface, inside endosomes, in the cytoplasm and in nuclei of cells incubated at 37°C as shown in Fig. 5. Incubation of cells with 2 m M 1,lO-phenanthroline, which inhibited IDE activity, resulted in an increase in the amount of the nG-I complex in the cytoplasm and nuclei. These ultrastructural results provided the

FIG. 5 Electron micrograph of nanogold insulin translocation to the cytoplasm and nucleus. H35 hepatoma cells were incubated for 30 min with 1.7 nM nanogold insulin and prepared for electron microscopic analysis (Shah et al., 1995). Silver-intensified nanogold particles (indicated by arrows) were observed in the nucleus (N), the cytoplasm, and on the plasma membrane. Bar = 0.5 pm.

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ROBERT M. SMITH E r AL.

first incontrovertible evidence that internalized insulin is translocated from endosomes to the cytoplasm, where it can be degraded by IDE, interact with other cytoplasmic proteins, and be translocated to nuclei. These findings also supported our hypothesis that the translocation of insulin, or a complex of insulin and cytoplasmic insulin-binding proteins (Harada et al., 1995b), may play a role in insulin’s regulation of gene transcription and cell proliferation. A. Mechanisms Involved in Translocation of Macromolecules into the Cytoplasm

The mechanism(s) by which exogenous ligands and macromolecules are transported through the lipid bilayer of the plasma membrane or endosome and enter the cytoplasm after internalization is not well understood. Studies by Papini et al. (1993) demonstrated that the translocation of internalized diphtheria toxin to the cytosol was a two-step process. Acidification in early endosomes resulted in a proteolytic-induced conformational change that could be inhibited by bafilomycin A1 (Baf) and permitted, in later Bafinsensitive endosomes, the reduction of the interchain disulfide bond joining the diphtheria toxin A and B promoters. These two steps are, at least in theory, compatible with the translocation of insulin. Insulin dissociates from its receptor and may be proteolytically processed in acidic endosomes (Hamel et al., 1988) and it has inter- and intrachain disulfide bonds that could be reduced. Recently, we began a series of studies using H35 hepatoma cells to determine the mechanisms by which insulin is translocated from the endosomes to the cytoplasm. In previous studies we and others (Marshall and Olefsky, 1979; Smith and Jarett, 1990) found that a number of acidotrophic agents, (e.g., chloroquine, ammonium chloride, Tris-chloride, etc.) and ionophores (e.g., monensin, nigericin, and valinomycin) increased intracellular insulin and inhibited insulin degradation when cells were incubated with physiological insulin concentrations. The increase in intracellular insulin along with the decrease in insulin degradation has been attributed to preventing acidification of the endosome, thus preventing the dissociation of the insu1in:receptor complex. However, this is probably not the full explanation. These agents also increase intracellular insulin and block insulin degradation at high insulin concentrations (>lo nM) in which significant amounts of insulin are internalized by fluid-phase endocytosis (Moss and Ward, 1991; Harada et al., 1992). Under these conditions, endosome acidification should not be needed to dissociate the insulin from its receptor. Many of these agents also prevent the normal recycling of insulin and other receptors and transporters to the cell membrane (Marshall and Olefsky,

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261

1979; Smith and Jarett, 1990) most likely by affecting membrane transport processes. In recent studies we have examined the effects of Baf, a specific inhibitor of vesicular ATPase in early endosomes (van Weert et al., 1995), on insulin degradation and accumulation of '251-insulin in H35 hepatoma cells. Pretreatment of H35 cells with 100 nM Baf did not result in decreased insulin binding as did monensin and other acidotropic agents (Marshall and Olefsky, 1979; Smith and Jarett, 1990) due to downregulating the insulin receptor, i.e., inhibiting the recycling of constitutively internalized receptors (data not shown). Baf had no significant effect on total cell-associated insulin when cells were incubated with either 0.7 or 17 nM '251-insulin and no effect on cellular insulin degradation, nuclear accumulation, or distribution of '2sI-insulin among membrane fractions when the cells were incubated with 17 nM insulin. In contrast, when cells were incubated with 0.7 nM 1251insulin, Baf decreased insulin degradation by more than 50% and blocked nuclear accumulation of insulin (R. Smith et al., unpublished observations). The cell fractionation scheme described previously was used to determine where Baf affected the insulin internalization pathway. As shown in Fig. 6, Baf increased the retention of insulin in the endosomes without affecting insulin association with the caveolae or plasma membranes. These results suggest that Baf may affect dissociation of the insu1in:receptor complex by blocking acidification of the endosome. By preventing insulin translocation from the endosome, Baf decreased insulin translocation to the cytoplasm and its degradation by IDE and the translocation of insulin to the nucleus. Studies are currently under way to characterize the mechanisms involved in the translocation of insulin from endosomes to the cytoplasm.

B. Interaction of Insulin with Cytoplasmic Proteins and Their Identification Previous sections of this chapter reviewed the data suggesting or demonstrating that internalized insulin enters the cytoplasm from the endosomes before entering the nuclei of various cells. We also demonstrated that the nuclear uptake of insulin is regulated in part by cytosolic I D E (Harada et al., 1994). These findings raised the question whether insulin interacts with other cytosolic insulin-binding proteins (CIBPs) that may be part of an insulin signaling pathway or mechanism to transport insulin to the nucleus. Insulin-binding studies were performed with cytosol isolated from H35 hepatoma cells, rat liver and muscle, and 3T3-Ll fibroblasts and adipocytes (Harada et al., 1995b; Lee et al., 1996). B26-1251-labeledinsulin (1.7 nM) was incubated in the cytosol at 4°C in the presence or absence of 4.2 p M unlabeled insulin for various times and cross-linked to cytosolic proteins

ROBERT M. SMITH ET AL.

262

Q

h

F

-

P I 0

E

Caveolae

Endosomes

Plasma

Membranes

Membrane Fraction FIG. 6 Effect of bafilomycin on insulin association with subcellular membrane fractions. H35 hepatoma cells were incubated with 0.7 nM (A) or 17 nM (B) 'Z51-insulinfor 15 min in the absence of I€!or presence of 100 nM bafilomycin at 37°C. '251-insulinassociated with each

subcellular fraction was determined in a gamma counter and the results were expressed as fmol insulin per milligram protein. Results are the mean of three experiments; the SD was too small to illustrate.

by disuccinimidyl suberate. The solubilized extracts were analyzed by reducing and nonreducing SDS-PAGE and autoradiography. The maximum cross-linking occurred at different times of incubation and seemed to correlate with the rate of insulin degradation in the different cell types (Harada et al., 1995b). Table I11 shows the molecular weights of the CIBPs in several insulin target tissues and cultured cells. B26-lz5I-1abeledinsulin was specifically cross-linked to both tissue-specific and common CIBPs. The 110-kDa CIBP was common to all cells and tissues. Muscle and 3T3-Ll adipocytes had only two CIBPs, the fewest of the cells examined. Rat liver had the most (eight), whereas CHO and H35 hepatoma cells had five and six, respectively. Studies were undertaken to identify the CIBPs. The 110-kDa protein was identified as IDE by an overlay technique consisting of Western blot analysis (ECL method) with anti-IDE antibodies and autoradiography of the B26-'251-insulincross-linked proteins was performed on the same nitro-

263

INSULIN INTERNALIZATION AND OTHER PATHWAYS TABLE Ill Comparison of Specific Cytoplasmic Insulin-Binding Proteins Affinity Labeled with B26-'251-lnsulin

Molecular mass (kDa)

H35 hepatoma cells

Rat liver

Skeletal muscle

CHO cells

3T3-Ll adipocytes

110" 82 78'

110" 82 78' 58 55' 45 27

110"

110" 82 78' 58

110"

55' 45 27

5.5'

27

27

Note. Cytosol fractions were prepared from the tissues, diluted to 1 mg proteinhnl, and incubated with 1.7 nM B26-'2sI-insulinin the absence or presence of 4.2 pM unlabeled insulin. Insulin was cross-linked to binding proteins with 0.5 mM disuccinimidyl suberate and subjected to SDS-PAGE analysis (Harada et al., 1995b). Insulin-degrading enzyme. Glucose-regulated protein 78. ' Cellular thyroid hormone-bindingprotein, glutathione insulin transhydrogenase,and protein disulfide isomerase.

cellulose membrane (Harada et al., 1995b). Other CIBPs, including CIBP p55 and CIBP p82, were purified by insulin-agarose affinity chromatography, SDS-PAGE, and electroelution.The proteins were subjected to amino acid sequence analysis. CIBP p55 was homologous with cellular thyroid hormone-binding protein (CTHBP), also known as protein disulfide isomerase (PDI) or glutathione insulin transhydrogenase (GIT). CTHBP is a cytoplasmic protein that binds intracellular thyroid hormone and plays a crucial role in regulating 3,3',5-triiodo-~-thryonine transcriptional responses (Ashizawa and Cheng, 1992). Varandani and colleagues (Vanadani et al., 1975,1978; Hern and Varandani, 1983) characterized insulin's inactivation by GIT, the regulation of GIT's expression or activity by various factors, e.g., insulin, other hormones, and phospholipids, and GIT's cellular distribution in endosomes. CIBP p78 was identified as glucose-regulated protein 78 (GRP78). The overlay technique, using antibodies to PDI, CTHBP or GRP78, confirmed the identity of these CIBP. Several lines of evidence support the hypothesis that some CIBPs may play a physiological role in the insulin signaling network. First, in H35 cells, serum deprivation for 24 h markedly reduced the amount of cross-linking of insulin to the various CIBPs, whereas the addition of fresh serum resulted in very strong B26-'251-insulincross-linking. In addition, insulin treatment of the serum-deprived cells for 1 h yielded cytosol in which B26-'251-insulin cross-linkingto CIPBs was maximal. Thus, the contents of the culture media

264

ROBERT M. SMITH ET AL.

(e.g., growth factors, cytokines, etc.) and insulin regulate the extent of insulin binding to the CIBP (Harada et al., 1995b). Second, Kupfer et al. (1994) identified IDE as the receptor accessory factor for glucocorticoid and androgen receptors and suggested that the ability of this protein to bind both insulin and steroid hormones may explain the competitive effects of the hormones on gene expression. Harada et al. (1996a) found that dexamethasone treatment of intact cells or isolated cytosol of H35 cells inhibited insulin cross-linking to IDE. Dexamethasone's effect on insulin binding to IDE may be involved in the ability of steroid hormones to modulate insulin signal transduction. Finally, an interesting pattern was observed in the expression of CIBPs during the differentiation of 3T3-Ll fibroblast to the adipocyte phenotype (Lee et al., 1996). In the fibroblast form, significant amounts of B26-'251-insulinwere cross-linked only to IDE; there was little or no insulin binding to CTHBP. However, in the differentiated adipocytes, insulin cross-linking to both proteins was readily observed and insulin-degrading activity significantly increased. These findings suggested the binding of insulin to CTHBPlGIT and the effects of CTHBP/ GIT on insulin may be involved in IDE's ultimate degradation of insulin. GRP78, also known as immunoglobulin heavy chain-binding protein, is a member of the heat shock protein family and a molecular chaperone expressed in the endoplasmic reticulum, cytoplasm, and nucleus (Hass, 1994). In the endoplasmic reticulum, GRP78 is involved in polypeptide translocation, protein folding, and protein degradation. Posttranslational modification (i.e., ADP-ribosylation and serinehheonine phosphorylation) of GRP78 regulates its affinity to cellular ligands (Hendershot et al., 1988). Although GRP78 has no known role in insulin action or processing, its expression levels are regulated by intracellular glucose and the development of diabetes in mice correlated with an increased level of GRP78 in liver and brain (Parfett et al., 1990). Immunocytochemical data from our laboratory suggest that insulin treatment masks the carboxyl terminus of cytoplasmic GRP78 in cells and tissues with wild-type but not kinase-deficient insulin receptors (Tezuka et al., 1996).The cause of the carboxyl-terminus masking and its relationship to insulin action or processing is unknown. It is not likely that the demonstration of specific CIBPs (Harada et al., 1995b; Lee et al., 1996) is due to insulin's well-known ability to absorb. First, although nonspecific or nonsaturable absorption to some proteins was observed, B26-'251-insulin cross-linking to the reported CIBPs was completely displaced by unlabeled insulin, which is a classic indication of specificityand saturability. Second, insulin was not cross-linked to numerous structurally similar proteins, e.g., HSWO, GRP94, ERp72, etc. Third, insulin's interaction with two of the identified CIBPs, IDE and CTHBP/GIT, has been demonstrated by other laboratories. These two proteins, as discussed previously, are known to bind steroid and thyroid hormones in the cyto-

INSULIN INTERNALIZATION AND OTHER PATHWAYS

265

plasm and play important roles in gene transcription. The translocation of insulin to the cytoplasm and its interaction with CIBPs provides additional potential signaling pathways that may account for insulin’s pleiotropic effects on cell growth and metabolism.

C. Interaction of Insulin with the Nuclear Matrix Having fully established that insulin was internalized, traversed the cytoplasm, and translocated into the nucleus in a variety of tissues and cells, it was important to characterize the nuclear insulin and its uptake route. The first question asked was whether the nuclear insulin was intact and did the plasma membrane insulin receptor go to the nucleus as well. Soler et al. (1989) studied these questions in H35 hepatoma cells and NIH 3T3 fibroblasts transfected with the human insulin receptor (HIR 3.5) using both biochemical and immunoelectron microscopy. Nuclear insulin uptake was time, temperature, and concentration dependent. Immunoelectron microscopy demonstrated the insulin in the nucleus was immunologically intact and associated with the heterochromatin as we had found in other studies (Thompson et al., 1989). Sephadex G-50 chromatography of insulin extracted from the nucleus showed only 1% eluted as [ L251]tyrosine,consistent with the insulin being structurally intact. Plasma membrane insulin receptors were easily detected by immunoelectron microscopy on the plasma membrane and in endosomes but were not found in the nucleus. Wheat germ agglutinin purified extracts of nuclei from control and insulin-treated cells were Western blotted and revealed that there was no insulin receptor present. Thus, insulin accumulates in the nucleus without its receptor. This finding is consistent with insulin escaping from the endosome and interacting with cytoplasmic proteins before entering the nucleus. Thompson et al. (1989) demonstrated that the vast majority of nuclear insulin was associated with the nuclear matrix. Nuclei were isolated from cells incubated with labeled insulin and extracted with DNase I, RNase A, and high salt. More than 75% of radiolabeled insulin was still associated with the nuclear matrix. Immunoelectron microscopy showed the insulin on matrix protein. SDS or high urea solubilized the matrix-associated insulin. Gel filtration revealed that half of the insulin eluted with intact insulin while half came out in the void volume associated with a high-molecular-weight matrix protein complex. This pattern was confirmed in subsequent studies by Harada et al. (1993). Next, Soler et al. (1992) studied the site of interaction of insulin with the nuclear membrane of isolated nuclei using 10-nm diameter gold particles containing five to seven insulin molecules and stabilized with BSA (Aulo-Ins) (Smith et al., 1988). Surprisingly, the Aulo-Ins entered the nu-

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ROBERT M. SMITH E r AL.

cleus through the nuclear pore and attached to the heterochromatin in the absence of ATP. This uptake was striking because the gold particles stabilized with BSA were =13 nm in diameter and should not have been passively transported through the nuclear pore. In control experiments, Aulo-BSA without insulin did not accumulate in the nucleus. However, when unlabeled insulin was added along with the Aulo-BSA,the gold complex accumulated in the nucleus. This result suggested that insulin dilated the nuclear pore allowing the 13-nm particle to enter the nucleus by diffusion. This effect was insulin dose dependent (0.02-17 nM). When Au15-BSA or AuZ0-BSA were used, no nuclear accumulation was observed in the absence or presence of insulin, suggesting there was an upper limit to the diameter of the nuclear pore. Other hormones, growth factors, and insulin A or B chains were ineffective in stimulating Aulo-BSA uptake. The insulin-induced uptake was blocked by concanavalin A and mimicked by wheat germ agglutinin, the opposite of the effects found with ATP-dependent transport of proteins with nuclear localization sequences. Interestingly, although insulininduced uptake of Aulo-BSA into the nucleus did not require ATP, efflux of the Aulo-BSA particles from preloaded nuclei required ATP. This study indicated that insulin causes dilatation of the nuclear pore, allowing certain macromolecules without nuclear localization sequences to enter the nucleus of insulin-treated cells. If insulin has the same effects in intact cells, these macromolecules could play a physiological role in insulin’s control of gene expression and cell growth. D. Potential Roles of Cytoplasmic and Nuclear Insulin in Biological Effects

Goldfine and colleagues (Goldfine and Smith, 1976;Vigneri et al., 1978) and others (Horvat, 1978; Goidl, 1979) identified insulin receptors on various intracellular organelles, including the cell nucleus. The receptors on the nuclear envelope, which display significant differences from receptors found on the plasma membrane (Goldfine et al., 1985), may be relevant if insulin were translocated to the cytoplasm. Several laboratories have investigated the effects of insulin in isolated nuclei and compared those to insulin’s actions in vitro. Insulin added to isolated nuclear envelopes stimulated nucleoside trisphosphatase activity (Purrello et aL, 1982), which is involved in the transport of mRNA out of the nucleus in wiro (Schumm and Webb, 1978). Schumm and Webb (1981), Goldfine et al. (1985), and Schroder et al. (1990) all demonstrated that the addition of insulin to isolated nuclei increased RNA efflux. Insulin added to intact cells increased the transport of macromolecules into the nucleus ( Jiang and Schindler, 1988). Insulin had the same effects on macromolecular

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nuclear uptake when added to isolated nuclei (Schindler and Jiang, 1987; Soler et al., 1992). Miller (1998) reported that insulin injected into the cytoplasm of Xenopus oocytes stimulated RNA and protein synthesis. These effects were subsequently attributed, in part, to the internalization of insulin into the oocyte cytoplasm because microinjection of anti-insulin antibodies into the cytoplasm reduced insulin’s effects by approximately 40% (Miller and Sykes, 1991). These studies strongly suggest that there are intracellular mechanisms by which insulin in the cytoplasm may affect nuclear processes, including gene expression.

IV. Insulin-Responsive Pathways Other Than IRS-1 Involved in Insulin’s Effects on Immediate-Early Gene Expression The previous sections have concentrated on the potential role of the internalization process and the intracellular translocation of insulin to the cytoplasm and nucleus in insulin signaling. However, as reviewed by Bevan et al. (1996), internalized hormones in endosomal vesicles participate in the same signaling process generally attributed to “plasma membrane” receptors. In fact, the signaling potential of endosomes may be much greater than a similar amount of plasma membrane protein, as illustrated previously. The remainder of this chapter deals with cytoplasmic signaling pathways responsible for insulin’s effects on immediate-early gene expression that are activated as a result of insulin receptor occupancy either at the plasma membrane or in endosomes. Notwithstanding the overwhelming evidence that tyrosine phosphorylation of IRS-1 plays a central role in insulin signaling (Sun et al., 1992; Rose er al., 1994), the sheer number of potential signaling circuits suggests that other mechanisms of signal transduction have yet to be resolved. As described in the following sections, we and others have begun to actively investigate the role of these substrates in insulin action. A. Identification of p p l 2 0 in Chinese Hamster Ovary Cells as the Principle Insulin-Sensitive Phosphoprotein and Its Potential Role in Signal Transduction

Insulin’s effects are initiated primarily by insulin binding to it surface receptor and the subsequent tyrosine phosphorylation of IRS-1, IRS-2, or Shc (Cheatham and Kahn, 1995). These substrates bind through their tyrosine phosphorylation sites to SH2 domains of various signaling proteins, includ-

ROBERT M. SMITH €T AL.

268

ing PI 3-kinase, GRB-2, or Syp. Activation of these proteins, and the subsequent cascading activation of other intracellular signaling cascade molecules such as p2lraS,raf-2, MAP kinase, and S6 kinase, lead to many insulin actions. One of insulin’s major effectsis the activation or inactivation of genes, especially immediate-early genes involved in cell growth and proliferation. Mundschau et al. (1994) showed that induction of expression of immediate-early gene egr-2, but not c-fos, c-myc, or JE, was independent of platelet-derived growth factor (PDGF) receptor autophosphorylation using three different conditions in which PDGF receptor autophosphorylation was blocked. Eldredge et al. (1994) reported that epidermal growth factor (EGF) induced c-fos expression in cells expressing kinase-deficient EGF receptors. These two observations indicate the existence of signaling mechanisms that operate independently of receptor kinase activity to affect gene expression. Harada et al. (1995a) tested the possibility of such a divergent pathway in insulin signal transduction mechanisms regulating immediate-early gene expression in CHO cells stably transfected with neomycin-resistant plasmids (CHON,,), with genes for wild-type human insulin receptors (CHOHIRc), or with ATP binding site mutated human insulin receptors (CHOAlOl8). Insulin binding was markedly lower in CHONeothan in any other cell type. Insulin stimulated tyrosine phosphorylation of the insulin receptor and IRS-1 only in CHOHIR~cells as shown in Fig. 7. Similarly, PI 3-kinase activity and c-fos expression were stimulated by insulin only in CHOHIRc cells (Harada et al., 1995a). In contrast, as shown in Fig. 8, all three cell types showed a similar insulin-induced increase of egr-I mRNA as determined by Northern blot. These findings indicate that insulin activation of egr-2 in CHO cells occurs through an alternative signal transduction pathway of

ppl20

+

IR+ Insulin

-

+

-

+

-

+

FIG. 7 Effect of insulin on tyrosine phosphorylation of the insulin receptor and IRS-1 in CHO cell clones. CHONe0,CHOHIRc.or CHOAIOLB cells were incubated in the absence (-) or presence (+) of 10 nM insulin for 1 min at 37°C. Cells were lysed and phosphotyrosinecontaining proteins were immunprecipitated and subjected to SDS-PAGE and Western blot analysis (Harada et al., 1995a).

CHON,,

C

I

CHO",,

S

C

I

CHO,,,,,

S

C

I

S

FIG. 8 Effect of insulin or serum on immediate-early gene expression in CHO cell clones. CHON,,, C H O H I Ror ~ . CHO,lols cells were incubated with no addition (C), 17 nM insulin (I), or 20%fetal calf serum (S) for 60 min at 37°C. Total RNA was isolated and 15 pg RNA subjected to Northern blot analysis (Harada el al., 1995a).

the insulin signal transduction network that is independent of insulin receptor and IRS-1 phosphorylation. Figure 7 illustrates that insulin caused a marked increase in tyrosine phosphorylation of insulin receptor /3 subunit and IRS-1 in CHOHIRc cells but not in CHONeo and CHOA1018 cells. Interestingly, insulin increased tyrosine phosphorylation of pp120 in CHONeoand CHOAlOlB cells. In CHOHIRccells, phosphorylation levels of pp120 were low in both the basal and insulin-stimulated conditions. These data suggest that the phosphorylation of pp120 might be the pathway involved in egr-1 induction. We have analyzed tyrosine-phosphorylated pp120 by immunoprecipitation and Western blot techniques to identify its components. Phosphorylated ecto-ATPase and c-cbl (protooncogene product) were not detected in any cells. Phosphorylated focal adhesion kinase was detected in all three cells but was not insulin sensitive at 1 or 2 min. Syp (protein tyrosine phosphatase-2)-associated ppll5 was markedly increased in CHOHIRccells but not affected in CHONeoand CHOA1018 cells. pp115 was associated at the basal state in CHONeoand CHOA1018 cells. Tyrosine phosphorylation of Gab-1 was increased in CHON,~ and CHOHIRccells but not in CHOAlOl8 cells. These results suggest the existence of cell clone-specific differences, particularly in insulin's effects on Syp-associated pp115 and Gab-1 phosphorylation, that may be responsible for insulin-induced egr-Z expression in CHO cells.

6. Identification of Shc in 32D Myeloid Precursor Cells as the Principle Insulin-Sensitive Phosphoprotein and Its Potential Role in Signal Transduction

In recently completed studies (Harada etal., 1996b,1996c),we compared the effects of insulin on egr-1 and c-fos expression to insulin-induced tyrosine

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phosphorylation of intracellular substrates in 32D myeloid precursor cells expressing insulin receptors (32D/IR), IRS-1 (32D/IRS), or both (32D/IR + IRS). These cells were chosen because of the almost total absence of native insulin receptor, IRS-1, and IRS-2/4PS in the parental 32D cell (Wang et al., 1993). The time course of insulin-induced egr-1 and c-fos was examined by Northern blot analysis. As shown in Fig. 9, 17 nM insulin had no effect on egr-1 or c-fos expression in the parental 32D or the 32DIIRS cells. However, we observed similar insulin-induced expression for both genes in 32D/IR and 32D/IR + IRS cells. Not surprisingly, these results demonstrated, that an insulin receptor was required for effects on immediate-early genes and also that IRS-1 was not necessary and may not be involved. An insulin concentration curve (1-100 nM) showed no significant differences in insulin sensitivity in 32D/IR or 32D/IR + IRS cells (Harada et al., 1996c), further suggesting that IRS-1 was not involved in the stimulation of these two genes. Insulin-induced tyrosine phosphorylated substrates were identified by immunoprecipitation with anti-phosphotyrosine antibodies (a-PY) and Western blot with a-PY, anti-insulin receptor p subunit, and anti-Shc antibodies. As shown in Fig. 10, the only tyrosine phosphorylated proteins observed in 32D/IR cells were the insulin receptor P subunit and Shc, whereas several proteins, including the insulin receptor, IRS-1, and Shc, were tyrosine phosphorylated in 32D/IR + IRS cells. We also examined the effect of insulin on Shc-GRB-2 association. Cell lysates were immunoprecipitated with anti-Shc or anti-GRB-2 antibodies and Western blotted with anti-PY, anti-Shc, or anti-GRB-2 antibodies. Figure 11 shows that Shc and GRB-2 expression levels were similar in

E I cfos

Ct-tubulin Min

0 30 60 90

0 30 60 9 0

0 30 60 90

0 30 60 90

FIG. 9 Effect of insulin on immediate-early gene expression in 32D cell clones. 32D, 32D/ IR, 32D/lRS, or 32DAR + IRS cells were incubated with 17 nM insulin for 0-90 min at 37OC. Total RNA was isolated and 15 pg RNA subjected to Northern blot analysis (Harada ef al., 1995a).

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bnmuno- Western precipitate Blot

c-IRS-1 UPY

+IR

aPY

+Shc apY

aIR

OPY

aShc

Mm

+IR 4-

0

1

5

0

1

Shc

5

FIG. 10 Effect of insulin on protein tyrosine phosphorylation in 32D cell clones. 32DnR or 32D/IR + IRS cells were incubated with 100 nM insulin for 0, 1, or 5 min at 37°C. Cells were lysed, and phosphotyrosine-containingproteins immunoprecipitated with antiphosphotyrosine antibody (aPY) and subjected to SDS-PAGE and Western blot analysis with antibodies against phosphotyrosine, insulin receptor p subunit (aIR), or Shc (aShc) (Harada ef al., 1995a).

hmuno-

precipitate Blot aShc

aShc

4-

a5 h c

aPY

+ Shc

Shc

+IRS-I aGRB-2

W Y

+Shc aGRB-2

+GRB-2

clGRB-2 Insulin

-

+

-

+

FIG. 11 Effect of insulin on protein tyrosine phosphorylation of Shc and Shc-GRB-2 association in 32D cell clones. 32D/IR or 32D/IR + IRS cells were incubated in the absence (-) or presence (+) of 100 nM insulin for 5 min at 37°C. Cells lysates were incubated with antibodies to Shc (aShc) or GRB-2 (aGRB-2) and the immunoprecipitates were subjected to SDS-PAGE and Western blot analysis with antibodies against phosphotyrosine (aPY), Shc, or GRB-2 (Harada et al., 1995a).

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32D/IR and 32D/IR + IRS cells. Insulin treatment caused tyrosine phosphorylation of Shc in both cell types, but the amount of phosphorylated Shc was greater in 32DIIR cells. Immunoprecipitation with GRB-2 antibody revealed that GBR-2 associated with both IRS-1 and Shc in 32D/IR + IRS cells but a much larger amount of Shc was immunoprecipitated with GRB-2 in 32D/IR cells. These results suggest that the insulin receptor, but not IRS-1, is required for Shc phosphorylation and association of Shc and GRB-2. The phosphorylation of Shc and its association with GRB-2 may be the IRS-1-independent pathway by which insulin stimulates egr-2 and c-fos expression in 32D cells. Lastly, to determine the downstream pathways involved in insulininduced immediate-early gene expression, we examined the effect of 25100 nM wortmannin, a PI 3-kinase inhibitor, or 30 pM PD 98059, a MEK inhibitor, on egr-2 and c-fos expression in 32D/IR or 32DIIR + IRS cells. Wortmannin had no inhibitory effect on insulin-induced egr-2 and c-fos expression. In contrast, PD 98059 almost completely inhibited insulin-induced egr-2 and c-fos expression (Harada et al., 1996b, 1996~). These results suggest that MEK and MAP kinase activation, but not PI 3-kinase activation, is involved in insulin-induced immediate-early gene expression via the Shc pathway in 32D myeloid cells. In combination with our previous studies with CHO cells (Harada et al., 1995a), these data demonstrate that the apparent proximal components in the signaling pathways resulting in insulin-induced immediate-early expression are different in 32D and CHO cells. These findings suggest that different cell types may utilize different cell-specific signaling mechanisms.

V. Summary The intent of this review is to support the hypothesis that the pleiotropic effects of insulin may result from multiple pathways: the signaling network, which is directly dependent on insulin-induced phosphorylation of the p subunit of the insulin receptor at the cell surface, and the internalization and intracellular translocation of insulin. We described reports from several laboratories of insulin signaling pathways that are independent of IRS-1. There now seems to be general acceptance of the concept of multiple, interacting, competing, and cell-specific signal transduction pathways. Ongoing research in many laboratories should add to our knowledge of the complexity of the pathways by which insulin regulates life-sustaining metabolic pathways. The potential role of insulin internalization and cellular processing in insulin action has been suggested for some time but received little accep-

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tance. We noted that studies with other growth factors, e.g., EGF, FGF-1, FGF-2, interleukin-1, prolactin, angiogenin, NGF, growth hormone, and IGF-1, have demonstrated endocytosis, translocation to the cytoplasm and accumulation in the nucleus, and linked the internalization to biological relevant roles in the effects of those agents on cell proliferation. We reviewed past and recent studies that clearly suggest that intracellular insulin may play a role in insulin action in endosomal apparatuses, which have phosphorylated insulin receptors and insulin signaling proteins, in the cytoplasm in which insulin specifically interacts with proteins that are known to associate with other hormones and growth factors and that play roles in gene transcription, or in the nucleus in which insulin associates with the nuclear matrix proteins. These observations, although falling short of proving a biological role for insulin internalization other than degradation of insulin, provide substantial support for the hypothesis that insulin internalization plays a significant role in insulin action.

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A Actin-binding proteins, regulation of MT-microfilament interactions, 226-230 Actin filament and MT, interdependence, 218-219 role in MT-microfilament interaction, 231-232 ADP, effect on flagellar beat cycle, 30-31 Amino acid sequence XRP catalytic site, 184-186 XRPs, 182-184 Amino terminus. collagen V, molecular structure, 96-97 Anchoring fibrils, role in collagen fibril anchoring, 136-137 Asian horseshoe crab, axonemal pattern, 5 ATP, effect on flagellar beat cycle, 30-31 ATPase, activity of dynein, 13-16 Auxin, regulation of XRP gene, 188-189 Axoneme physical model, 39-46 role in flagellar beat cycle, 29-31 structural components, 2-5 structural variations, 5-9 Axons, mitochondria1 transport in, 223-225 Azuki bean. endoxyloglucan transferase enzymatic reaction, 173-174 localization, 181-182 pH dependency, 176-177 substrate specificity, 174-176

B Basal bodies, associated fibrillar networks basal foot in ciliated epithelium, 208-209 ciliated protozoa centrosomes, 214 ciliary motility, 213 MT-microfilament complex, 211-213 proximal structures, 211 neurosensory epithelium cochlea hair cells, 210 MT-microfilament interactions, 210 photoreceptor cells, 209-210 Basal foot, in ciliated epithelium, 208-209 Basement membrane, as specialized ECM, 75 Beaded fibrils, role in collagen fibril anchoring, 136 Beat cycle, flagella effect of nucleotides, 30-31 mechanics, 31-34 minimal requirements, 29-31 role of Ca2+,30-31 Brassinosteroids, regulation of XRP gene, 189-191

C Calcium effect on flagella, 20-21 role in beat cycle, 30-31 role in flagellar motility, 25-29 Caldesmon, regulation of MT-microfilament interactions, 228-229

281

282 Calmodulin, regulation of MT-microfilament interactions, 228-229 Carboxy terminus, collagen V, molecular structure, 99-100 Cell development, MT-microfilament interaction centriole migration, 216-217 centrosome movement, 214-215 cortical movement, 215-216 cytoplasmic streaming in ring canals, 220-221 role of actin filament arrays, 218-219 role of growth cones, 221-222 role of nuclear migration, 219-220 spindle orientation, 216 spindle positioning and cytokinesis, 217-218 Cell division, MT-microfilament interaction centriole migration, 216-217 centrosome movement, 214-215 cytoplasmic streaming in ring canals, 220-221 role of actin filament arrays, 218-219 role of growth cones, 221-222 role of nuclear migration, 219-220 spindle orientation, 216 spindle positioning and cytokinesis, 217-218 Cell lines CHO, pp120, role in signal transduction, 267-269 32D myeloid precursor, Shc protein, 269-272 Cellulose microfibrils, 162 interaction with xyloglucans, 195-196 fucosylation, 165-166 pectic polysaccharide network, 167 synthesis, 168-169 -xyloglucan framework, 162-165 Cell walls, plant architectural models, 160-162 classification, 159-160 construction cellulose microfibril synthesis, 168-169 cleavage of load-bearing crosslinks, 192- 194 dynamic aspects, 167-168 EXGT-mediated molecular grafting, 194-1 95

INDEX xyloglucan-cellulose microfibril interaction, 195-196 loosening, cleavage model, 169-171 molecular grafting model, 171-172 Centrioles, migration in cell division and development, 216-217 Centrosomes in basal body-associated fibrillar networks, 214 movement in MT-microfilament interactions, 214-215 Chlamydomonas axonemal dynein diversity, 19-21 dynein ATPase, 13-14 Cilia, see Flagella Cochlea hair cells, in neurosensory epithelium, 210 COLl domain, collagen V, molecular structure, 97-99 COL2 domain, collagen V, molecular structure, 96-97 Collagen I assembly into fibrils, 83-85 as connective tissue component, 78-80 disorders, 85-86 encoding genes, 81-82 molecular structure, 80-81 posttranslational modification, 82-83 Collagen I11 encoding genes, 87-88 during morphogenesis, 86 role in fibrogenesis, 87-88 during tissue remodeling, 86 Collagen IV a(IV) chains, length polymorphism, 90-92 and collagen V, codistribution in vivo, 127 gel, 94 homotypic interaction, 92-94 interaction with laminins, 107-108 and lamina densa skeleton, 120-123 molecular structure, 88-90 Collagen V chain structure, 95-96 and collagen IV, codistribution in vivo, 127 on fine collagen fibrils, 113 molecular structure COLl domain, 97-99 COL2 domain, 96-97

INDEX C-terminal region, 99-100 NC2 domain, 96-97 NC3 domain, 96-97 role in collagen fibril growth in v i m , 116-120 role in regulation of collagen fibril diameter, 110-1 11 significance in vivu, 130-131 subtypes function, 101 preparation. 100-101 Collagen VII gene structure, 102-103 molecular structure, 101-102 roles in vivo, 103 supramolecular structure, 101-102 Collagen XVll gene structure, 104-105 molecular structure, 103-104 Collagen fibrils anchoring to lamina densa anchoring fibrils, 136-137 beaded fibril role. 136 microfibril role, 134-136 microthreads, 137-138 role in organogenesis. 132-133 architecture in tissues, 131-132 diameter regulation in different tissues, 109-110 regulation in vivo. 110-1 I 1 direct physical connection with lamina densa, 127-130 fine collagen V on, 113 pNcollagen 111 on, 111-112 growth in v i m role of collagen V, 116-120 role of pNcollagen 111, 114-116 -lamina densa connection, light microscopy, 125-126 -lamina densa interaction, related molecules collagen 1. 78-86 collagen 111, 86-88 collagen IV, 88-94 collagen V, 95-101 collagen VII, 101-103 collagen XVII, 103-105 laminins, 105-108 nidogen, 108

283 osteonectin, 109 perlecan, 108-109 species, 77-78 Connective tissue collagen I as component, 78-80 interactions, role in organogenesis. 132-133 role of fibrillin fibrils, 134-135 role of fibronectin fibrils, 135-136 Cortical flow, in cell development, 215-216 Cross-bridge cycle, role in dynein function, 38-39 Crosslinks, load-bearing, cleavage in plant cell walls, 192-194 Cyclic AMP, role in flagellar motility, 25-29 Cytoplasm insulin role in biological effects, 266-267 translocation, 258-260 macromolecule translocation, mechanism, 260-26 1 proteins, interaction with insulin, 261 -265 streaming in ring canals, 220-221

D Development cell, see Cell development plant cell wall cleavage of load-bearing crosslinks, 192-1 94 EXGT-mediated molecular grafting, 194-1 95 xyloglucan-cellulose microfibril interaction, 195-196 Disease, collagen I disorders, 85-86 Dynactin, regulation of MT-microfilament interactions, 232-233 Dynein ATPase activity, 13-16 axonemal, diversity, 19-21 inner arm, effect of dynein regulatory complex, 7-9 role of cross-bridge cycle, 38-39 -tubulin interaction. role of t-force, 46-47 -tubulin sliding, dynamics, 17-19

284

INDEX

Dynein motor in eukaryotic flagella, 4 and geometric clutch model, 51 isolation, 18 Dynein regulatory complex effect on inner arm dynein, 7-9 regulation of MT-microfilament interactions, 233

E ECM, see Extracellular matrix Electron microscopy, plant cell wall architecture, 160-162 Elliptio cornplanatus, dynein-tubulin sliding in, 17-19 Endocytosis, insulin and insulin receptors, 249-250 Endosome-associated insulin, role in biological effects, 253-258 Endoxyloglucan transferase azuki bean enzymatic reaction, 173-174 localization, 181-182 pH dependency, 176-177 substrate specificity, 174-176 fluorescence detection, 177-179 identification, 172-173 mediated molecular grafting, 194-195 mediated molecular grafting in muro, 179-180 purification, 172-173 xyloglucan disproportioning reaction, 179 xyloglucan endotransglycosylase activity, 177 Environmental signals, in XRP gene regulation, 191-192 Epithelium ciliated, basal foot in, 208-209 neurosensory cochlear hair cells, 210 MT-microfilament interactions, 210 photoreceptor cells, 209-210 polarized cells, vesicular transport in, 225 EXGT, see Endoxyloglucan transferase Extracellular matrix associated molecules, interactions with laminins, 107-108 development, 75 major structures, 76-77

Ezrin, regulation of MT-microfilament interactions, 229-230

F Fibrillar networks, basal body-associated basal foot in ciliated epithelium, 208-209 ciliated protozoa centrosomes, 214 ciliary motility, 213 MT-microfilament complex, 21 1-213 proximal structures, 211 neurosensory epithelium cochlea hair cells, 210 MT-microfilament interactions, 210 photoreceptor cells, 209-210 Fibrillin fibrils, role in connective tissue, 134-135 Fibrils collagen, see Collagen fibrils collagen I assembly into, 83-85 microfibrils, see Microfibrils role in collagen fibril anchoring anchoring fibrils, 136-137 beaded fibrils, 136 role in connective tissue fibrillin fibrils, 134-135 fibronectin fibrils, 135-136 Fibrogenesis, role of collagen 111, 87-88 Fibronectin fibrils, role in connective tissue, 135-136 Flagella axonemal variations, 5-9 basic axenome, 2-5 beat cycle effect of nucleotides, 30-31 mechanics, 31-34 minimal requirements, 29-31 role of Ca2+,30-31 compound, natural development, 9-10 effect of nickel ion, 21 motility, 213 motility, regulation effect of vanadate, 27-28 role of Ca2+,25-29 role of CAMP, 25-29 role in living cell, 23-25 signal pathways, 21-23 physical model, 39-46

INDEX

285

physical parameters of movement, 34-39 structure, 1-2 Fluorescence. in detection of EXGT, 177-179 Fodrin, regulation of MT-microfilament interactions, 229-230 t-Force in geometric clutch design, 46-47 role in geometric clutch model, 53 Fucosylation, in xyloglucan-cellulose microfibril interaction. 165-166

G Gel, collagen IV, 94 Genes collagen I. 81-82 collagen 111, 87-88 collagen VII, 102-103 collagen XVII, 104-105 xyloglucan-related protein family, 182-184 xyloglucan-related proteins, regulation by auxin, 188-189 by brassinosteroids, 189-191 environmental signals, 191-192 by other hormones, 191 spatial and temporal regulation, 186-188 Geometric clutch model and experimental data, 50-54 flagellum, 39-46 t-force in, 46-47 oscillations in, 48-50 role of t-force, 53 as rudimentary model, 54-55 Grafting, see Molecular grafting Growth collagen fibrils in vitro role of collagen V, 116-120 role of pNcollagen 111, 114-116 plant cell wall cleavage of load-bearing crosslinks, 192- 194 EXGT-mediated molecular grafting, 194-195 xyloglucan-cellulose microfibril interaction, 195-196

Growth cones, role in celI division and development, 221-222 Growth factors, internalized, signaling mechanisms, 247-248

H Hair cells, cochlea, in neurosensory epithelium, 210 Hormones internalized, signaling mechanisms, 247-248 regulation of XRP gene expression, 191

I Insu 1in endocytosis, 249-250 interactions with cytoplasmic proteins, 261-265 with nuclear matrix, 265-266 internalization effect of insulin receptor mutations, 250-252 mechanisms, 247-248 role in biological effects cytoplasmic and nuclear insulin, 266-267 endosome-associated insulin, 253-258 sensitive phosphoprotein pp120 as, 267-269 Shc as, 269-272 signal transduction network, 244-247 translocation to cytoplasm and nucleus, 258-260 Insulin receptor endocytosis, 249-250 mutations, effects on insulin internalization, 250-252

L Lamina densa collagen fibril anchoring to anchoring fibrils, 136-137 beaded fibril role, 136 microfibril role, 134-136 microthreads, 137-138

286 Lamina densa (continued) role in organogenesis, 132-133 -collagen fibril connection, light microscopy, 125-126 -collagen fibril interaction, related moIecu1es collagen I, 78-86 collagen 111, 86-88 collagen IV, 88-94 collagen V, 95-101 collagen VII, 101-103 collagen XVII, 103-105 laminins, 105-108 nidogen, 108 osteonectin, 109 perlecan, 108-109 direct physical connection with collagen fibrils, 127-130 formation in absence of macromolecules, 123-125 skeleton, and collagen IV, 120-123 structural components, 108-109 tissue location, 75-76 Laminins interactions with ECM molecules, 107- 108 molecular structure, 106 superfamily, roles in vivo, 106-107 Light microscopy, collagen fibril-lamina densa connection, 125-126

Macromolecules absence in lamina densa formation, 123-125 translocation into cytoplasm, mechanisms, 260-261 Membranes, basement, as specialized ECM, 75 Microfibrils cellulose, 162 interaction with xyloglucans, 195-196 fucosylation, 165-166 pectic polysaccharide network, 167 synthesis, 168-169 role in collagen fibril anchoring to lamina densa fibrillin fibrils, 134-135 fibronectin fibrils, 135-136

INDEX

Microfilaments -MT complex, 211-213 -MT interaction, in cell division and development centriole migration, 216-217 centrosome movement, 214-215 cortical movement, 215-216 cytokinesis, 217-218 cytoplasmic streaming in ring canals, 220-221 role of actin filament arrays, 218-219 role of growth cones, 221-222 role of nuclear migration, 219-220 spindle orientation, 216 spindle positioning, 217-218 -MT interaction, regulation by multisubunit complexes, 232-233 role of different motors, 232 role for MT and actin filament dynamics, 231-232 role of posttranslational modifications, 230 by tubulin- and actin-binding proteins, 226-230 organelle transport, model systems, 222-226 Microscopy collagen fibril-lamina densa connection, 125- 126 plant cell wall architecture, 160-162 Microthreads, role in collagen fibril anchoring to lamina densa, 137-138 Microtubule-associated proteins, regulation of MT-microfilament interactions, 226-228 Microtubules accessory MT, 10 in basic axoneme, 2-3 in eukaryotic flagellum, 1-2 -microfilament complex, 211-213 -microfilament interaction, in cell division and development centriole migration, 216-217 cen trosome movement, 2 14-2 15 cortical movement, 215-216 cytokinesis, 217-218 cytoplasmic streaming in ring canals, 220-221 role of actin filament arrays, 218-219 role of growth cones, 221-222 role of nuclear migration, 219-220

INDEX

spindle orientation, 216 spindle positioning, 217-218 -microfilament interaction, regulation by multisubunit complexes, 232-233 role of different motors, 232 role for MT and actin filament dynamics, 231 -232 role of posttranslational modifications, 230 by tubulin- and actin-binding proteins, 226-230 organelle transport, model systems, 222-226 role in dynein-tubulin interactions, 18-19 Migration centrioles in cell division and development, 216-217 nuclear, role in cell division and development, 219-220 Mitochondria1 transport, in axons, 223-225 Models architectural, plant cell wall, 160-162 cleavage, for cell wall loosening, 169- 171 flagellum physical model, 39-46 physical parameters of movement, 34-39 geometric clutch, see Geometric clutch model molecular grafting, 171-172 organelle transport, 222-223 Molecular grafting as cell wall model, 171-172 mediation by EXGT, 194-195 mediation by EXGT in muro, 179- 180 Molecular structure collagen I, 80-81 collagen IV, 88-90 collagen V COLl domain, 97-99 COL2 domain, 96-97 C-terminal region, 99-100 NC2 domain, 96-97 NC3 domain, 96-97 collagen VII, 101-102 collagen XVII, 103-104 laminins. 106

287 Morphogenesis, collagen 111 during, 86 Motility cilia, 213 flagellar, regulation effect of vanadate, 27-28 role of Ca”, 25-29 role of CAMP, 25-29 role in living cell, 23-25 signal pathways, 21-23 Motors dynein, see Dynein motor role in MT-microfilament interactions, 232 types in organelle, 225-226 MT, see Microtubules Mutations, insulin receptor, effects on insulin internalization, 250-252

N NC2 domain, collagen V, molecular structure, 96-97 NC3 domain, collagen V, molecular structure, 96-97 Nexin, role in t-force, 47 Nickel, effect on flagella, 21 Nidogen, as lamina densa component, 108 Nuclear matrix, interaction with insulin, 265-266 Nucleus insulin role in biological effects, 266-267 translocation, 258-260 migration in, role in cell division and development, 219-220

Organelle transport different motors, 225-226 model systems, 222-223 Organogenesis, role of connective tissue interactions, 132-133 Oscillations, in geometric clutch model, 48-50 Osteonectin, as lamina densa component, 109 Outer dense fibers, structure and function, 10-13

288

INDEX

P Perlecan, as lamina densa component, 108-109 pH, dependency of azuki bean EXGT, 176-177 Phosphoproteins, insulin-sensitive pp120 as, 267-269 Shc as, 269-272 Photoreceptor cells, in neurosensory epithelium, 209-210 Plants cell wall architectural models, 160-162 classification, 159-160 construction cellulose microfibril synthesis, 168- 169 cleavage of load-bearing crosslinks, 192-194 dynamic aspects, 167-168 EXGT-mediated molecular grafting, 194- 195 xyloglucan-cellulose microfibril interaction, 195-196 loosening, cleavage model, 169-171 molecular grafting model, 171-172 cytokinesis in, 217-218 pNcollagen I11 on fine collagen fibrils, 111-112 role in collagen fibril growth in virro, 114-116 Polymorphism, length, collagen a(IV) chains, 90-92 Polysaccharide, pectic network, 167 pp120 protein, role in signal transduction, 267-269 Proteins, cytoplasmic, interaction with insulin, 261-265 Protozoa, ciliated basal body cage, 211-213 basal body proximal structures, 211 centrosomes, 214 ciliary motility, 213 Pyridylamino oligosaccharides, in characterization of EXGT, 174-176

R Ring canals, cytoplasmic streaming, 220-221

S Sea urchin, sperm, flagellar motility, role of Ca2+,27 Shc protein, role in signal transduction, 269-272 Signal transduction by growth factors, 247-248 by hormones, 247-248 network for insulin, 244-247 for regulation of flagellar motility, 21-23 role of ~ ~ 1 2 0 , 2 6 7 - 2 6 9 role of Shc, 269-272 Skeleton, lamina densa, and collagen IV, 120-123 Sperm flagellar motility, 23-25 role of Ca2+,27 mammalian, special adaptations, 9-13 Spindles, orientation in cell division and development, 216 Supramolecular structure, collagen VII, 101-102 Synapsin, regulation of MT-microfilament interactions, 228-229

T Tetrahymena pyriformis, dynein ATPase, 13-14 Tissues collagen fibril diameters, 109-1 10 connective, see Connective tissue fibrillar collagen architecture, 131-132 remodeling, collagen 111 during, 86 Translation, posttranslational modification collagen I, 82-83 role in MT-microfilament interactions, 230 Translocation insulin to cytoplasm and nucleus, 258-260

289

INDEX

macromolecules into cytoplasm, mechanisms, 260-261 Transport mitochondrial, in axons, 223-225 organelle, model systems, 222-226 vesicular, in polarized epithelial cells, 225 Tubulin -dynein interaction, role of t-force, 46-47 -dynein sliding, dynamics, 17-19 Tubulin-binding proteins, regulation of MT-microfilament interactions, 226-230

v Vanadate, effect on flagellar motility, 27-28 Vesicular transport, in polarized epithelial cells, 225 Viscous drag, effect on flagella movement, 35-38

X XRP,see Xyloglucan-related proteins Xyloglucan endotransglycosylase, activity of EXGT, detection, 177 Xyloglucan-related proteins amino acid sequence, 182-184 catalytic site, 184-186 gene expression environmental signals, 191-192 other hormones, 191 regulation by auxin, 188-189 regulation by brassinosteroids, 189-191 spatial and temporal regulation, 186- 188 gene family, 182-184 Xyloglucans -cellulose framework, 162-165 disproportioning reaction for EXGT, 179 interaction with cellulose microfibrils, 195-196 fucosylation, 165-166 pectic polysaccharide network, 167 xyloglucan forms, 166-167

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