Membrane-cytoskeleton interactions in animal cells

B i o c h l m i c a et B i o p h ~ i c a Acta.

988 (1989) 147-171

147

FAse',Ser

Membrane-cytoskeleton interactions in animal cells Kermit

L. Carraway

and

Coralie

A. Carothers

D e p a r t m e n s o f A n a t o m y a n d C e l l Biolog~ a n d B i ~ h e m i ~ t m ' a n d ~ o l e c M a r B i o l o ~

Carraway

b ' m ~ r s i ~ ' o f Miama School o f M e d l t i n e ,

M i a m i , F L CU S . A . )

( I ~ e i v e d 10 Ma~ 1988)

Contellts I.

Introduction

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IL The elements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A, M e m l : ¢ ~ ...................................................... B. Cytoske.leton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

148

148 148

Ill. PrototYpe: the ¢D'throc)tc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. E r j t h r ~ y t e raembr~ne s k d e m n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

149 149

t. Isolation of the skeleton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. CharactellzatJonof skeletal components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

149 150

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3. Association of the skeleton with the ~ e m b r a n e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Assembly ~f the ~kaleton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. E r y d u ~ t ¢ c y t ~ k ) l e t a l analogs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

]51 152 153

IV. Interaction of plmsmamembraneswith specific cytoskaletal smactures . . . . . . . . . . . . . . . A. Micro~amenls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . L Microvilfi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . &

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154 154 154 154

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2. Ceil activation and transmembrane signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a. Platelets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b. Neutrophils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Signal transducfion mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Cell a d h t ~ o n and eeU~extracellalar matrix interactions: junctions . . . . . . . . . . . . . . . . . . . a. Junctional components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i, Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ii. Characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i l l Orsanization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b, R ¢ ~ l a f i o n of cen contacts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Micrombules . . . . . . . . . . . . . . . . . . . . . . . . . . . C, Intermediate fdaments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 'V, FulUte studies

148

159 160

1~0 160 t~)t . . . . . .

162 162 163 165 166

Ac knowledsemems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

166

Refer*races

166

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Abbreviations: MAP, mletotub~e-associatod prolein: SDS-PAGE. sodium dodccyl null'ate p¢lyac~lamide gal electrophoresis; NE specttin. non-etyll~old spcctfin; BB spccmn, brush-border spcctnn; CAG, ¢ytoskaleton-assoClaled glycoprotein; ABP, actin-binding protein; EGF, epidermal growth factor; MABs, ~ n ~ l o n a l antibodies; CSAT. cdl-sul~lrate attachment antigen. Correspondence: C,A•. Canaway, Department of Biochemistt,/ and Molecular Biology, University of Mimni School of Medicine, Miami, FL 33101, U.S.A. 0 3 0 4 4 1 5 7 / 8 9 / $ 0 3 . 5 0 © 1989 Elsevier

Science

Publishers B.V. (Biomedical D i v ~ , o , )

148 I. Intreducllon One of the important problems in cell biology is understanding how cells respond to their environment and how these resp,3nses are converted into information leading to dynamic normal cellular processes such as activation, growth (or arrest of growth), differentiation and organization into tissues, as well as malignant transformation. The last two of these also involve the question of describing cell-cell and cell-matrix interactioos. Howew:r, recent studies have emphasized the strong relationship between the strueturc of the cell itself and its extracelhilar interactions. Thus, the plasma membrane has become recognized as the focal point for two sets of interdependent interactions: extracellular and intracenular. Clearly, a critical element in this problem is understanding how the plasma membrane is organized. In this article we shall examine one side of this problem, the cytoplasmic side, by reviewing work on membrane-cytoskeieton interaefians. Our objective in this effort will be to describe these interactions at the molecular level. Because of the state of the field, this goal is still elusive. We do not at,erupt in this review to cover in detail many sy',tems or processes in which membrane-cytoskeleton interactions have been implicated but not substantiated. Such topics as cell motility, endo. cytosis, differentiation, transformation and others require more extensive treatment than can be provided in the context of this review. It. 7he elements II-A. Membranes

Cellular membranes provide permeability barriers wl.Jch regulate the composition of specific compartments and separate synthetic and degradative aspects of 'cell function. The intracenular membranes of eukaryotic cells also provide surfaces upon which metabolic reactions are organized. Moreover, they are involved in the intracelhilar transit of molecules during secretion, endocytosis and expression at the cell surface. The plasma membrane is the external limiting membrane ol the animal cell. As such, it serves not only as the permeability barrier between the cell and its external environmeat, but also as a sensor for that environment. Recognition functions of the plasma membrane are particularly important. Regulation of cell growth, proliferation and metabolism often depend on the binding of growth factors, hormones, neurotransmitters and other biological figands, as well as drugs, to specific receptors on the cell surface. Receptors on the plasma membrane are also responsible for the recognition of other cells, for forming call-ceil contacts or junctions, and for forming associations with the extraeeUular matrix which are involved in cell motility or orientation.

One important aspect of plasma membrane receptor mechanisms is that the functional behavior of those receptors often depends on their topography, as well as their concentration in the membrane. Induced dustering of membrane receptors has been implicated in transmembrane signaling [1], as well as receptor-mediated endocytosis [2]. Receptor clustering appears to be modulated by cytoplasmic cytoskeletal elements via interactions of the cytoskeleton with integral membrane proteins [3] through a transmembrane linkage. II-B. Cytoskeleton

The cytoskeleton can be defined as the cytoplasmic network of fibrillar structures wifich provides a framework for the cell. Three types of fiber have been defined in molecular terms: microfilaments, intermediate filaments and microtubules. A fourth possible cytoskeletal element, the microtrahecdiar lattice [41], has not been as well characterized and remains controversial. Thus, the cytoskeleton can be described as being composed of these elements and their associated proteins. The eytoskeleton is often operationaily or experimentally defined as the insoluble residue remaining when cells are extracted with nonionie detergent [5]. Although this approach and definition have value as a first approximation to studies of some aspects of the cytoskeleton, two caveats must be kept in mind. First, some tyroskeleton components can be solubillzed by the detergent extractions. Even if the fibfillar components themselves remain, some of their important associaied proteins may be lost. Second, some cellular compotLents, such as remnants of organellcs, which are neither part of nor associated with the cytoskeleton, are inherently insoluble. Miecofilaments arc composed of actin subunits in linear homopolymers approx. 7 nm in diameter. In the living cell actin can exist in several forms [6,71. For example, in a fibroblast growing on a substratum, stress fibers composed of bundles of microfilameats (F-atria) are located near the cytoplasmic surface of the ventral plasma membrane. The stress fihea's appear to attach to the membrane at sites of cell adhesion to the substratum. On the dorsal surface of the same call, mierovilli may be found which also contain bundles of microfilameats. In contrast, the leading edge of the fibrolilast contains meshworks of microBlaments which are not highly organized [8]. The entire cortical region of most types of cell contains such meshworks. These diverse polymeric forms of artin are in dynamic equilibrium with each other and with monomerie G-aetin. Moreover, cellular membranes may have associated forms of activ which are not readily defined as microfilaments or G-aetin ]9,10]. The state of the acfin in a cell and the organization of the microfilaments depend on a large and diverse group of aetin-binding proteins [7,11,12]. Included among these are proteins which bind G-aetin

149 and which cap. sever, stabilize, crosslink and bundle microfilaments. Intermediate filaments are IG nm fibrillar structures of the cytoplasm which are both more stable and more diverse than microfilaments. Five classes of intermediate filament have been described, based on their cell of origin and the nature of their subunits [13]. (1) Vimentin filaments have a 53 kDa subunit and are found in cells of rnesenchymal origin or cells in culture. (2) Desmin, found predominantly in myogenic cells, has a 52 kDa subunit. (3) Glial fibrillary acidic protein is a 50 kDa protein found in astroglial cells. (4) Neurofilaments, composed of varying proportions of three subunits of 70, 140 and 200 kDa, are found in neuronal tissue. (5) Cytokeratins, or tonofilaments, have heterogeneous subunits of 40-70 kDa and appear to be correlated with the differentiated state in epithelial cells. Patterns of cytokeratin subunits are proving to be useful classification and diagnostic marker for epithelial tumors [14]. All of these classes of intermediate filaments are morphologically similar beca~!se ,h,~; ~ b units contain a conserved central helical rod domam as a core [15]. In higher eukaryotic cells, intermediate filaments have been proposed to serve as mechanical integrators of cellular space [16]. Microtubnies are 25 nm tubular filaments composed of beterodimers of a- and .8-tubniins, which are 54 kDa subunlts [17]. Miecotubules are found in cilia and flagella in highly organized structures in which nine outer microtubule doublets are arranged around two central mierotubules [18]. This organization, which is maintained by several associated proteins, is involved in generating the bending movements of the cilia and flagella using the energy provided by ATP hydrolysis by the mierotubule-associated ATPase dynein [19]. Microtubules are also found in the cell cytoplasm, radiating from the cell center during interphase [20]. During mitosis these microtubales are disassembled and microt u b u l ~ of the mitotic spindle are formed. Assembly and disassembly of micrombules durin 8 m, tosis or formation of cilia involve growth of microtubnies from microtubule organizing centers in the regions of cantrioles or at basal bodies, respectively. Polymerization and stabilikation appear to be aided by two classes of microtubule-associated protein 1211: MAPS, which are large polypeptidas ( > 250 kDa), and tan proteins (60-70 kDa), MAPS may also be involvea in the association of miecotubules with microfilamants [22,23] and of microtubules with neurofilaments [24].

III, Pl~ot~pe: the erythrocyte 111-.4. Erythrocyte membrane skeleton III-A.I. Isolation of the skeleton Erythrocytes catty oxygen and carbon dioxide between the lungs and the tissues. For this function they

must sur, ivc the rigors of the circulation, passing through capillaries whose diameters are smaller than the erythrocy~e diameter. Thus, the erythrocyte must be smail, flexible and extremely resilient. Thc~e last two qualities are achieved by virtue of the fact that the erythrocyte has an elaborate membrane skeleton associated with its plasma membrane [25]. In many ways erythrocytes are an ideal experimental system for the study of plasma membranes [26]. They can be obtained in large quantity from many different species. Mature mammalian erythrocytes contain no intracellular memSran¢~ rnicrombales or intermediate filaments to contaminate membrane preparations. They are easily lysed to release soluble intracellalar contents. They can be resealed after lysis to reconstitute a sealed ghost which maintains the shape and many of the permeability properties of the original cell [271. By appropriate experimental manipulations, sealed erythrocyte membrane vesicles can be obtained which are in an inverted orientation (inside-out vesicles), with their cy:~plas~Jc surface:, exposed [28]. Likewise, sealed vesicles can be obtained in the normal orientation (right side-out vesicles). Thus, both surfaces of the membrane are accessible to experimental manipulation. Erythrocyte membranes obtained by hypotonic lysis contain both a lipid bilayer with associated integral membrane proteins and a membrane skeleton, Since an important component of the skeleton is the cytoskeletal protein actin, the erythrocyte membrane has been used as a prototype for membrane-cytuskeleton interactions. The bilayer- and skeleton-associated fractions of the membrane tan be separated by simple extraction techniques. If erythroeyt¢ membranes are extracted with nonionic detergent, the membrane skeleton remains as an insoluble residue, which can be isolated by centrifugation [29]. In cnntrast, if the membranes are extracted with strong alkaline solutions, the membrane skeleton and other peripheral meh'.brane proteins are so!abilized, leaving the integral membrane proteins (primarily glyeophorins and band 3) with the bilayer [30]. The major proteins of the membrane skeleton and stripped Inurebrahe are given in Table I. The organization of the detergent-isolated er/throcyte membrane skeleton has been studied by a number of techniques. Analysis by sodium dodecyl sulfate polyacrylamide gel eleetrophoresis (SDS-PAGE) reveal: the major protein components: spectdn, actin and band 4.1 [26]. Minor components inehide t .:omyosin and band 4.9. Ultrastructurally, the skeleton appears as a dense reticulum or network of filaments subjacent to the membrane bilayer [31], Incubation of iso =:cd ~kdclons with dithiothreitol at low ionic strength causes their expansion and permits visualization of the reticulum as a network of long, fine filaments connected by shorter, thicker filaments [32] (Fig. 1). From compositional analyses and ultrastructu*al studies of isolated proteins

150 TABLE I Components of tile erythroeyte membrane and membrane cyt~lceleton Protein Integral components Band 3 Oly~phvrins gkeletoncomponents Spee,rin B~d 4.1 Ba:ld 4.9 Aetin Tropomy~in

Fig. l. El~tron micrograph of erythrocyte skelet~s prepared by Triton X-100 exlraetion and expansion in hy0otonic buffer. {From gef. 32 with permission.) the fine filaments are presumed to be spectrin tetramers and the thicker filaments to be actin protofilaments (oligomers). III-A,2. Characterization of skeletal components Spectrin, the major component of the erythrocyte membrane skeleton, was first isolated from low ionic strength extracts of erythrecyte membranes [33]. It is a heterodirner composed of 240 (e¢) and 220 (/8) kDa polypeptides [34,35], The two subtmits are homologous, with approx. 30% identity [36]. In the hetetodimer, they are afigned in an antiparallel side-to-side orientation to give a flexible. 100 nm rod-shaped molecule [37] with the amino and carboxyl termini near the ends of the rods. Heterodimers can furthet associate head-to-head to form 200 nm tetramers [37,38] and higher oligomers [39]. Chemical crosslinking [40], extractico under mild conditions [41] and the dimer-tetramer equilibrium [421

Sttbulfit M,

Structure

Copies/cell

90000 dimer/'tetramert000O00 23000-3100Odimer 5001100 240000 22O00O 78000 450(]0 4300O 2900O 27OOO

tetramer

100000

monomer trimer oligomer dimer

10o0~o 50000 5OOO00 70~O

suggest that the form of spectrin in the erythrecyte is primarily tetramerie. Sequence analysis of the spectdn subunits indicates that both contain homologous, repeating 106-resldue sequences which comprise more than 90% of the mass of the heterodlmet [36[. Purified spectrin binds specifically to a number of proteins, including aetin, band 4.1, enkyrin and ealmodulin [43]. Most important for reticolum formation is the lateral association of spectrin with F-actin [44], which provides the basis for the flexible spectrin. aetin network beneath the erythrocyte membrane. Rotary shadowing indicates that the site of actin interaction on the speetrin is near the ends of the tetramer [45,46]. This interaction is strengthened by addition of band 4.1 [45,47,481, which can also bind independently to purified spectrin with a stoichiometry of 2 reel of band 4.1 pet 1 mol of spectfin dimer [38]. The sensitivity of the spectrin-actin interaction to band 4.1 is apparently a property of the spectrin ,8.subunit [49]. The interaction site for 4.1 is also near the ends of the spectrin tetramer [50], which suggests the possibility of a ternary spectrin-aetin-4.1 complex [511. Since the actin of the network is in the form of protofilaments or oligomets, multiple speclfin tetramers can be associated with the actin to form a more condensed and stronger network [32] (Fig. 2). Complexes (26-30 S) can be isolated from low ionic strength extracts of erythrocyte membranes which contain primarily actin, spectrin di-

Fig. 2. El~lmn mlcrograph of expanded skeletons showing acdn protofihmenls with associated spectrin molecules. (From Ref. 32 with permission.)

151

brane form of the spectrin binding protein was identified as a 2[0 kDa protein by immunological analyses with anti-72 kOa [61] and by comparative peptide maps [62.63] The 210 kDa protein was named ankyrin to indicate its role in linking the spectrin network to the membrane [431.

Fig, 3. Model for e,"ythrocyteskeletalnetwork. mar and 4.1. By rotary shadowing these exhibit a spider.like ultrastructure containing three to six spectrin dimer arms [52-54]. The presence of tropomyosin in the erythrocyte membrane suggests that the actin ofigomers might be stabilized by this protein, since the molar ratio of tro~myosin to actin is appropriate for the saturation of F-actin with nonmuscle tropomyosin [55]. One additional protein, band 4.9, has also been identified as a minor ~,omponent of the network and of the isolated complexes [52,53]. Purified 4.9 is a wimer which is an F-actin bundling protein [56]. Its role in the network is uncertain. A second F-actin bundling protein from the erythrocyte skeleton has been termed addncin [57]. Purified adducin, a hetarodimar of 100 and 105 kDa subuints, not only binds actin but also stimulates spectfin binding to actin in a calmodulin-dependent manner [58]. Fig. 3 shows a schematic version of the skeletal network with the actin protofilamants as vertices with which multiple speetrin-4.1 complexes interact. The role of the membrane skeleton in stabilizing the arythrocyte depends on its ;ntaractions with the membrane. An involvement o~ spectrin in this interaction was suggested by the high-affinity binding (Kd, 10 -7 to 10 - s ) of spectrin to inside-out erythrocyte membrane vesicles which had been stripped of spectrin and actin [59]. Chymotrypsin treatmant of the vesicles inhibited the binding with concomitant release of a 72 kDa pniypeptide [60]. This fragment was identified as a binding site for spectrln on the membrane by its ability to inhibit spectrin binding to unproteolyzed inside-out vesicles and by the ability of anti-72 kDa to similarly inhibit spectrin binding. Morcow,', the intact mere-

III-A.3. Association of the skeleton with the membrane Ankyrin can be purified as a water-soluble monomer which forms a 1 : 1 complex with spectrin heterodimer. Rotary shadowing of the complex indicates that ankyrin binds about 20 nm from the head of the spectrin molecule at whi2h the hetcrodimers associate to form tetramers [38,6~¢]. The number of ankyrin molecules in the erythrocyte membrane (100000 per cell) is sufficient for binding one ankyrin per speetrin tetramer [43]. The ankyrin binding site on the membrane was identiffed by immunoprecipitation of detergent extracts of spectrin-depleted vesicles with anti-ankyrin [65]. Band 3, the anion transport channel, was coprecipitated with ankyrin in a 1 : 1 complex. This r~ult was consistent with the observations that a fraction of hand 3 is associated with isolated membrane skeletons. Moreover, extracted band 3 will rebind to ankyrin-containing, but not ankyrin-depleted, skeletons. A 43 kDa cytoplasmic domain of band 3 can be released by mild protcolysis [66]. Ankyrin binds to this fragment with a K a of 5 . 1 0 9. Antibody against this fragment inhibits binding of ankyrin to ankyfin-depleted inside-out ee3ieles. Finally, ankyrin binds specifically to liposomes reconstituted with band 3 [67]. interestingly, only a small fraction of the erythrocyte band 3 molecules are bound to the cymskeleton. This observation is consistent with the fact that there are about 10-fold more band 3 molecules than ankyrin in a cell. N o differences have been noted between bound and free forms of band 3 [431. The association of the membrane skeleton with band 3 via ankyrin does not explain one other membrane skeleton-related phenomenon. Ba~ld 4.1 remains associated with the inside-oas membrane vesicles even after depletion of speettin [43]. Thus, band 4.1 must also be associated with the membrane by another mechanism. The most likely candidate for this association is glycophorin, the major sinioglycoproteln(s) of the erythrocyte membrane. Liposomes containing glycophofin A, the predominant glycophorin, associate specifically with purified band 4.1 [68]. The interaction between 4.1 and glycophorin is enhanced by the presence of polyphosphoinosilides [69]. Band 4.1 can also bind to hand 3 of inside-out erythrocyte vesicles [70], an interaction that is inhibited by the 43 kDa fragment of band 3 or anti-43 kDa fragment. The 43 kDa fragment will also bind directly to purified 4.1. A modal for the association of the erythrocyte skeleton ,'dth the membrane is shown in Fig. 4.

152 ~

Glycopho~n •

Fig.4. Modelfor associationof crylhrocyl¢skeletonwi[hmemhran~

The erythrocyte membrane skeleton is involved in the maintenance of cell shape and the restriction of membrane protein mobility [71,72]. Erythrnaytes and their membranes undergo ATP-dcpendent shape changes [73]. Although phosphorylation of the B-chain of spectrin was shown to occur under the conditions of the shape change [74], subsequent studies found no requirement for specttin phosphorylation for the shape change [75] and no relationship between spcctrin phosphorylation levels and spectrin binding to erythrocyte membranes [76]. Interestingly, the phosphorylation of 4.1 h~.~ recently been shown to lower its affinity for spectrin by 5-fold [77], Phosphorylations by both cAMP-dependent kinase and kinase C can reduce band 4,1 stabilization of spectrin binding to F-actin [78], The multiple intcxactions of 4.1 in the membrane skeleton suggest a possible role for its phosphorylation in erythrocyte shape changes, but additional studies will be necessary to establish such a role. In vitro phosphorylation of ankyrin reduces its affinity for spectrin tetramer, suggesting that this interaction with the skeletal matrix may also be regulated by Idnases [79]. Similarly, the 43 kDa fragment of band 3 can be phosphorylated, but the modification did not affect its interaction with ankyrin [80]. Many other ¢rythrocyte proteins are phosphorylated, but the roles of these phosphoryladoos in erythrocyt¢ function have not been ehieidated. Other putative mediators for controlling the erythrocyte membrane skeleton structure have been described, including inositol phosphatides [81] and calmodulin [58,82,83]. Calmodulin not only interacts with adduein, as described above., but also binds directly to spectrin. In vitro, this association inhibits the interaction of spectrin with aetin [82,83]. How calmodulin affects the cellular skeleton in response to calcium changes is uncertain, but provides an intriguing potential control mechanism.

III-A.4. Assembly of the skeleton Since mature mammalian erythrocytes do not synthesize significant amounts of the meanbra,~e or skeletal protdns, synthesis and assembly of the membrane skeleton have been studied in nucleated chicken embryo ery.throid cells [84]. Pulse-chase and immunoprecipita. tion studies indicate the presence of a- and ~-spectr:ns and ankyfin (called gobhn in avian erythrocytes) in both soluble and cytoskeletoo-assoclated pools within 10 rain of labeling [85]. However, the proteins are 'rot present in the ratios expected if regulation of protein synthesis determined their stoichiometry. Instead, newly synthesized a-spectrin is present in a 3-fold greater amount than ~-spectrin [86,87]. Analysis of the soluble forms of the newly synthesized specttins by gel filtration shows that a-spectrin is present as a homodimer and fl-spectrin as a homotctramer [88]. Since equal amounts of the two spectrin subanits are assembled into the cytoskeleton, these results suggest that newly synthesized spectrin suhunits are rapidly assembled into heterodimers, a homodimer (a), or a homotetramcr (/]). Assembly onto the membrane is postulated to occur by binding of ankyrin to the anion channel (band 3), followed by association of the spectrin heterodimer with ankyrin [84], By this p n ~ s s , the limiting factor in assembly of spectrin onto the membrane is the extent of heterodimerization [88]. The overall assembly process is postulated to be limited by the amounts of band 3 available for binding ank~in [gS]. Soluble forms of spectfins and ankyrin are rapidly degraded. Support for this mechanism was obtained from studies of trans. formed erythroid cells, which do not express band 3. In these cells the membrane skeletal components assemble only transiently, and then are degraded [89]. Induction of terminal differentiation and expression of band 3 results in an increase in the levels of the membrane skeletal components [84,89]. One questian raised by this mechanism is how band 3 can be limiting when it is present in substantial excess over nakyrin. Perhaps band 3 binding sites are blocked by ofigomerization or binding to othcr proteins. Mouse erythtoleukeania cells induced to differentiate show different behavior [89a]. At day three, ,8-spectdn synthesis is 3-fold greater than a-synthesis, and B-spestrin is more rapidly degraded than a-spectrin. A balance of the two forms may thus be maintained by compensatory synthesis and degradation mechanisms. Band 3 synthesis begins at day 2, and it is relatively stable to degradation, suggesting that it provides a site for anchoring and stabilizing the cytoskeleton in these cells. The synthesis and assembly onto the membrane of protein 4.1 in chicken erythrocytes is rapid and efficient, rexluiring less than 1 rain for assembly of 95~ of the molecules [90]. Although chicken 4.1 is more haterogeneous than the mammafian erythrocyte protein. which has two major isoforms, all of the variant forms

153 show similar kinetic behavior. The amounts of the variants assembled at different stages of erythroid differentiation are determined at the transcriptional or translational stage, not postranslationally. Moreover, assembly of 4.1 onto the membrane skeleton increases relative to spectrin during tenrdnal differentiation [90].

lll-B. Erythrocyte cytoskeletm analogs

plasma membrane glycoprotein (GP 180) has been described [99]. If it can be shown that no other proteins are present in this complex, this observation would provide evidence for such a direct linkage of spectrin with the membrane. Binding of HeLa cell spectrin to HeLa plasma membranes also supports the possibility of a direct biudiag mechanism, since other acti~ binding proteins (filamins) failed to bind to the membranes

[1001. Analogs of the erythrocyte membrane skeletal proteins are found in most cell types and over a wide span of evolution [91]. Immunological, biochemical and ultrastructoral analyses have identified three classes of spectrin-like molecule in animal cells. All three classes have a similar 240 kDa subunit (~ subunit of erythroid speetrin). ED,throid spectrin, as noted earlier, has a f l subunit of 220 kDa. Non-erythroid spectrin (NF ¢~-'z=trim also caUed fodrin) from most cell *_)pes has a fl subanit of 235 kDa, but a ~ : ialtzed spectrin from intestinal bnlsh-border spectrin (BB spectrin, also called 260/240) has a second subunit of 260 kDa [92,93]. NE spectrin is the most widely distributed is~form in the chicken, being found in all tissues examined except erythrocytes and skeletal muscle, both of which contain erythsoid spectrin. BB spectrin appears 'imited to intestian. The story is further complicated by the presence in brain of two distinct subtypes of NE spectfin [94,95]. Subtype 240/235E is found in neuronal cell bodies, dendrites and some glial cells and cso,~-reacts with anti.eD'throcyte spectrin. Subtype 240/235 is found in neuronal axons and does not cross-react with antierythrocyte spectrin. Cloning and sequencing of a-spectrio cDNAs demonstrated that the nonerythrnid polypeptides ate conserved across species, while the erythroid counterparts diverge significantly [96]. The function of the non-erythrnid spectrins is a subject of considerable interest [911. Clearly, in most cells the amount of spectrin present is insufficient to form the kind of spectrin skeletal network found in the erythsocyte, An association with the plasma membrane is suggested by immunofluorescence localization studies [97] and by capping of the speetrin with cell surface molecules [98,99]. However, both of these properties might also be explained by association of spectrin with the submembeane or cortical mierofilatnents. Such considerations raise the possibility that spectrin could link microfilaments to the plasma membrane as it links actin oligomers to the plasma membrane via ankyrin, 4.1 and glyeopborin in the erythrocyte. Two feasible mechanisms for this linkage have been proposed, In the first, spectrin would link mierofilaments to an ankyrin-like molecule as in the erythrncyte. As described below, ankyrin analogs have been found in several cell types. In the second mechanism, spectrin would link microfilameats directly to a plasma membrane glycoprotein. A complex containing HE spectrin and a lympbomt~

One possible consequence of Nt~ spectrin links between microfilaments and the plasma membrane is that spectrin may be involved in the regulation of receptor mobility and function. NE spectrin does undergo redistribution along with actin during ligand-indueed capping of lymphocytes [98,101]. A possible role has been proposed to- spectrin in the regulation of the glutamate r~eptor ia i'rain [102], based on a correlation between spectrin content and glutamate binding to membranes subjected to culcium-dependent eaipein proteolysis. Moreover, calmodulin stimulates caipain degradation of brain spectrin [103]. Whether spectrin is dire~tl~ ia volved in receptor functions remains to be determined. However, a putative role for spectrin in calcium-dependent neuronal function is suggested by the observation that spectriv is the major calmodulin-binding protein in postsynaptic density preparations [104]. How the calmodalin binding may contribute to spectrin functions remains uncertain [105]. One propnsai for the function of nonerythroid spectrios and their associated proteins is that they play a role in the development el polarity in polarized eclk such as epithelial cells [106]. Both NE, spectriu [107] and ankyrin [108] exhibit such polarized distributions, localizing at the basolateral surface of epithelial cells. Spectrio undergoes significant changes in distribution and turnover during development of polarity of MDCK epithelial cells [107]. Moreover, the establishment • f call-cell contacts appears to play an important role in the formation of a spectrin-containing membrane skeleton during developn'~m of polmJty [109]. How this skeleton may function is uncertain, but one possibifity is that key functional components of this membrane domain may associate with it. In support of such a mechanism is the fact that ankyrin will bind to purified kidney Na+/K÷-ATPasc in a membrane-bound form

0]o1. Functions of nonerythsoid spectrins fie not appear to be limited to membrane-eytoskeleton linkages, BB spectrio (TW (260/240) does not bind to erythrnc3,te insideout vesicles or to ankyfin, suggesting that it may serve only as a microfilament crosslinking protein in the brush-border terminal web [111]. However, it may bind to a brush-border membrane component(s) not found in the crythrcoyte. NE spemrlns have also been shown to associate with microtuhales [112], tan protein [113] and dasmin [114] and are present with aclin, tubulin and

154 neurolilaments in the slow-moving group of axonally transported proteins [97]. Mieroinjeetion of anti-spectrin into cells leads to formation of intracelhilar spectrin aggregate~ and distortion and condensation of vimentin-containing intermediate filaments, with no apparent effects on mierofilament or microtubule organization [115]. Clearly, the role of spectdns in cells is complex, and much more work is needed to clarify their various functions. Analogs of erythreoyte ankyrin [91] have been detected immunologically in brain, HeLa cells [116], lymphonla cells [117] and chicken skeletal muscle [118]. Proteins comigrating with mierotubule-associated prowins MAP1 and MAP2 were found to be cross-reactive with anti-erythrocyte ankyrin in brain. These studies suggest that brain ankyrin has a structural segment related to the MAPs; moreover, anti.ankyrin distributes with microtubules in cells, and erythroeyte ankyHn binds isolated microtubules [116]. Purified brain ankyrin (210 and 220 kDa) can be cleaved to produce a 72 kDa speetrin,binding domain [119]. The purified, intact ankyrin binds spectrin, tubulin and the cytoplasmic domain of erythrocyte band 3 [120]. It en'ild feasibly serve as a linker between membranes and microfilaments, membranes and microtubaies, or microtubules and microfilaments. Linkage to membranes would require a membrane molecule with an ankyrin binding site such as band 3. Nonerythroid analogs of band 3 have been identified by immunological cross-reactivity [121] and gene cloning [122]. Recently, a 72 kDa polypeptide with properties of the 72 kDa eryth-oeyte ankyrin fragment has been isolated with an 85 kDa glycoprotein from lymphoma ceils [117]. Whether this truncated ankyrin-like molecule could act as a cytoskeleton linkage site to the plasma membrane remains to be shown. Immanologically cross-reactive analogs of erythro. eyte protein 4.1 [91] have been found in brain [123], lens [124], fibroblasts [125] and se,verul blood cell types [126]. The fibroblast protein is f.~und on stress fibers, indicating that it is located primarily on micrnfilaments rather than at the membrane of the fibroblast. Interestingiy, protein 4.1 may be immunulogieally cross-reactive with synapsin I [127], a major phosphorylated protein of synaptic vesicles [128,129]. Synapsin I binds to speetrin [43], suggesting a fanetinnal site in common with protein 4.1. However, there appear to be only marginal sequence similarities between the two proteins [12~a]~ and evidence has now been presented for a band 4.1 analog in neurons [129b]. A domain of synapshi was shown to cross-react with antibodies against villln, an F-actin bundling and fragmenting protein of intestinal brush-border microvilli [130]. Moreover, purified synapsin caused a phosphorylation.dependent bundling of F-aetin, suggesting that synapsin and villin share both functional and structural similarities [131]. Since syn-

apsin I is associated with microtubule and neurofilamerit preparations from rat brain [132], it may provide linkages among the different neuronal eytoskeletal elements or link them to synaptic membranes. That protein 4.1 analogs can be associated with plasma membrane proteins in nonerythroid cells is suggested by observation of a complex containing a 4.1 analog and lymphocyte Thy-1 in detergent extracts of lymphoma membranes [133]. IV. Interactions of plasma membranes wi~h speclfie cytoskeletal slraetures IV-A. Microfilaments IV-A.I. Microvilli Microvilli are finger.like projections on the surfaces of many cell types. Considerable variability is observed among different cells in the number and packing density of microvilli [134]. The densely packed microvilli of the intestinal brush-bordar and other surfaces involved in the exchange of nutrients provides a vastly increased surface to iacilitate this exchange. The function of other microviili is less readily appment. One possibility is that they provide a reservoir of membrane [135] and actin for rapid changes in cell morphology associated with cell movements and divis~un. They are bound by a membrane and contain a core of microfilaments which provides structural support. Maintenance of this relatively rigid structure p~su.'nably requires associations of the core with the membrane at multiple points. Thus, microvilli provide an extremely useful system for investigating the association of organized mierofilaments with membranes. IV-A.I.a. Brush border Brush border rnicrovilli are densely packed special. izarions of the apical surface of intestinal epithelial cells. Their microfilament cores are hishly organized and orlenW.d such that their arrowhead complexes with heavy meromyosin point away from the microvillus tip [136]. Individual microfilaments extend from the microvillus tip into the terminal web region of the epithelial cell body. At the microvillns tip, the mierofilaments of the cores terminate in an ill-defined dense plaque apparently associated with the plasma membrane [136]. The terminal web consists of two regions. The region into which the core is inserted contains fine filaments bridging the rootlets of the cores. These filaments ,:.mtaln myosin [137] and nonerythrnid speotrin [138]. The second region contains circumferential bundles of microfilaments of mixed polarity perpendicular to the rootlets. Associated with these microfilamants are myosin [139], t~-aetinin [140] and tropomyosin [141], all of which are absent from the microfilaments '_n the

155

I10K,Ca~l

I

W,

. J HiILf Utit Fig. 5. Modelfor filamentsand associated pro~ins in intestinal brush border. C.B., circumfe~ntial bundles; MF, microfilaments; Tin, tropomyo~in;CaM.~lmoduli~ tubular regions of the microvilli. The microfilaments insert into the membrane at adhesion plaques at the level of the zonula adherens [142], which contains aactinln and vincufin. Below the zonula adherens, a dense meshwork of eytokeratin.containing intermediate filaments is nssociated with the macola adhereals, These filaments often appear to interact with the microvillar core rootlets [137I. The microvillar cores of the brush hordel have highly organized bundles of m i c ~filaments [143]. Thus, the microvilli are a particularly amenable system to ultrastructural studies. This highly organized structure is apparently formed and maintained by specific distribution of cytoskeleton-assoeiated proteins (Fig. 5). Microfilament cores contain three major proteins which are not present on the fdamems located in the terminal web region: fimhrin (68 kDa), villin (95 kDa) and a 110 kDa protein [144I. In addition, tropomyosin is present in the rootlet regions of the core which extend into the terminal web [145]. A model for organization of the intestinal brush border and its microvilli is shown in Fig. 5. Fimbrin is a ubiquitous protein present in a wide variety of tissues. Purified fimbrin assembles mierofilaments into bundles [146]. Viilln is a caleium-sansitive protein which has a limited distdbuiion. At low calcium concentrations (0.1 ItM), villin bundles microfilaments. At higher calcium concentrations villin acts as a severing agent, a tliament-cepping protein and a nucleator of filament formation. These Ca2+-scosltive -villin effects have been reviewed by Mooseker [144]. These observations suggest that fimbrin and villin function in the formation and stabilization of the filament bandies. VLllin may also p ~ iicipate in filament formation; however, a more 1Lkeiy role is in the breakdown of filaments in dying cells which have lost their ability to exclude calcium. The 110 kDa protein, which is found in only the tubular regions of the microvilli of the brush border

(Fig. 5) has been implicated in the lateral association of microfilaments with the membrane. These lateral connection~ e:~=~ be ,Asual~ed by electron microscopy as periodic projections from the microfilament core to the membrane in microvi|li of brush borders [147]. These projections remain in microvilh demembranated with Triton X-100, but are specifically extracted by treatment with ATP [148]. The l l 0 kDa protein is also released by ATP and can he purified as a complex with calmoduiin [149]; this complex is stable in the absence of calcium. The 110 kDa calmodulin complex exhibits actin binding and ATPase activities similar to those of brush-border myosin, but is dissimilar by immunological and peptide-mapping analyses [150]. To show that the 110 kDa protein 7s the link between the microfilaments and the plasma membrane, the site and mode of interaction with the cytoplasmic face of the membrane must be established, Although 110 kDa protein isolated with detergents has hydrophobia properties [1511, l*. is probably not associated with the membrane as an t'ttegral membrane proteia [152], The most likely alternative mode of association is via an integral membrane protein, Triton extraction of brushborder microvillar v~icles prepared in the presence of calcium yielded a cytoskeieton fraction which contained a major giyenprotein component of 140 kDa [153l. Antibodies raised against this protein also cross-reacted with a 200 kDa component. The immunological cratereactivity and pcplide mapping indicate that the 140 kDa component is a fragment of the 200 kDa component [154]. Overlays of SDS gels of microvillar cores, using a crude preparation of 140 kDa protein and antibody blot analysis, suggested binding of the 140 kDa protein to the 110 kDa protein. Direct binding studies are n~:ded to verify this interaction. Moreover, the differences between Triton co~'es of microvilli, which appear to contain little if any 140 kDa component, and Triton residues of microvillar vesicles, which contain substantial 140 kda component, need to be resolved. The roles of ATPase and calmodulin binding in 110 kDa protein function are yet to be resolved. The protein can also be phosphoryiated by an endogenous protein kinase [155], which might provide a regulatory mechanism similar to the regulation of myosin contraction. However, the analogy to myosin should be evaluated carefully. A mecbanochemical role for 110 kDa protein is difficult to visualize, if its localization is restricted to the region between a relatively static microvillar membrane and mierof'dament core. IV-A.Lb. Ascites tumor cell Microvilli of many other cell types are much less highly ordered and much more dynamic than those of the brash border. These micruvilli probably play important roles in t~-gulating changes in the topography of cells with dynamic cell surfaces, changes which are

156 involved in cell motility, division, endoeytosis and other surface-related functions. Understanding these changes first requires a knowledge of the microvi]li at the molecular level, particularly of the association of the microfilaments with the membrane and the organization of the microfitament core. For such studies we are using ascites sublines of the 13762 mammary adenocarcinoma [156], which offer particular advantages. We have selected a number of sublines with different cell surface properties and have concentrated on two of these sublines (MAToB] and MAT-C1) because they represent the extremes of the properties of interest. MAT-C1 cells have been studied more thoroughly because of their unusual surface properties. Their cell surfaces have abundant, highly branched mierovilli extending from the cell body [156,157]. Cell surface receptors on the MAT-C1 cells are immobile, as assayed by fluoiesc~nt concanavaiin A. In contrast, MAT-BI cells have rather ordinary microvilli and mobile cell surface receptors. One other striking difference between these sublines is noteworthy. MAT-C1 microvilli are very stable to treatments of the cells with cytochaiasins or hypotonic solutions [157]. MAT-B1 cells and other cells grown in suspension have microvilli which are lost during these treatments. We have postulated that the restriction of cell surface receptor mc;bility and branched mierovillus structures of the MAT-C1 cells are a consequence of this unusual microvillus stability, because of the inability of the highly stabilized cell surface to interconvert between mierovilli and ruffles. Thus, the stability of the microvilli freezes the surface into a configuration which limits its mobility [157]. Clearly, knowledge of the factors regulating cell surface organization in this system could contribute significantly to understanding how cells regulate their cell surface dynamics. For these studies, we have developed a gentle procedure for isolating microvilli from these sublines which does not disrupt the cell bodies. After passing cells through a wide-gauge syringe needle, the microvilli were isolated by Perco]l gradient centrifogation [15B] or by differential centrifugation [159]. Isolated MAT-C1 microvilli retain substantial branching. The microvilli are sealed rightside out and are free of contaminating intracellular organelles, since the cell bodies are not disnlpted by the shearing. By electron microscopy, the mierofilament core of the i~olated microvilli appears intact and uraperturbed [159]. Thus, the isolated ascitas tumor cell nllerovilli are a plasma membrane preparation with its associated cytoskeleton which is comparable in purity to the erythrocyte membrane. The aseites microvilli differ substantially from brush.border microvilli. Ultrastructurally, their filament bundles are not as highly organized and tightly bundled [lfig]. No evidence of lateral cross-bridges between microfilaments and membrane have been observed. Bicchenlieally, the

ascites microvilli appear to have little or no fimbrin, villin or 110 kDa protein [158,160]. In contrast m brush-border microvilli they do have a-aetinin [158,160] and tropomyosin [161] in substantial quantities relative to their actin contents. Thus, in composition as well as microfilamant organization !hey resemble the cortical region of cells much more than they do the brush-border microvilli. Our research has concentrated on two questions, which have proved to be related. Why are MAT-C1 mierovilll more stable than MAT-B1 microvilfi? How are the microfilaments associated with the membrane? One possible reason for the difference in stability might be due to a structural difference at the molecular level, most likely a protein or proteins. SDS-PAGE of the two types of microvlili identified a 58 kDa polypeptide which was present in MAT-C1 mierovilfi but undeteeta. ble in MAT-B1 microvilli [159]. The 58 kDa component was pelleted at 100000 X g with microfilament cores prepared by extracting microvilli with Triton X-100 under conditions which stabilize the microfilaments [159,160].: It was also found in microvillar membranes depleted of mierofilamentx by homogenizing the microvilli under actin-depolymerizing conditions [%159]. These membranes retained a large amount of actin which behaved differently from either F-actin (i.e., was stable under microfilament depolymerizing conditions) or O-actin (did not inhibit DNase) [9]. Like actin, 58 kDa protein was inaccessible to labeaing by lactoperoxidase in the sealed mlerovilli. These results suggest that the 58 kDa component is present at the cytoplasmic face of the membrane at the membrane-microfilament attachment site in the microvilli. However, this component cannot be solely responsible for the membrane-mierofilament linkage, because it is absent in MAT-B1 microvilli. The extraction and membrane preparation studies provided the first clue to a lir~kagu mechanism. Examination of microfilament cores by two-dinlensional isoele2tric focusing-SDS-PAOE demonstrated the presence of a 75-80 kDa glycoprotein [162], which we named C A G (¢ytoskelet'~n-associated #ycoprotein). CAG was also present in the microvillar membranes. By lactoperoxidase labeling, neuraminidase treatment and glucosamine metabolic labeling, CAG was shown to be a cell surface glycoprote'm. Myosin affinity precipitation and DNase treatments suggested that CAG was associated with the microfilament core, rather than nonspecifically co-pelleted [162]. Triton X-100 extraction of M A T - e l microvillar membranes from cells metabolically labeled with lancine left a residue containing CA(}, 58 kDa protein and actin in a ratio of approximately 1 : 1 : 1 [163], suggesting that these components might be present in the membrane as a complex. Velocity sedimentation sucrose density gradient centrlfugation and gel filtration of these

157 extracts demonstrated co-migration of the three proteins in a species of molecular mass > 106. Concanava]in A, which binds to CAG but not to 58 kDa protein or actin, co-precipitated the three components from the gel filtration ehiam. Complex was isolated by the same procedure from MAT-B1 microvillar membranes and shown to contain actin and CA(;. These complexes have been designated transmembrane complexes, because CAG has a segment exterior to the membrane, and actin and 58 kDa protein are both located inside the membrane [159]. To demonstrate that the transmembrane complex is associated with microfilaments, we have dot,eloped a new method, phalioidin "shift' on velocity sedimer,talion gradients, for identifying strongly bound microfilament-associated proteins [164]. This procedure employs the enhanced resolving power of velocity sedimentation sucrose density gradient centrifugation over peiicting procedures in fractionating Triton exl~2:t~ ,:__"microvilli. In addition, the specificity of phalioidin, the c~crofilament-stabilizing toadstool toxin, provides a means by which microfilaments can be shifted on the gradients to aid in identifying microfilamcnt-associated proteins. Under normal extraction conditions, microfilaments are partially fragmented and migrate more slowly than intact cores. The fragmentation can be essentially eliminated by including phalioidin in the extraction buffers [160,164]. Thus the microfilaments incubated with phallnidin during extraction shift farther into the gradients than those extracted in the absence of phalInidin. The double dilution (extraction and centrifugation) eliminates trapping and causes dissociation of all but the most strongly bound prctalns. For example, a-actinin of the cores is largely dissociated during the procedure [164], unless specifically stabilized [160]. Thus. this phalloidin shift procedure can identify strongly bound mlcrofilament core proteins. Both CAG and 58 kDa protein are found associated with the mic~ofilamerit fractions on the gradients in the presence and absence of phalloidin, indicating that they are strongly associated with the microfilaments. These results indicate that the transmembrane complex is a linkage site for microfilaments at the membrane [165]. To understand the nature of that linkage, we have investigated the properties of CAG. Further fractionation studies have demonstrated that the 75-80 kDa species comprises two different components. One is a disulfide-linked multimefic glycoprotein [166] which cannot be metabolically labeled. Immunological analyses have shown that this is bovine IgM. which is present in serum used to stabilize the cells during preparation of microviili. The results presented above indicate that the IgM is strongly bound to a component on the cell surface, since it survives the low ionic strength and high p H conditions (pH 9.5) used for preparation of microvillar membranes [159]

and the extraction and fractionation pro:edures for preparation of transmembrane complexes [163]. Moreover. bound IgM survives the extraction and phalioidin gradient procedures for preparing the microfilament cores, indicating that it is strongly associated with a cell surface component bound to the core. Thus, it is a non-native, but strongly bound, microfilament~ore-associated protein. The second 75 80 kDa component (CAG) is the one metabolically labeled with glucosamine and leucine. This component is poorly detected by protein stains on gels and is best detected by fluorography of glucosamine-labehid preparations or by concaaavalin A staining of gel transfers. It is strongly bound to and co-isolates with microfilament cores (phalhiidin shift) [164] and uansmembrane complex i163]. Moreover, it remains associated with microvillar membranes prepared by extraction of mierovilli with carbonate (pH 11), a procedure which removes IgM. This 75-80 kDa CAG can be extracted from microfilament cores with high salt, a procedure which also removes 58 kDa protein. Gel filtration of these extracts showed co-ehition of 75-80 kDa CAG with two other concanavalin A-binding glycoproteins, of apparent molecular mass of 60 and 50 kDa. which can also be metabolically labeled. These glycoprotelns co-purify with transmembrane complex prepared under several sets of conditions. We have termed the glycoprotein trio of the transmembrane complex the CAG domain and the three components of the domain, CAG-1 (75-80 kDa), CAG-2 (approx. 60 kDa) and CAG-3 (approx. 50 kDa). Since the putative CAG complex can lie isolated from mlcrofilament co~.s, the glycop~oteins of this domain are prime candidates for the transmembrane linkage to micsofilaments. Whether this linkage is direct or indirect has yet to be demonstrated. However, preliminary experiments with the isolated glycoprotein trio suggest that they retard actin polymerization. These results suggest that one or more of them can bind to actin directly. The role for 58 kDa protein at the microfihimentmembrane interface is being actively investigated. Recently, we have purified 58 kDa protein from high :alt extracts of microlilamant cores. It is isolated as a mono. tactic, elongated molecule, which binds to F-acfin and retards actin polymerization in the manner of a capping protein [167]. It also binds to phuspholipid vesicles. Thus it has the properties required to stabilize memlirane-miarofilament interactions, as we previously proposed [168], by binding to both the membrane and microfilaments. From the results described above we can propose working models (Fig. 6) for the association of mierofilaments with the transmembrane complex. The CAG domain is envisioned as a transmembrane glycopsotein complex of at least three components with actin-binding

158 ation of metabolism by hormonal stimulation, the action of neurotransmiuers, and the stimulation of the egg during the fertilization process are examples of these activation processes [174]. Association of cell surface molecules with the cytoskeleton is widely believed to be one of the early consequences of cellular activation for many systems [175!.

Fig. 6. Models for the ~s~iafion of microfilamentsof MAT-CI ascites lumor ~11 microvilliwith the microvillarmembrane. sites on its cytoplasmic segment. The 58 kDa component can then associate in the MAT-C1 ceils with the CAG complex a n d / o r the membrane lipid and with proximal monomers of the filament 1o stabilize the mierofilament-membrane links in MAT-C1 microvilli. We suggest that dissociation of mierofilaments from the membrane initiates depolymerization and collapse of the microvilli, as observed with MAT-B1 cells. Prevention of this dissociation by 58 kDa protein will freeze the cell surface and prevent conversion of microvilli into ruffles and other dynamic cell surface structures, thus restricting mobility of cell surface molecules on MAT-Cf cells. The absence of 58 kDa protein in MATB1 cells permits dissociation of the mierofilamcots from the membrane and allows mobility of cell surface components. These hypotheses are under investigation. The observations described above suggest a possible direct linkage between microfilaments and integral plasma membrane components. Direct linkages have also been suggested in other systems. The laminin receptor has been proposed to be linked to mierofilaments by virtue of the fact that the purified receptor binds actin [169]. The membrane glycoproteins G P I I b / G P I I I in platelets are induced to associate with actin after concanavalin A activation, as demonstrated by crosslinking studies [170]. A subfraetion of the actin also appears to he bound in unstimulated platelets [171]. The ability of actin to bind to membrane components of Dictyostelium has been shown by binding of inside-out vesicles to an F-actin affinity column [172]. An aetinbinding membrane component of 17 kDa, named ponticulin, has been identified [173].

IV-A.2. Cell activation and transmembrane signaling Molecular recognition at cell surfaces and transmission of signals from the external milieu across the plasma membrane are key steps in activation of many cell types. Specific binding of ligands to their receptors is the first step in a cascade of molecular events culminating in altered cellular behavior. Lymphocyte, neutrophil and platelet activation, stimulation of cell proliferation by growth factors, alter-

IV-A.2.a. Platelers. Platdet activation triggers a complex and remarkably rapid sequence of cellular changes, which result in increased adhesivity of the platelets to vascular endothelinm and to other platetets. These include shape change, extension of filopodia, aggregation, secretion of granule eoutcots and clot retraction. Each of these involves the plasma membrane [176] a n d / o r the cytoskeleton [177]. Platelets are an excellent system for studying cytoskeleson organization or memhrane-cytoskeleton interactions, because they undergo these activation phenomena and because they contain an abundance of cytoskeletal proteins [178]. They have a high content of actin (15-20~) and of proteins which bind actin, such as myosin (2~), actin-binding protein (ABP) (2~), and talin (2%). Unstimulated platelets contnin about 60~ of their actin as monomeric actin, as assessed by DNase inhibition assay of Triton extracts, while activated plateiets have only 30~ [179]. Differential ceutrifugation of the Triton extracts of unstimulated platelets, lyseA in the presence of inhibitors of plafelot calcium.activated protetnas¢ [177], yields two eytoskeleton fractions. The bulk of the cytoskeleton is sedimented at low speed, but a fraction requires high-speed sedimentation. The major c o m p o n ~ t s associated wlth the high-speed pellet are OPIb, a major c¢11 surface glycoprotein, actin filaments and ABP. This fraction has been proposed to contain the plateiet membrane skeleton [180]. If platelets were lysed in the absence of inhibitors of the platelet calcinm-activated protease, GPlb was no longer associated with the skeleton fraction. The loss of GPIb occurred concomitantly with de.avage of ABP by the proteinase [177]. Further evidence for the association of ABP and GPIh was obtained by immunoprocipitation studies. When Triton lysates of platehits were treated with DNese to depolymvrizo miceofilamcalts and treated subsequently with anti-ABP, both ABP and GPIb were precipitated [181]. These results suggest that microfilameuts arc associated with GPlb of the unstimulated plateiet plasma membrane. However, these linkages axe broken upon activation, as a consequence of the influx of calcium and activation of the protoase to cleave ABP. Activation has a second apparent effect on platelet membrane-cytoskeleton interactions: association of glycopruteins GPIIb and IIIa with the cytoskeleton of aggregated or activated platelets [182,183], as described in the previous section. This effect can be demonstrated

159 by Triton extraction of concanavalin A-stimulated plateiets [184]. After stimulation 80-95% of the two glyenproteins are associated with the Triton cytoskeletal residue, enmpared to 10-15% in unstimalated platelets [171]. Cross-linking studies suggest that the linkage of the $1ycoprotein~ to actin is direct [1701. The studies de-edbed above suggest an interchange of membrane-eytoskeleton interactions during platelet activation which may be important during shape changes, aggregation, filopodia formation and secretion. Since GPlb is believed to be the receptor for both thrombin [185] and the van Willeb-and factor [186.187] involved in platelet aggregation, changes in its association with the cytoskeleton may alter its receptor actbity [188] as part of the mechanism for priming platelets to aggregate. A caleium.dependea~t complex of GPIlb and GplIla [189] acts as a receptor for four proteins: fibrinogen [190,191], fibronectin [192,193], van Willebrand factor [186,193,194] and vitronectin [195]. O P f l b / i l l a appears to be involved in the association of fibrin with platelet eytoskeletons [196]. The mechanism by which the putative exchange of cytoskeleton interactions of GPlb for GPIIb-III affects platelet physiological functions may be quite complex and will require further investigation. IV-A.2.b. Neutrophils Neutrophils are phagocytic leukceytes involved in the acute inflammatory response. Exposure of these cells to formylated peptides, such as formylmethionyllencineph~ylalanine (tMLP), induces chemotactic and secretory responses, and transiently activates a plasma membrane redox system responsible for the production of superoxlde radicals for defense against infection. These chemotactic peptides act through a receptor. mediated mechanism t~, trigger aetin polymerization and concomitant increases in cell locomotion [197], followed by actin depolymerizarion [198]. New peptideinduced actin nucleation sites have been suggested to determine the cellular localization of actin polymer~ arian, which could datermine directional extension of filopodia [199]. Chemotaetie peptide-induced tyro. skeletal alteration in the neutrophil has been reviewed by Omann and co-workers [200]. Binding of peptide to its cell surface receptor modulates the number of available receptors [201]. At least two kinetically different GTP-sensitive [2021 bound forms of the peptide can lie observed [203,204]. Moreover, some [2031 or all [204] of these bound poptides are not released by treatment of the cells with Triton X-100. Since the peptides remain associated with the cytoskeletal residue from the coils, 3esaitis and co-workers have suggested that the receptors are associated with the cytoskaleton [203]. A similar association with nentrophil eytoskeletal residues has been observed for an 80 kDa

cell surface glycoprotein [205] and leukotriene B4, which is presumably receptor bound [2061. The mechanism and physiological consequences of the putative association of the peptide receptor with the cytoskeieton remain unclear. However. the association with the cytoskeletal residue can be prevented by dihydrocytochalasin B with a concomitant augmentation of the rate and duration of superoxide production I207]. Thus. the binding of the receptor to the cytoskeleton may represent an off switch in the mechanism for activating the neutrophils. IV-A.3. Stgnal transduction mechanisms Cell activation is initiated by some form of transductiou of si~.a~is gel~eiatral by ligauds th~oug,h the plasma membrane [17,1]. Five general mechanisms for signaling have been proposed: (1) transfer of the signaling molecule across the membrane; (2) formatiorl of a e_ytoplasmic second messenger; (3) signal-induced reversible covalent modification of cytoplasmic proteins or protein domains, such as phosphorylatinn and rnethylation; (4) transmembrane conformational coupling (still a hypothetical mechanism, although it has been suggested as part of the neutrophil activation scheme) [200]; and (5) transmembrane protein clustering. A modification of tile clustering mecha:t;~m has been proposed which specifically addressed cmsterin$.induced enhancement of submembrane actin-binding affinity [208]. A number of recent reviews 1209-222] describe, refine and provide evidence or suggestive evidence for these mechanisms. Transduction by some ligands appears to combine more than one of these general mechanisms. Interestingly, membmne-cytoskcleton interactions have been implicated in four of the five mechanisms. Indeed, the cytoskeleton can fulfd the requirements of signal transductinn of second messengers by signal amplification via physical forces [211]. The best examples of direct transfer of the signaling molecules are the ion channels, in which the receptor itself forms a channel or ionophoee. The most thoroughly studied ion channel is the acetylehniine-activated channel, which upon stimulation by llgand allows the selective entry of ions. In adult muscle, most of the acetylcboline receptors controlling these channels are clustered in the postsynaptic membrane of the neuromuscular junction. These are highly specialized structures containing many of the proteins associated with other types of specialized junct,an; a-actinin, vinenlin [223], and talin [224]. Receptor clusters which are immobile are also resistant to non-ionic detergent extraction, suggesting an association with the submembrane cytoskeleton [225]. Such eiustca's appear to be stabilized by a class of non-myofibrillar actin filaments [226}. Receptor clusters isolated from cultured rat myotubes by extraction with seponin contain associated actin [227]. Receptors redistribute within the plane of the

160 membrane when actin is depleted by selective extraction. How the receptors arc Inked to microfilaments is still unclear, but isolated receptors have been reported to contain actin [228] and a 43 kDa aetin-binding protein [229], which appears to be specifically associated with the acetylcholine receptors [230]. Endocyto_sls [2], an alternative mechanism for delivery of a signal molecule directly to the cell interior to organelles or to the cytoplasmic face of the membrane itself, is an extensive field which is beyond the scope of this review. The first mode of transmembrane signaling to be characterized was the production of cyclic AM D in response to ligand binding to a cell surface receptor [209,210]. The receptor-dependent mechanism is mediated by GTP-binding proteins (G-proteins), a family of related proteins, including transducin of retinal outer rods, which couple a wide array of hormones, neurotransmitters and other regulatory molecules to adenylate cyelase [222]. The G-proteins then translate the information of ligand binding into the activation (G,) or inhibition (G i) of the catalytic subunit of adenylate cyclase [209,214,218]. A possible role for G-proteins in mediation of signals via another system, that involving inositol phospholipid, has also been suggested. G-proteins are heterotrimeric, consisting of an ~-subunit (39-45 kDa or lar~,r), a common /~-stdiudit (35 kDa) shared by G s- and Gi-proteins, and a y-subuhit (8-10 kDa), which may be similar for the different G-protelns [215]. The a-subanit provides both the receptor specificity and effector functions of the G-proteins. The mechanism for coupling of ligand apparently involves translation of one or more of the three components of the system in the plane of the membrane [231]. Thus, mobility of the components is important to the hormone response. Evidence has been presented that the G-protein and cydase are partially associated with the detergent-insoluble cytcekeleton of erythrocytes [232-234]. Activators of cyelase release this apparent eytoskeleton association, suggesting that the association may re,strict hormone activation. Microtubu!~ disrupting agents can also enhance hormone-medlated eyclase activation [235]. The mechanism of the effect has not yet been elucidated. Transmembrane phnsphorylatian as a result of ligand binding can also play a key role in signal transduetion. M~togens such as epidermal growth factor, platelet-derived growth factor, insulin and somatomedin, upon binding to their receptors, appear to induce tyrosinespecific kinase activities in the jTeceptors themselves, resulting in ligaod-stimulated autophosphorylation of a cytoplasmic segment of the receptor [215,216]. For example, the receptor for epidermal growth factor (EGF) phosphorylates both its own cytoplasmic surface domain and a 35 kDa cytoplasmic protein when calls or membranes are treated with E G F [236]. EGF binding

also triggers receptor clustering [237] and internalization [238]. Evidence from both non-ionic detergent extraction [239] and uhrastructural [240] studies suggest an association of the E G F receptor with the submembrane cytoskeleton, although the data do not preclude association with detergent-insdiuble clathrin of coated pits. How this association might contribute to phosphorylation, endocytosis and the ultimate proliferation response of the cells has not been determined. Another mechanism by which EGF and insuhn receptors, as well as cytoplasmic proteins, are phosphorylated and are thought to mediate signal transduction is by activation of the Ca2+/phospholipid-dependent protein kinase C [217]. A number of stimuli promote increased turnover of phosphatidylinnsltol and ealeium mobilization by a cascade of processes starting with activation of a membrane phospholipase C. This activation leads to increased production of diacylglyc, erol, which activates protein kinase C, and of inositol trisphosphate (IP3) which mobilizes Ca 2÷ by releasing it from the eadoplasmie reticuhim [219-221]. An attractive scheme involving this pathway has been hypothesized for cytnskeleta] alterations in chemotuetie peptide-stimulated neutrophils, but certain aspects of the model require verification [2001. IV-A.4. Cell adhesion and ceU-extraeellular matrix interactions: junctions The term 'cell adhesion' encompasses two types of interaction: eaU-cell and cell-substratum, where the substratum can be the natural extracellular matrix or an artificial substance. Clearly, these two types of adhesion are different. However, there are different das,ses of both ceil-fell and cell-substratum adhesion, some of which have remarkable similarities. In this section, we will focus on studies of intercellular junctions [241] or focal substratum contacts 1242,243] which exhibit two characteristics: first, microfdaments have been observed to be associated with the structure in question and second, some molecular entities of the structure have been identified and characterized. Most of the work to date in this area has addressed three questions. (1) What are the components present in the region of the microfilament attachment to the membrane? (2) What are the properties of those components which contribute to their role at the membrane-cytoskehiton interface? (3) How are these components organized? IV-A.4.a. Junctional components IV-A.4.a.L Identification. Identification of components present in adherens junctions, which have microfilaments astociatod with the membrane [244], is generally made by immunoeytochemieal means. One of the first components to he found in a wide variety of junctions was a-actinin. Table I! shows its localization in a number of non-muscle and muscle junctions. The

161 TABLE II Components of cellularjunctions

Protein

Fibroblast focal ~nlaet

MDCK junction

Brushborder junction

Myolendinous junctiog

Cardiac muscle di~

~Actinin Vinculin Talin geU~latin 200 kDa

+ + +

+ +

+ + ?

+

+

+

+

?

+ + + +

original impetus for studying a-actinin was its localization in the Z band of striated muscle, the site of apparent attachment of microlilaments to the sarcolemma. The same rationale led to the isolation of other proteins from smooth muscle. Amisesa prepared against such prote~ns were used to assay the presence and localization of these proteins in other tissues and cells. In this way vinculin [245] and talin [246], which are present in some types of junction, were identified. A more direct approach to identifying components in adhesens junctions has been to isolate specific junctional complexes and use these to prepare a hatter-/ of monocinnal antibodies (MAbs). Antibodies are then used for identification and localization e l specific components by immunocytochemistry or immunoblotting. Maher and Singer [247] have isolated fascia adherens domains of the intercalated disk membranes of chicken cardiac muscle and identified a 200 kDa protein present in non-muscle junctions (Table il). MAbs have identified a large muscle-specific protein named zeugmatin [2481. All of the components mentioned above are present at the cytoplasmic surface of the membrane. A similar approach has been used to identify integral membrane components. MAbs against detergent-extracted components of membranes of chicken cardiac muscle rich in intercalated discs idcatified a 135 kDa glycoproseln component (A-CAM) specifically associated with intercellular adhemns-type junctians [249]. In cultured chick lens cells A-CAM is associated with caicium-dependem junctions and becomes accessible to proteinase cleavage after EGTA treatment [250]. Addition of Fab fragments of anti-A-CAM to EGTA-treated cells prevents calcium-induced reformation of cell-cell adherens junctions and leads to apparent deterioration of ~" ess fibers [25!1. Biochemical studies suggest its similarity to other cell adhesion molecules (CAMs) from other systems [252]. An 83 kDa desmosomal protein common to intercellular junctions, but absent from cell substratum adhesion sites, has bean identified by means of a monodonut antibody and a eDNA done [2531. Studies on the identification and localization of junction-specific molecolas dearly show biochemical differences among dif-

-

+

$rllt~lh m~Ie dense plaque + + + + ?

Striated muscleZ b~d + + ?

ferent classes of adherens junctions. Vinenlin and ACAM are present in intercellular juncfions, but A-CAM is absent from focal adhesion sites [254]. Talin is absent from intercellular junctions but present in focal contacts and smooth muscle plaques, Adding fur'her to the complexity are the interactions of cell surfaces with the extracellular matrix and basal lamina, For example, the fibronexus appears to be an interaction site at which fibers of extracellular fibruneetin and cytoplasmic F.actin are directly apposed across a segment el plasma membrane [255]. Although the membrane components involved at the fibronexus have not been specifically identified, one of the cell surface binding sites for fibronectin is a cellsubstrate attachment antigen (CSAT) 1256.257]. CSAT is a complex of three glycoproteins. The primary sequence of one of these components has been determined by eDNA sequencing [258]. Inte~stingly, CSAT also binds to laminin [257], one of the prominent components of the basement membrane [259]. CSAT is one of a family of receptor proteins, called integrins [260], which recognize the Axg-GlyoAsp sequence on fibronectin and a number of other extracellular gly¢oproteins [261]. Thus, CSAT qlay be involved in cell-matrix junctions with both baseme:t membrane ~ d the extracellular matrix. However, a different receptor for laminim which may also play a role in the formation of transmembrane junctions [169], has been isolated and par~.ially characterized [259]. Moreover, evidence has been presented for direct interaction of beparan sulfate proteoglycans with the cyloskeleton [262l. Such interactions may also be involved in the organization of microfilament cables [263,264]. IV-A.4.a.iL Characterization. Only three of the cytoplasmic components mentioned above have been purified and characterized [242]: a-acfinin, vinenlin and latin, a-Actinins from both skeletal and smooth muscle, and non-muscle sources are dimers of 100 kDa subunits, which act as cross-linking and gelfing agents for F-actin. Binding of non-muscle a-actinin, but not the muscle protein, to F-actin is inhibited by calcium [265]. This calcium sensitivity may play a role in the cortical organizalion of filaments in non-muscle cells.

162 Vinculin was first isolated from chicken gizzard as a 130 kDa monomer [2451 and found by immunofiuorescenee to be located in focal adhesions of non-muscle cells at the termini of stress fibers. A second larger (152 kDa) form of vincutin was also isolated from chicken gizzard, but appeared to be restricted to smooth muscle [266]. The larger form, termed mela-vincu!in, was reported to have more hydrophohic properties, which might permit its direct association with membranes [267]. Both vineniin and rneta.vincuFm have asymmetric structures with a globular head attached to a red-shaped tail [268] and both tend to self-aggregate. Purified vinculin was reported to associate with F-actin to decrease its low shear viscosity [265,269-271] by the formation of filament bundles [272]. Subsequent studies on more highly purified preparations attributed the effects on actin to contaminants in the original vincobn preparations [273-276]. Vinculin has been shown to interact with talin by direct velocity sedimentation analyses [277] and by gel overlays [277-279]. Talin is a 215 kDa monomer also isolated from chicken gizzard [246,280]. By rotary shadowing talin was observed to exist in two different conformations, depending on the ionic strength. At low ionic strength a globular conformation was observed; at high ionic strength the molecule was extended [268]. The extended form is the one which probably interacts with vincelin [268]. Preparations of talin usually contain smaller polypeptides, most notably a 190 kDa species, which appears to be a proteolytic fragment of intact talin [281]. Although the amounts of this putative cleavage product can be reduced by using proteinase inhlhltors, it can still be detected, suggesting that cleavage may occur within the intact cell. Interestingly, a relationship has been shown between talln and p235 [282], one of the major proteins of platelets which is cleaved by a calcium-activated proteinase during platelet activation. Whether talin, which is a component of adhesion sites in fibroblasts, plays an important role in platelet adhesion is an intriguing question. IV-A.4.a.iii. Organization. The studies described indicate that the junctions at which microfilameuts insert into membranes are complex and vadegated. Dctermin. ing the organization of these complexes has only recently begun to yield results as the various components have heen isolated and studied. Definition at the ultrastructural level has not provided a great deal of information, although immunoeytoehemieal localization studies indicated that vinculin is more closely associated with the membrane in smooth muscle dc-qse plaques than is ct-actinin [141]. One method for testing whether a protein might be involved in mierofilament attachment to the membrane is to remove either the protein or actin and assay for the presence of the other. Microfilament fragmenting protein can be used to remove all immunologieally detectable acfin from adhesion plaques

FIb;onlotln

Vi

n

Fig. 7. Model for the association of micTorilaments(MF) with the plasma membranein adherensjuncaona] complexes. without the loss of vincufin [283]. Thus, vincufin appeers not to be associated solely with the microfilamants. However, when M D B K cells are incubated with EGTA to disrupt intercellular junctions, microfilaments associated with those junctions are released from the cytoplasmic side of the membrane in association with vincelln [284|. Thus, in these intercellular junctions vinculin appears to be located between the membrane and microfilaments. How might vinenlin be associated with the membrane? The interaction of vinenlin and teiin has been demonstrated by several techniques [275]. Recently, talin has also been shown by equilibrium gel filtration to hind CSAT [285], a plasma membrane integrin, at attachment sites [286]. Thus, a vincolin-mlin-CSAT complex [242] (Fig. 7) could act in these cells in a manner similar to speetrin-4.1-ankyrin-band 3 complex in the erythrccyte in linking ectin to the membrane. However, the molecule(s) associated with F-actin and linking the microfilaments to such a membrane complex reanains unidentified. ll/-A.4.b. Regulation of cell contacts The fact that cell contacts (call-substratum or cell-cell) are modified in tumors has promoted interest in mechanisms by which these contacts may be regulated [242]. Transformation of fibroblasts causes cell rounding and loss of their stress fibers. Analysis of these effects has been aided by studies of Rous sarcoma virus, which contains a single transforming gene src. This gene specifies a 60 kDa tyrosine kiuase [287] which is localized beneath the plasma membrane and enriched in focal adhesion sites [288]. One of the targets for this Idnase is viuenlin, whose tyrosine phosphate content increases about 10-fold upon transformation [289]. However, studies with partial transformation mutants

t63 of the virus show no direct correlation between transformation and vinculin phosphorylatinn [290,291]. If there is any relationship betv een vinculin phosphorylation and ,.raasformation-induced changes, it must be complex. The fact that talin can also be phosphorylated on tyrosine in Rues-transformed cells [292] may provide an answer t~ the role of the tyrosine kinase in the morphological and cytoskelelai changes. Ahernatively, phosphorylation of both talin and vinculhi might be required. Protein kinase C, which can be activated by phorbol esters, has also been implicated in controlling the organization of junctional components. Capping of lymphocytes with anti-integrin causes redistribution of latin in phorbol ester-treated, but not untreated, cells [293]. Neither vinenlin nor a-actinin was affected by the capping in the presence or absence of the perturbant. In contrast, fluorescent t~-acrinin and vinculin injected into cells underwent reorganization with phorbol treatments [294]. How klnase C might be involved in such changes is unclear. Another major subslrate of the Rous sarcoma tyrosine kinase is a 36 kDa protein ]295] which has a submembrune distribution similax t~ nc, ncD'throid speetrin [296]. This protein, named calpactin [297], binds actin [298], spectrin [298], and phospholipid liposomes [297] in a Ca2+-dependent manner [299]. Its function is unknown, but it appears to be one of a family or families of calcium-binding proteins [300] which include epidermal growth fac~r receptor klnase substrate [216]; phospholipas¢ A 2 inhibitor ]301]; phenothiazine-bio4ing proteins called calcimedins [302]; proteins which bind cytoplasmic transport vesicles [303,304]; and AMV-p35, a calpactin homolog which is a major protein of 13762 tumor cell microvilli, AMV-pg5 binds microfdaments and liposomes in a calcium-dependent manner ]305] and phenothiazines in a Ca~÷-indepen dent manner [306]. The properties of calpnctin and its analogs l~dght suggest a role at the mombranc-cytoskeieton interface, These clas~es of calcium-binding proteins have been implicated variously in a number of cellular functions, including regulation of cell growth (EGF kinase substrate [236] and p p t 0 *~ klnase substrate) [216,295], mediation of inflammatory response (lipo. curtius) [301], and secretion (granule proteins) [303,304]. Further studies arc necessary to define their various functions. One interesting property of calpactin is the presence of covalently bound fatty acid [307]. An interaction with lipid appears to be important to a number of cytoskcietal proteins [308], including vinenlin, ankyrin and band 4.1, which have attached fatty acid, and a-actinin, profilin, gelsolin, vinculin, spectrin, band 4.1 and the 110 kDa microvillus protein, which bind specific lipids [309]. These lipid-binding proteins, which may be associated with or recruited to the cytoplasmic region of

the plasma membrane, have been termed amphitropic proteins by Bum [309]. who suggests that their properties may be important for orgamzationat and regulatory functions. Support for this hypothesis is suggested by the fact that myristylation of calpactin is sensitive to cell trans~3nnation [307]. An alternative mechanism for regulating cell contacts is through modulation of the synthesis of proteins. Expression of genes encoding cytoskeletal proteins is affected by cell morphology and contacts [310]. Moreover, vinculin synthesis is regulated by cell contact- and cell shape-dependent mechanisms [311]. Although the molecular basis for this regulatinn is not understood, it provides a means for cellular response to its environment and for increase in cell contacts with substratum or with other cells by increasing cellular vinenlin synthesis.

IV-B. Microtubulez Studies of microtubule-membrane interactions have revolved around Lhrce questions. How are the microtubules in cilia and flagella associated with the membrane? ,~"c n-Acrolubules and tubufin associated directly with cellular membranes? How are microtuhules involved in intracel|ular transport of membrane vesicles? The first two questions have been considered extensively in reviews by Dentler [312] and Stephens [313]. Cilia and enkaryolic flagella ate motile organelles extending from the cell surface. Eukaryolic cilia and flagella have a substructure composed of nine doublet microtubules surrounding two single central microtubules. As in the case of microfilaments in microvilli, microtubules in the cilia and flagella must be attact,,~.l to the organell¢ membrane at the llps and along the sides for the organelle t~ be stable. Uhraslructural studies, summarized by Dentler [312], have shown the presence of complex, specialized structures at the locus of attachment of the microtubules to the tip [314]. Unfortunately, very little is known of the molecular composition of these structures. Lateral interactions between the outer doublet micrombules and plasma membrane appear to involve fdamentoos bridge structures [312]. In most organisms they me apparently connected direcdy to the membrane. These attachment sites may also be sites of localization of mastigonemes, extramembrane halr-llke appendages such as those found on the flagellar membrane of Eaglena, In some organisms, the bridge from the mlerotnbules is connected to a lasg~ membrane-attached structure, such as the paraflagellar rod of trypanosomes or compartmenting lamella of ctenophor¢ swimming plates. Cilia also appear to have attachment sites at their bases where they attach to the cell body. Two different types of structure have been implicated in these attachments, the ciliary necklace [315] and the ciliary granule plaques

164 [316]. Both are best observed in freeze-fracture replicas [312]. By electron microscopy of conventional sections, they appear to attach to the doublet micrombuies by a fibnllary bridge [312,317]. Electron microscopy has also shown in trypanosomes a close and stable contact between a tight, helical array of single pellicuiar microtubules and the cell membrane [318,319]. The molecular description of membrane-mlerotubaie interactions in cilia and flagella has been confounded by two problems, the complexity of the components of the organdies and the question of the presence of membrane mbulin. Stephens [320] found tubuiin associated with membranes isolated from scallop gill cilia but not with membranes from scallop sperm flagella. Comparative amino acid analyses and peptide maps suggested that the membrane-associated tubuiin is different from tubulin of the outer doublets [321]. Membrane vesicles from Tetrohymena cilia, prepared by three different techniques, eont'~An a tobulin-like protein as a major protein [322]. Other workers, using different isoiation procedures, have found much smaller amounts of tubulin in Tetrahymena Ciliarymembranes [323,324]. Ciliary membrane tubalin will associate strongly with lipid vesicles [325]. Axone~qal tubulin does not associate under similar conditions [.~26]. However, in contrast to many integral membrane proteins, membrane tubulin is not partitioned into Triton X-114 above the detergent cloud point [326]. Instead, the tubulin appears to be present as a detergent complex with other proteins in the aqueous phase. These combined studies clearly show the association of tubulin with, but not necessarily intercalation into, ciliary membranes. Definitive answers to the questions of where this putative membrane tubulin is located in the cilia and how it functions must yet be provided. Cross-linking cilia with a photochemically sensitive, cleavable agent stabilizes filamentous bridge structures between the outer doublets and the membrane to detergent extraction [327] and links together a dynein-like ATPase, tubulin, and other proteins. Cleavage of the cross-linker released the bridge-membrane complex and the ATPase. These results suggest that the ATPase may be the filamentous bridge between the membrane and the outer doublet. The ane.logy between this proposal and the observation of a 110 kDa ATPase as the linker between microfilements and the brush-berder microvillus membrane is interesting, but its significance is unknown. Tubuiin has also been reported to be a significant component of membranes from other sources, particularly neural tissue [328-330]. The membrane tobulin resembled microtubule or soluble mbulin in specific colchicinc binding, vinblastine precipitability,subunit size, elcctrophoreti¢behavior and peptide maps [329]. One noteworthy difference was the increased licat stabilityof membrane tobulin,possibly reflectingan effect

of the association with membrane [330]. A second potentially important difference between membranebound and cytoplasmic tubaiins in brain is that the a-subunit of membrane tubulin contains no C-terminaJ tyrosine, whereas cytoplasmic tubulin contains a large fraction which is tyrosinylated [331]. A possible role for tyrosinylation of cytoplasmic mbofin in maintaining membrane excitability in squid axon has been proposed [332], but no role for non-tyrosinylated me,mbrane tubuiin has been found. Tabu[in has also been reported as a component of post~:ynaptic densities [329,333], myelin [334], coated resides [335], and thyroid membranes [336]. Stephens has reviewed membrane tobulins in brain and other tissues and compared their properties to those of cytoplasmic tubulins [313]. The mode of association of tubulin with membranes remains unclear [308]. a-Tuba "lin is more tightly associated than ~-tubulin [337,338]. Several lines of evidence suggesting that membrane tubulin is an inmgral membrane protein include extraction studies [338,339], partitioning into Triton X-114 [340], and synthesis on membrane-bound polysomes [341,342]. None of these studies is completely compelling b ~ a u s e of the vagaries of the individual techniques, as illustrated in studies described earlier for brush-border 110 kDa protein [151,152[. As aught be expected for an integral membrane protein, membrane tubulin has been reported to have associated carbohydrate [343,344]. Feit and Shclanski found that total mouse brain tubulin labeled metabolically with glucosamine and isolated by vinblastine precipitation contained bound radioactive sugars, recoverable as gluensamine and galaclosamine [344]. However, Eipper, using a different isolation technique, found that rat brain tubalin contained virtually no bound amino sugars and a minute amount of neutral sugars [345]. Moreover, no oligosaceharidee have been isolated from membrane tubulin preparations. The basis(es) for these discrepant reports is not certain. The qvestion of giycosymtion of membrane tubulin is an intriguing one and b e ~ s more intensive investigation. The issue of membrane tubuiin is further confounded by reports of tubuiin bound to the cell surface [346,347]. On leukemic cells this tubuiin appears to be associated with a cell surface mbuiinbinding protein [348]. The origin of this tubuiin and its function are unclear. The role of microtubules in intracelhilar transport has been most thoroughly investigated in the case of axonal transport. Use of disrupted axoplasm from the giant axan of the squid has permitted the observation of bidirectional orgenelle movement along microtubuias [349-351]. Much of the recent work has been directed toward identifying the motor(s) responsible for this movement. A prime candidate is a 110 to 140 kDa protein(s) which can generate ATP-dependent movemeats of microtubules across glass surfaces and of polystyrene beads along microtubules (kinesin) [352-

165

f:

i!i!!m ,

:

Fig. 8. Electron rmcroggaphof stable squid brain microtubulesand axoplasmicvesiclesfrom squid ~iant axon. Scalebar is 250 nm. (From Pratt, M.M.,J. Cell Biol.,103,957,1986,with permi~ion.) 354]. However, this protein(s) effects movements in only one direction [352]. Moreover, other proteins may he required in the association of organeiles with the microtubules. Microtubul¢ complexes with axoplasmic vesicles can be px~pared by mixing squid axoplasm with taxobstabilized squid brain microtubnies (Fig. 8) [355]. The complexes contain a discrete set of anoplasmic proteins and a dynein-like ATPase activity, all of which appear to be associated with the vesicles. The possibility that the ATPase acts as a membrane-micrutubnie link, as proposed from the esOes-linking Sntdies O0 allia [327~ , intriguing and deserves further investigation.

IV-C. lnter,~ediate filaments Intermediate filaments inteTact with the plasma membrane at desmosomes. These intercellular junctions ere laminated structures with an inte~ellular core, cross-bridges between this core and the plasma membrane extracelhiler surface, and a thick plaque at the cytoplasmic surface of the membrane. Intermediate iliamenUs appear to insert into this plaque. As mentioned previously, classes of intermediate fdamanus are specifically associated with particular differentiated tissues and can serve as markers for differentiated cells. Likewise, different intermediate filament classes have been identified with the desmosomes of different tissues. Cytnkeratins are found with epithelial desmosomes [356], desmin with cardiac myocytes [357,358], and vimentin with ereningiomes [359]. Glycoprutein components of the intercellular core of the desmosomes of bovine muzzle have been obtained

by extraction with non-ionic detergent in citrate buffer (pH 2.6) containing proteinase inhibitors and density gradient centriingation in metrizamide [360]. Highly glycosylated components of 150,115 and 100 kDa were identified. The major components of the desmosomal plaques are 250 and 215 kDa protons, named desmoplaklns [361]. These ere particularly insoluble proteins which can be hic~,dized immunocytochemically to the desmosomes. Another desmosome-associated component has been identified by autoantthodies in the serum of patiems suffering from the skin disease pcmphigus vulgans [362]. This 140 kDa glycoprotein is localized not only in desmosomes, but also aiong the whole epidermal cell surface. Anti-140 kDa protein is able to disrupt cell-cell contacts in cultured kcralinocytes. Plakoglobin, an 83 kDa component of desmosomes, has also been observed by means of a monoclonal a n t i b ~ v and a eDNA clone to be associated with other types of call-call junction, including vinculin-act in-ass 0ciated junctions [363]. As in the case of other junctional complexes which a ~ being cheractarized, the molecular organization of the desmosoma[ components involved is still uncertain. However, the fact that major components can he isolated suggests that reconstitution studies with the proteins or their proteolytic fragments may be feasible. Interactions between intermediate filament bundles and the plasma membrane have been observed also in cells which do not contain desmosomal plaques [364]. It has been recently proposed that these bundles may serve as cell surface-nucleus signal transducers [365], although there is currently no published evidenec to support this intriguing hypothesis. Vimentin has been shown to bind to the in.let surface of erythrccytes thx~,ugh a ifigh-,df'mii~/ interaction with ankyrin [366[. Vimentin-type intermediate filaments can also interact directly with the lipid bilayer, as shown by electron microscopic observations on binding of the filaments in vitro to vesicles prepered from Ehrllch ascites tumor cell lipids [367]. As mentioned previonsly, intermediate filaments have been proposed to serve as mechanical integrators of cytoplasmic space [16[ and to link the nucleus to the plasma membrane [368,369]. Thus, it is important to know the sites of interaction at the nuclear membrane and at the plasma membrane. Recent studies have shown that the nuclear lamina, a meshwork lining the inner surface of the nuclear inner membrane, is composed of intermediate-type filaments of lamin polypeptides A, B and C [370]. Lamins A and C, but not B, have merked homologies to intermediate filament polypeptides [371,372]. However, in vitro studies indicate that vimentin binds by its C.terminus to lamin B [373,374]. In the nucleated chicken erythrocyte, the intermediate filaments are attached to the membrane skeleton only in a limited area, an ellipsoidal patch [3691. Two candi-

166 dates for the attachment site have been suggested: ankyrin [366], as mentioned above, and one of the forms of band 4.1 [841. How the specificity of localization of the attachment to the membrane skeleton is established and maintained is presently unclear. V. Future studies Analysis of membrane-cytoskclcton interactions at the molecular leval requires a multifaceted approach. Oltrastruetural approaches have not been very successful to date because of limited resolution. A likely exception is the application of freeze-etch techniques to the examination of membrane-cytoskeletal interfaces (Fig. 9) [375]. In addition to yielding high resolution images of the structures involved, the technique can be combined with irrLmunOCytochemical labeling methods to identify proteins involved in those structures. Reconstitutian studies provide an ultimate test for understanding complex structures. However, in attempting to understand dynamic systems, one potential caveat should be kept in mind: weak or transient interactions may play important roles in the structures of interest. Whereas strong interactions can be detected by isolation of stable complexes or by binding studies, weak

interactions are much more difficult to detect. The use of equilibrium gel filtration can circumvent problems of studying weak interactions, if sufficient material is available. Quantification of the i~;teractions can he achieved by the Hummel and Dreyer [376] modification of this method. Successes in understanding membrune-cytosheleton interactions have resulted largely from ultrastrucmral, biochemical, and immuno!ogical approaches. Only recently have modern molecular biological techniques been applied to these rroblems. However. the knowledge acquired from the eDNA scqueness of such molecules as spectrin [3FI, integrin [2581 and calpactin [377] demonstrates the power of these methods. Application of eDNA sequencing techniques along with site-directed mutagenesis, expression methods and the use of antisense inhibitors [378] promises exciting revelations in understanding call and tissue structure in the near future. Acknowledgements We thank Drs, B. Shan, M. Pratt and N. Hirnkawa for providing the photographs used in the figures, and Drs. W. I~ntler, IL Stephans, S. Lin, J. Fox, J. Collins and J. Glanney for providing reprints, preprints or abstracts of their work. We are also grateful to Dr. Pratt for critically reading the manuscript. Unpublished work cited in this manuscript was supported by grants from NSF (PCM 8300771) and N I H (GM 33795) and by the Papanicolaou Comprehensive Cancer Center of the University of Miami (CA 14395). References

Fig. 9. I~tron micrograph of mlcrofilament-m~br~e inter~ti~ re,ion in a quick-fr~n, deeply etchedrotary-shadowed replicaof an unL,.~l macola of chick ear hair cells. (From Hirokawa, N. and Tilney,L.G, J. Cell Biol,95, 249,1982.with permission.)

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