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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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).

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0

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I I I 1

0

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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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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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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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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).

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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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ROBERT M. SMITH ET AL.

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.

252

ROBERT M. SMITH ET AL.

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

254

ROBERT M. SMITH FT AL.

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

255

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.

258

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

259

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.

260

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,

INSULIN INTERNALIZATION AND OTHER PATHWAYS

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

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(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-

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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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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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Blackard, W. G., Smith, R. M., and Jarett, L. (1986). Insulin processing by cultured hepatocytes. Am. J. Physiol. 250, E148-E155. Blazer-Yost, B. L., Shah, N., Jarett, L., Cox, M., and Smith, R. M. (1992). Insulin and IGFl receptors in a model renal epithelium: Receptor localization and characterization. Biochem. Int. 28,143-153. Burgess, J. W., Bevan, A. P., Bergeron, J. J. M., and Posner, B. I. (1992a). Intracellular trafficking and processing of ligand-receptor complexes in the endosomal system. Exp. Clin. Endocrinol. 11,67-78. Burgess, J. W., Wada, I., Ling, N., Khan, Mn., Bergeron, J. J. M., and Posner, B. I. (1992b). Decrease in 0-subunit phosphotyrosine correlates with internalization and activation of the endosomal insulin receptor kinase. J. Biol. Chem. 267, 10077-10086. Burwen, S. J., and Jones, A. L. (1987). The association of polypeptide hormones and growth factors with the nuclei of target cells. Trends Biochem. Sci. 12,159-162. Carpentier, J.-L. (1992). Insulin-induced and constitutive internalization of the insulin receptor. Hormone Res. 38, 13-18. Carpentier, J.-L., and McClain, D. (1995). Insulin receptor kinase activation releases a constraint maintaining the receptor on microvilli. J. Biol. Chem. 270, 5001-5006. Carpentier, J.-L., Gorden, P., Freychet, P., Le Cam, A., and Orci, L. (1979). Lysosomal association of internalized 1251-insulinin isolated rat hepatocytes. Direct demonstration by quantitative electron microscopic autoradiography. J. Clin.Invest. 63, 1249-1261. Carpentier, J.-L., Paccaud, J. P., Backer, J., Gilbert, A., Orci, L., and Kahn, C. R. (1993). Two steps of insulin receptor internalization depend on different domains of the betasubunit. J . Cell Biol. 122, 1243-1252. Cheatham, B., and Kahn, C. R. (1995). Insulin action and the insulin signaling network. Endocr. Rev. 16,117-142. Clevenger, C. V., Altmann, S. W., and Prystowsky, M. B. (1991). Requirement for nuclear prolactin for interleukin-2-stimulated proliferation of T lymphocytes. Science 253,77-79. Corvera, S., Graver, D. F., and Smith, R. M. (1989). Insulin increases the cell surface concentration of alpha-2-macroglobulin receptors in 3T3-Ll adipocytes; Altered transit of the receptor between early and late endocytic compartments. J. Biol. Chem. 264,10133-10138. Czech, M. P., Klarlund, J. K., Yagaloff, K. A., Bradford, A. P., and Lewis, R. E. (1988). Insulin receptor signaling, activation of multiple serine kinases. J. Biol. Chem. 263, 11017-11020. Di Guglielmo, G. M., Baass, P. C., Ou, W.-J., Posner, B. I., and Bergeron, J. J. M. (1994). Compartmentalization of SHC, GRB2 and mSOS, and hyperphosphorylation of Raf-1 by EGF but not insulin in liver parenchyma. EMBO J. 13,4269-4277. Eldredge, E. R., Korf, G. M., Christensen, T. A., Connolly, D. C., Getz, M. J., and Maihle, N. J. (1994). Activation of c-fos gene expression by a kinase-deficient epidermal growth factor receptor. Mol. Cell. Biol. 14,7527-7534. Eveleth, D. D., and Bradshaw, R. A. (1992). Nerve growth factor nonresponsive phechromocytoma cells: Altered internalization results in signal disfunction. J. Cell Biol. 117, 291-299. Fan, J. Y., Carpentier, J.-L., Van Obberghen, E., Blackett, N. M., Grunfeld, C., Gorden, P., and Orci, L. (1983). The interaction of 12SI-insulinwith cultured 3T3-Ll adipocytes: Quantitative analysis by the hypothetical grain method. J. Histochem. Cytochem. 31, 859-870. Ford, T., Graham, J., and Rickwood, D. (1994). Iodixanol: A nonionic iso-osmotic centrifugation medium for the formation of self-generating gradients. Anal. Biochem. 220, 360-366. Gibbs, E. M., Lienhardt, G. E., Appleman, J. R., Lane, M. D., and, Frost, S. C. (1986). Insulin stimulates fluid-phase endocytosis and exocytosis in 3T3-Ll adipocytes. J. Biol. Chem. 261,3944-3951. Goidl, J. A. (1979). Insulin binding to isolated liver nuclei from obese and lean mice. Biochemistry 18,3674-3679.

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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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