The Mechanics of Vascular Cell MotilityC.B. SHUSTER* AND I.M. HERMAN†
*Department of Biological Sciences, University of Pittsburgh,Pittsburgh, PA, USA
†Department of Physiology, Tufts University School of Medicine,Boston, MA, USA
ABSTRACT
Alterations in vascular cell shape and motility occur during developmental pro-cesses and in response to injury. Similarly, during tumor vascularization andatherogenesis, endothelial and smooth muscle cells undergo motile and prolif-erative responses to extracellular cues. Recent inroads into our understanding ofsignal transduction have identified several candidate pathways by which theextracellular matrix- and growth factor-mediated stimulation of vascular cellmotility may be mediated. The multiple and divergent extracellular stimuli thatstimulate vascular motile responses may converge on the cytoskeleton via afamily of ras-related GTPases. Biochemical analyses as well as examination ofcytoskeletal dynamics in vivo indicate that actin polymerization at the forwardaspects of spreading cytoplasm is capable of driving forward protrusion forma-tion in the absence of a conventional actin motor. Actin polymerization at theplasma membrane of leading lamellae may be mediated both by de novo nucle-ation of actin filaments and the generation of free filament ends by uncappingthe barbed ends of existing actin filaments. This review summarizes the mostrecent findings in extracellular–cytoskeletal-signal transduction, therein, provid-ing a framework to explain the remarkable remodeling seen in the vasculatureduring developmental and disease-related processes.
The cells of the vasculature respond to a wide varietyof extracellular cues that potentiate proliferation,changes in vascular permeability, vasoconstrictionand relaxation, as well as stimulation of cell motility(1). Facilitating each of these processes is a dynamicreorganization of the vascular cytoskeleton. The ini-tiation of motile responses has received a great dealof scrutiny, not only as a basic question in cell biol-ogy, but because of the central role motility plays invascular development and disease. Yet, despite therapid advancement in our understanding of signaltransduction, per se, there remain large gaps in our
detailed appreciation of the mechanisms regulatingthe transformation of extracellular signals into thestimulation of a motile response. Additionally, al-though the components and dynamics of the actincytoskeleton have been studied in minutia, much re-mains to be elucidated regarding the controlled ex-pression of contractile protein isoforms and their as-sociating proteins, as well as how these isoproteinarrays contribute to vascular cell-specific functions.
This minireview focuses on mediators of signal-transduction pathways that converge on the cyto-skeleton to regulate actin assembly and cell motility.These points of biochemical convergence may pro-vide novel insights and possible targets for the de-velopment of therapeutics that inhibit the growth ofnew blood vessels during tumor vascularization andproliferative diabetic retinopathy. In the followingdiscussion, the mechanics of cell motility are consid-ered in the context of the molecular and cellular pro-cesses driving vascular cell motility in large and
Supported by the National Institutes of Health Grants Nos. GM55110 and EY 09033 to IMH.For reprints of this article, contact Dr. Ira Herman, Departmentof Physiology; Tufts University School of Medicine, 136 HarrisonAvenue, Boston, MA 02111 USA; e-mail: [email protected] 10 October 1997; accepted 25 June 1998
small blood vessels. There is also a brief discussionfocusing on novel inroads recently made in under-standing the molecular mechanisms regulating theselective expression and cytoplasmic targeting ofcontractile protein isoforms in microvascular peri-cytes.
In vitro analysis of living cell behavior revealed theforward movement of cells as a transient, but delib-erate elaboration of cytoplasm. Thin veils of mem-brane (ruffles) were extruded prior to their retrac-tion from the forward aspects of the cell, while si-multaneously, cellular contacts with the substratumat the trailing edge were engaged and disengaged asthe advancing cytoplasm seemingly pulled the pos-terior forward. (2–4,9). This actively ruffling do-main of cytoplasm appeared by video microscopy tobe the primary ‘‘motile apparatus’’ of the cell. Thus,two-dimensional motility involved three basic pro-cesses: the forward extension of cytoplasm, the es-tablishment of contacts with the substratum, and theretraction or disengagement of the trailing (edge)portion of the cells. This was also true in the vascu-lature where EC and SMC motility had been assessedin response to soluble and matrix-bound growth fac-tors (11,61,66,131), as well as vasoactive cytokines,which positively and negatively influenced endothe-lial cell motility (17). Thus, from these early char-acterizations of endothelial cell motility, links hadalready been established between soluble cytokines,the extracellular matrix (105,145) and the regula-tion of cell migration.The development of antibodies against contractileproteins allowed investigators to place contractileproteins previously characterized in muscle withinthe spatial context of a nonmuscle cell, and attemptswere made to extrapolate the intracellular arrange-ment of actin and myosin within current models ofmuscle contraction and force production. The local-ization of actin, a actinin, tropomyosin, and myosin(49,93–95) to bundled actin filaments or stress fi-bers prompted speculation that stress fibers may in-deed be contractile, possibly generating tension withthe substratum or actually providing protrusiveforce (75,88). However, studies of non-muscle cellmotility in vitro argued against a myosin-basedmodel for force generation during non-muscle cellmotility. Observation of fibroblast and endothelialcell motility in vitro by video microscopy followed bysubsequent actin and myosin localization indicatedthat while actin is enriched in motile cytoplasmiccompartments (i.e., ruffles), myosin is virtually ab-sent (65,66). From these studies, it was concluded
that there is actually an inverse correlation betweenthe elaboration of myosin-containing stress fibersand motility. As shown in Fig. 1, the retention ofmyosin II in non-motile cellular compartments canbe clearly seen in retinal pericytes, where myosin isenriched in stress fibers (panel B), but absent in do-mains of spreading cytoplasm rich in F-actin (panelA). These in vitro observations were supported laterby in situ analyses of endothelial cytoskeletal dy-namics in cells lining large blood vessels, wherestress fibers are elaborated in response to fluid-shearstress (63,180). Thus, the spatial organization ofmyosin to ‘‘non-motile’’ domains provided circum-stantial evidence against myosin-based models ofprotrusive force production.
Figure 1. Myosin is excluded from actively spreading do-mains of cytoplasm. Bovine retinal pericytes were platedonto glass coverslips, then fixed in a buffer containing 4%formaldehyde, briefly permeablized with 0.1% TritonX-100, and processed for actin localization with NBD-phallicidin (A) and myosin localization with anti-nonmuscle myosin IgG (B). Note that myosin II is local-ized primarily to stress fibers and orthogonally arrangedactin bundles in the perinuclear region of the cell. In con-trast, the spreading fan lamellae (arrows) contain diffuseF-actin staining, but no myosin. Bar, 10 mm.
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The most direct evidence against myosin II-basedmotility came from studies in which myosin expres-sion or function was inhibited in living cells usingeither cell biological or genetic approaches. Injectionof antibodies against myosin II inhibited progressionof the cleavage furrow, but did not inhibit fibroblastmotility (70,83,99). Additionally, disruption ofmyosin II expression in Dictyostelium results in cellsthat display normal cell spreading and lamellar ex-tension, but fail to undergo cytokinesis or efficientlyrespond to chemotactic gradients (40,85). These re-sults indicate that while myosin II plays an essentialrole in the contractile-ring formation and cytokine-sis, its role in motility may have more to do withsteering than with protrusion and extension.
In the absence of a force-generating motor, can theregulated polymerization of actin alone drive leadingedge extension? One model system that invokes sucha mechanism is the formation of the acrosomal pro-cess in Thyone sperm (74,168,169). Upon fertiliza-tion, a long bundle of actin filaments is formed denovo from a pool of actin monomers activated uponsperm–egg binding, resulting in an explosive poly-merization of actin filaments and elongation of theacrosomal process. Similar observations have beenmade by polymerizing monomeric actin containedwithin liposomes causing the extension of long, thinprocesses resembling microspikes (35,76). Based onthese observations, a ‘‘Brownian ratchet’’ mecha-nism has been invoked to explain how actin–filament polymerization can drive the extension ofthe plasma membrane in the absence of a myosin-like motor (128). In this scheme, actin–filament as-sembly itself can act as a ratchet by directly contact-ing the membrane, and as monomers diffuse to thefilament–membrane interface, they add to the fila-ment end when Brownian motion creates a gap. Ef-fectively, rearward contraction would yield a localmembrane deformation where actin filaments insertin an end-on manner. During the moment that themembrane deformation occurs, new actin monomerscould add, thus extending the membrane forward.While not accounting for the actions of actin-bindingproteins, the Brownian ratchet model proposes thatactin-filament formation provides the energy andforce required for the forward protrusion of theplasma membrane.Isoactin Filament Dynamics
The kinetics of actin filament nucleation, elongation,and depolymerization have been carefully studied invitro, and the rate constants of these respectiveevents calculated (131). Actin polymerization pro-ceeds under four basic steps: 1) a monomer activa-tion by divalent cation binding; 2) a rate-limiting
nucleation step where three monomers join to form anucleus; 3) filament elongation that occurs by mono-mer addition to the nucleus; and 4) filament anneal-ing in which two filament ends join together. Actinmonomers assemble into a polarized filament, whichmay be visualized by binding of myosin head frag-ments, resulting in an arrowhead-like decorationalong the length of the filament as seen by electronmicroscopy. ATP-bound monomers have a log–orderhigher affinity for the barbed (+) end than for thepointed (−) end, resulting in a biased elongation ofthe filament from the barbed end (181). The char-acterization of actin-filament dynamics in solutionhighlight the requirement for exquisite controlmechanisms to spatially regulate actin polymeriza-tion within living cells. Under steady-state condi-tions, the amount of free monomer in a solution ofactin filaments will approach the critical concentra-tion required for addition to the barbed end. How-ever, the extraordinarily high concentration of actinin living cells can be as high as three log–ordersgreater than the critical concentration required forbarbed–end assembly (19). This strongly suggeststhat mechanisms exist not only to prevent spontane-ous polymerization, but also to regulate monomeraddition to the barbed end.
The chemistry of pure actin solutions may not, how-ever, accurately predict the kinetics of filament po-lymerization in vivo, where a host of actin-associatedproteins coordinately act to regulate the form andorganization of actin filaments. Additionally, itshould be noted that the kinetics of actin polymer-ization were determined almost exclusively usingskeletal muscle actin. Indeed, nonmuscle isoactinshave distinct structural features, and a large numberof expression and biochemical analyses have deter-mined that these actin isoforms are functionally dis-tinct (60). And in light of the fact that one isoform inparticular (nonmuscle b–actin) is enriched in lead-ing lamellae (71), whereas the non-muscle g–isoac-tin is globally distributed (126), the numbers de-rived for skeletal muscle actin may not necessarilyreflect the kinetics of actin polymerization at theleading lamellae of endothelial cells, pericytes, andsmooth muscle, each of which express b–actin dur-ing motile responses.Observations In Vivo
The development of fluorescent derivatives of actinafforded the first opportunity to directly study thedynamics of the actin cytoskeleton within livingcells. Injected actin rapidly incorporates into fila-ments at the leading edge of the cortical cytoskele-ton, as well as into stress fibers (8,89,171–173).Fluorescence recovery after photobleaching (FRAP)
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reveals that actin appears to remain stationary rela-tive to the advancing front, resulting in a net cen-tripetal movement towards the nucleus at rates rang-ing from 0.8–1.5 mm/min (171–173). Similarresults were obtained by observation of actin poly-merization in nerve-growth cones following cytocha-lasin treatment (47). Upon removal of cytochalasinfrom the culture media, filament formation is firstobserved at the leading edge, and the formed fila-ments move centripetally over time. From thesestudies a treadmilling mechanism has been invoked,whereby actin monomers are added at the leadingedge where barbed ends contact the membrane anddissociate from the pointed end of the filament, pro-viding monomer for further addition at the leadingedge (114,172). This model implies that filamentsspan the entire length of the cortex (153). However,in macrophages as well as other nonmuscle cells, thisis not the case (58).
The development of caged fluorescent derivatives ofactin and tubulin has been instrumental in the studyof filament dynamics by virtue of the ability to lo-cally activate fluorescence and measure the rates ofmovement and decay of the fluorescent foci (114).By injecting caged resorufin actin into motile epithe-lial keratocytes and fibroblasts, and then activatingfluorescence in labeled filaments at the leading edge,the rates of centripetal flow and filament disassem-bly could be assessed by measuring the signal decayover time (165,166). The calculated half-lives forfilaments in the cortex were not consistent with thetreadmilling model described above, where an actinmonomer treadmilling through a filament wouldtransverse the entire lamellapod. From these obser-vations, Theriot and Mitchison proposed a ‘‘nucle-ation-release’’ model for actin dynamics, where thenucleation and depolymerization of short filamentsoccur throughout the process, but that nucleatingactivity is enriched threefold at the leading edge(159,166). Additionally, while centripetal move-ment and filament decay occur at constant rates indifferent cell types, the rates of forward extensionand motility vary (166). As is the case for the in vitrokinetic studies, these studies were performed by us-ing a heterologous muscle actin in non-muscle celltypes. Studies in our lab using fluorescent deriva-tives of b–actin injected into microvascular pericytesreveals that like muscle actin, b–actin first incorpo-rates in domains of spreading cytoplasm. But asshown in Fig. 2, flourescent b–actin appears to berestricted to these domains with only a fraction in-corporating into more metastable domains such asstress fibers. Similar results have been obtained in
endothelial cells recovering from monolayer injury(Strauss and Herman, manuscript in preparation).Thus, while further analyses of isoactin dynamicswill reveal the exact role that b–actin plays in facil-itating protrusion formation, these combined in vivoanalyses suggest that the factors regulating actin po-lymerization at the leading edge are the primary de-terminants of the rate at which forward protrusionsform during cell motility.
The study of actin filament dynamics in vitro and invivo predict that factors regulating actin-filamentpolymerization in motile cytoplasm are concentratedat the leading edge, and act by regulating filamentpolymerization from the barbed end. This notion isfurther supported by morphological studies indicat-ing that the predominant actin filament–plasmamembrane interaction occurs at the barbed end.Morphological examination of microvilli from intes-tinal columnar epithelial cells and fertilized sea ur-chin eggs reveal that the bundled filaments contactthe membrane via their barbed ends (16,24,117); atthe leading edge of crawling fibroblasts and kerato-cytes, F-actin also contacts the plasma membranevia their barbed ends (153,154). However, exami-nation of the leading edge of growth cones fromAplysia neurons indicate that end-on interactionsoccur via both ends (97). The notion that barbed-
Figure 2. Rhodamine-labeled b–actin distributes to mo-tile cytoplasmic domains. Erythrocyte b–actin was cova-lently labeled with rhodamine isothiocyanate and injectedinto bovine retinal pericytes. Following recovery, the lo-calization of the labeled protein was observed in the livingcells by confocal microscopy. Arrows denote positions ofmotile cytoplasm where rhodamine b–actin is enriched.
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end-membrane interactions represent an importantregulatory mechanism for controlling filament for-mation is further supported not only from the kineticanalyses discussed above, but also from studies of live,permeabilized cells, where incorporation of exogenousactin at the leading edge is blocked if permeabilizedcells are incubated with capping protein (159).
Barbed-End Regulation of Filament Assembly
The functional consequences of barbed-end regula-tion have long been appreciated, and to date, thereare as many as six proteins that may interact withthe barbed ends of actin filaments in vascular cells:gelsolin, capping protein, cap G, radixin, tensin, andbcap73 (140,150). The in vitro filament capping ac-tivities of gelsolin, capping protein, and cap G havebeen extensively studied (140), and all three bindacidic phospholipids such as PIP and PIP2, whichresults in a loss of filament capping activity. Indeed,in cells such as platelets and polymorphonuclear leu-kocytes (PMN), as much as 90% of all capping ac-tivity may be accounted for by gelsolin and cappingprotein (13,43,57,142). In these cells, it is proposedthat the calcium-activated severing activity of gelso-lin creates new filament ends which are, in turn,released by increased levels of PIP2 (57). It has beencalculated that the levels of capping protein in cellsare sufficient to account for the majority of actin-filament ends (43).
Whether capping protein is the most functionallysignificant capping activity in cells other than plate-lets and neutrophils remains to be seen. Both celltypes respond to extracellular stimuli with rapid in-creases of actin polymerization, often within 30 sec-onds of ligand binding. The other capping proteinactivities present in vascular cells may play equallyimportant, if not more specialized roles in regulatingand organizing the actin-based cytoskeleton. Thedifferential localization of tensin, radixin, andbcap73 to focal adhesions, junctional adherens andleading lamellae, respectively, argue that these pro-teins may perform more specialized roles in regulat-ing actin dynamics in motile cells. Originally puri-fied from detergent-extracts of retinal pericytes,bcap73 binds to the barbed ends of b actin fila-ments, and co-localizes with b–actin filaments in theleading lamellae (150). Because the form and orga-nization of b–actin as seen by immunoelectron mi-croscopy reveals that b–actin is found in the form ofshort oligomers just subjacent to the plasma mem-brane (Shujath and Herman, unpublished observa-tions), bcap73 may serve to cap these b–actin oligo-mers that anchor to the plasma membrane through
the ERM protein, ezrin (151). Indeed, ezrin associa-tion with b–actin filaments is sensitive to calpain-mediated proteolysis (151), and this cleavage mayserve to disengage the capped b–actin oligomer fromthe membrane and lead to filament uncapping andfilament elongation. Revealing the mechanism bywhich ezrin and bcap73 regulate b–actin filamentdynamics should prove invaluable to our under-standing isoactin dynamics during cell motility andprotrusion formation.
De Novo Filament Nucleation
As mentioned above, studies of actin dynamics invivo predict that in addition to regulation of freebarbed ends, actin filaments are nucleated de novoin leading lamellae, which are then presumablycapped and crosslinked as filaments undergo cen-tripetal flow and eventual turnover. Because the for-mation of an actin nucleus (trimer of actin mono-mers) is thermodynamically unfavorable, it has longbeen presumed that actin-associated proteins facili-tate nucleation. There are several proteins, includingfilament capping proteins, talin, and ponticulin(from Dictyostelium) that have been demonstratedto nucleate actin polymerization in vitro, yet untilvery recently, the identity of in vivo nucleating fac-tors has remained a mystery. Of interest has been thebehavior of the intracellular pathogen, Listeriamonocytogenes, which propels itself through hostcell cytoplasm by nucleating a long ‘‘comet’’ of actinfilaments. This has recently become a model bywhich in vivo actin nucleation can be studied(101,166). Results from these studies have revealedtwo nucleating factors that may play important rolesin controlling actin-filament formation in motilecells: vasodilator stimulated phosphoprotein (VASP)and the ARP2/3 complex. Vasodilator stimulatedphosphoprotein was orignally characterized as acGMP-and cCMP-dependent kinase substrate highlyexpressed in platelets as well as in vascular cells(54). Recently, it was revealed that the Act A gene ofListeria monocytogenes contains a polyproline-richtandem repeat that binds VASP (155); and a similarVASP-binding, polyproline-rich domain has beendiscovered in the focal adhesion protein vinculin(22). These protein–protein interactions may medi-ate the association of VASP with the bacterial sur-face, to focal adhesions in cultured cells as well asdense bodies and dense plaques in smooth muscle invivo (107). The actual role that VASP plays in fa-cilitating actin polymerization, the binding or re-cruitment of profilin:actin complexes (133), and thepromotion of actin-filament nucleation is under in-
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tensive investigation. In turn, VASP may represent akey component in the signaling cascade mediatingdiverse actin-based cytoskeletal reorganizations.
A second important actin-nucleating factor identi-fied by studies of Listeria motility is the actin-relatedprotein (ARP) complex (175). First isolated as acomplex that co-purifies with profilin from Acanth-amoeba extracts (102), this ubiquitous complexcontains Arp2 and Arp3, and polypeptides of14,18,19,35, and 40 kd. Actin-related protein rep-resents a new class of cytoskeletal proteins that shareseveral structural similarities with conventional ac-tin isoforms (40,60), yet do not appear to copoly-merize into conventional 7 nm filaments. Actin-related protein 1, which shares the most homologywith conventional actin, forms homotypic filaments35 nm in length, and forms part of the dynactincomplex (141). The seven components of the Arp2/3complex have a stoichiometry of 1:1:1:1:1:1:1, andform a horseshoe-like structure as revealed by rotaryshadowing (119). This complex is capable of deco-rating actin filaments, and crosslinking studies sug-gest that p35 and Arp3 constitute the actin-bindingsite (119). Supporting evidence for filament-nucleating activity of the Arp2/3 complex has beenreported in yeast, where the homolog of the 40 kdpolypeptide can suppress profilin mutations (12).More direct evidence has come from studies of Lis-teria motility in human-platelet extracts, where theArp2/3 complex is identified as the major compo-nent of the actin nucleating machinery interactingwith the Listeria gene Act A (176). Studies of Arp2and Arp3 in Acanthamoeba as well as cultured cellsreveal that these proteins are abundant polypeptidesthat are localized to the actin cortex (81,176). It hasbeen proposed that this complex interacts withprofilin and may participate in either delivering pro-filin:actin complexes to free filament ends, or mayaffect profilin–actin interactions to directly facilitateactin-filament nucleation. To date, there is no infor-mation regarding how the Arp2/3 complex is in-duced to bind profilin and nucleate actin filamentformation when cells crawl.
Role of Monomer-Sequestering Proteins inRegulating Filament Assembly
Studies examining how cells regulate their pool ofmonomeric actin also point to the crucial role thatregulation of the barbed end plays in controlling iso-actin filament dynamics. As mentioned above, esti-mations of the relative amounts of filamentous ver-sus monomeric actin indicate that monomeric poolsmay be 2.3–5 × 103 above the critical concentration
for barbed-end addition (19,183). Monomer-binding proteins, such as profilin and thymosin b4,are believed to regulate polymerization by prevent-ing de novo filament formation (156). Both thymo-sin and profilin bind actin monomers in a 1:1 molarratio, and can inhibit the polymerization of actinfilaments in vitro. Profilin also binds phosphatidyli-nositides PIP and PIP2 with high affinity, and thisbinding inhibits profilin-actin binding (92). In ad-dition to sequestering actin monomers in a phospha-tidylinositide-sensitive manner, profilin performsadditional roles in regulating actin dynamics. Pro-filin-binding accelerates actin-nucleotide exchangeby four log–orders, possibly serving to facilitate po-lymerization by regenerating ATP-bound monomersfrom the less polymerization-competent ADP-boundform (52). In addition, it has been shown recentlythat the critical concentration for the addition ofprofilin–actin complexes to the barbed end in vitro isgreatly decreased compared to free-monomer addi-tion, suggesting that profilin might directly addmonomers to the filament end (127,130).
While profilin–actin binding is negatively regulatedby phosphatidylinositides, no such regulatorymechanism exists for thymosin b4. A ubiquitous5-kd polypeptide found in great abundance (580mM in platelets), thymosin b4 binds actin monomersand prevents filament formation (31,120,139). Be-cause the affinity of actin monomers for thymosin b4is roughly equal to or less than the affinity of mono-mers for the barbed end of the filament, the ability ofthymosin b4 to sequester actin monomers is in-versely proportional to the number of free barbedends (120). Recently, it has been proposed that thy-mosin b4 and profilin can together facilitate theelongation of uncapped filament ends (127). Underthis mechanism, profilin–actin complexes would adddirectly to the barbed end of the filament, and uponATP hydrolysis by actin, profilin would then disso-ciate. Through mass action, thymosin b4-actin com-plexes would dissociate to supply monomer for pro-filin-mediated addition to filament ends. Taken to-gether, these two proteins not only act to sequestervirtually all the free monomer in the cell, but arecapable of rapidly responding to barbed-end uncap-ping to supply monomers for filament elongation.
Filament Turnover: Regeneration of theActin-Monomer Pool
The turnover of actin filaments in vivo occurs atrates approximately 100-fold greater than what ispredicted by studies in vitro (186); and this differ-ence cannot be explained solely by the ability of
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monomer sequestering proteins to remove monomersfrom the pointed or ‘‘−’’ end of the filament. Howthen, are actin filaments recycled and monomers re-generated for further incorporation at the leadingedge? Results of recent studies suggest that the ac-tin-depolymerizing factor (ADF)/cofilin family ofproteins may provide the answer to this interestingquestion. Actin-depolymerizing factor/cofilins are aubiquitous family of low-molecular-weight polypep-tides (15–22 kd)(116,164) that accelerate actin de-polymerization up to 22-fold (28). Because ADF/cofilin binds ADP-bound actin with greater affinitythan ATP-bound actin (103), it is a likely candidateto remove actin monomers from the pointed end ofthe actin filament. Additionally, these proteins arenegatively regulated by phosphorylation (39,118,125); and although the kinases which downregulateADF/cofilin activity remain unidentified, several sig-nal transduction pathways have been identified (PKC,adenylate cyclase, free calcium)(116). Thus, ADF/cofilin represents an additional level of regulation bywhich actin filament formation and turnover mightbe regulated in a sensitive and responsive manner.
Signaling Pathways and The Cytoskeleton: Pointsof Convergence
As illustrated in Fig. 3, the forward protrusion ofcytoplasm at its most elemental level presumably in-
volves the regulated uncapping of filament ends(capping protein, cap G, bcap73) in combinationwith de novo nucleation of actin oligomers (Arp2/3complex, VASP) to increase net-actin polymeriza-tion at the leading edge of spreading cytoplasm. Mo-nomeric actin is delivered to these sites of polymer-ization by a ‘‘monomer delivery’’ system (thymosin,profilin) and is regenerated through the regulatedturnover of actin filaments (ADF/cofilin) as fila-ments flow centripetally toward the cell body. Notincluded in this model is the consideration of thenumerous filament crosslinking proteins and mem-brane-associated proteins that provide structuralintegrity to the cortical actin cytoskeleton, whichenables the net polymerization of actin to drive for-ward protrusion. Additionally, there is no consider-ation of how extracellular cues activate these pro-cesses. Smooth muscle migration during athero-sclerotic lesion formation, for instance, may bestimulated not only by the substratum (73), but bysoluble cytokines such as platelet-derived growthfactor (PDGF)(1). But while much is known aboutthe multiple and diverse ligand/receptor systemsthat stimulate vascular cell proliferation and motil-ity, how these different signal transduction mecha-nisms elicit a common motile response is still un-clear. In many cases, the direct link between thesecond messenger (protein kinase or G protein) and
Figure 3. Model of actin dynamics in leading lamellae. At the leading edge of the forward protrusion, actin monomersare either added to free filament ends via profilin: actin complexes, or actin filaments are nucleated de novo by bcap73or the ARP2/3 complex. The nascent filaments are then crosslinked or incorporated into myosin II containing actinbundles. Monomers are regenerated by the action of ADF/cofilin.
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their respective cytoskeletal effector proteins remaincompletely unknown. But there is growing evidenceto suggest that modulation of the actomyosin-basedcytoskeleton stimulated by cell-matrix interactionsand growth factor stimulation may be facilitated bya single family of mediators: the rho-familyGTPases.
Cell-Matrix Interactions
There is an expansive body of literature to indicatethat the extracellular matrix plays an integral role inregulating vascular cell biology and dysfunction. En-dothelial cell spreading and motility (61,184,185),as well as pericyte and smooth muscle cell prolifera-tion/ contractile phenotype (38,62,64,121) are allaffected by either purified or cell-derived extracel-lular matrices in vitro. Whereas it has been recog-nized for some time that the extracellular milieuplays an integral role in angiogenesis (145), endo-thelial cell motility in vitro is actually retarded whencells are plated on extracellular matrices (61). None-theless, inhibition of FGF-stimulated angiogenesisusing antibodies against the avb3 integrin arguesstrongly that cell-matrix interactions are crucial forvascular motility in vivo (23).
Cell-matrix interactions are mediated by a family ofheterodimeric receptor molecules termed the inte-grins (72,146). In cultured cells, ligand binding andreceptor clustering induce the assembly of focal ad-hesions (FA) and stress fibers(SF). While a hallmarkof vascular cell-matrix interactions in vitro, the ac-tual functional significance of these structures in vivorequires further clarification. Stress fibers have beendocumented in endothelial cells in vivo (180), wherethe primary determining factor of stress fiber forma-tion is fluid-shear stress (26,63,177). As was men-tioned earlier, SF expression actually correlates in-versely with endothelial motility (66). Thus, whileSF assembly per se may not reflect matrix-mediatedcytoskeletal remodeling in vivo, the assumption hasbeen made that the regulatory factors mediating SFassembly in vitro also promote actin-based motilityin vivo.
Identifying the exact sequence of events that result inactin assembly at sites of cell-matrix contact hasbeen complicated by the fact that there are now over20 structural and signaling molecules that are re-cruited to focal adhesions upon matrix binding andintegrin clustering (115,182). However, use of fibro-nectin- or RGD peptide-coated beads on endothelialcells (129) or fibroblasts (115) has enabled investi-gators to study the early events surrounding ligand
binding and focal adhesion assembly. Results ofthese studies suggest that before ligand binding, thecapping protein tensin and focal adhesion kinase(FAK) are found in association with the cytoplasmictails of integrins. Upon ligand binding, vinculin, ta-lin, and actinin are recruited to the nascent plaque,followed by activation of tyrosine kinase activity. Arole for tyrosine kinases in adhesion and motility hasbeen supported by studies using tyrosine kinase in-hibitors, where focal contact formation (25) andsmooth muscle cell chemotaxis (149) are inhibited invitro. Just where tyrosine kinase activity fits in thecascade of events leading toward SF-assembly for-mation, however, remains unclear. Focal adhesionkinase has been implicated as the major scaffold towhich both signaling and cytoskeletal proteins arerecruited (115). Indeed, in addition to paxillin-, ta-lin-, and integrin-binding, FAK serves as a bindingsite for adapter proteins of the ras-activation path-way, Src family-tyrosine kinases, and phosphoi-nositide 3-kinase (PI 3-kinase) (143). Autophos-phorylation of FAK at tryosine 397 serves as a bind-ing site for the non-receptor tyrosine kinase Src(143), which in turn phosphorylates FAK, creatingbinding sites for the adaptor proteins Grb2 andp130cas. While Grb2 goes on to stimulate the ras-mediated activation of the MAP kinase pathway(144), the downstream effectors of p130cas are lesswell-known. Expression of FAK deletion constructssuggest that FAK-promoted motility requiresp130cas binding and tyrosine phosphorylation (30).Thus, FAK activation by integrin binding appears tobe capable of mediating two bifurcating signals: onethat regulates cell proliferation and another that me-diates cytoskeletal reorganization.
There are, however, several lines of experimentationthat call into question the role that FAK plays instress fiber formation or even its role as the masterregulator of focal adhesion assembly. Stimulation ofstress fiber formation in smooth muscle cells platedon fibronectin occurs in spite of low FAK activity(178), suggesting that FAK activity may not be nec-essary for stress fiber assembly. Additionally, experi-ments in 3T3 cells indicate that both stress fiberformation and FAK activation may be under thecontrol of a small GTP-binding protein p21rho. In-jection of constitutively active or dominant negativeforms of rho into cells treated with lysophosphatidicacid (to induce SF formation) reveal that while ty-rosine kinase activity is required for FA and SF for-mation, FAK activation lies downstream of rho(136). While the relationship between rho and FAKactivation in the course of FA assembly remain
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a point of controversy (115,136), the multitude ofresponses elicited by integrin-matrix binding(146,182), suggests that FAK serves more of a scaf-folding role analogous to the cytoplasmic domains ofreceptor tyrosine kinases than as direct mediator ofSF and focal adhesion assembly.
Growth Factor-Induced Actin Polymerization
Just as the extracellular matrix affects the actin cyto-skeleton and proliferation of endothelial cells, peri-cytes, and SMC, vascular cells undergo both mito-genic responses and cytoskeletal remodeling in re-sponse to growth factor stimulation (37). Inparticular, vascular cell responses to PDGF and ba-sic fibroblast growth factor (bFGF) have been a fo-cus of intense scrutiny because of the roles thesemolecules play in lesion formation and repair in thevessel wall (1). Activation of these growth factor re-ceptors results in the recruitment of adapter proteinsof the ras-signaling pathway, non-receptor tyrosinekinases, and phospholipase C to cytoplasmic do-mains of the activated receptor. In addition, there isoften a concomitant stimulation of actin polymeriza-tion and membrane ruffling (34,109,110). Studiesof EGF-stimulated membrane ruffling in A431 cellssuggested that actin polymerization may be facili-tated by phosphorylation of cytoskeletal proteins byreceptor tyrosine kinases (20). More recent studiessuggest that another signaling molecule recruited byreceptor tyrosine kinases may be particularly rel-evant to endothelial cell and smooth muscle cell mo-tility. Phosphoinositide 3-kinase (PI 3-kinase) wasidentified as a lipid kinase that phosphorylates phos-phatidylinositide 4,5 bisphosphate (PIP2) to phos-phatidylinositide 3,4,5 trisphosphate (PIP3). Phos-phoinositide 3-kinase is a heterodimer consisting ofa 85-kd-regulatory subunit and a 110-kd-catalyticsubunit (68), and is thought to be activated throughthe coupling of the 85-kd subunit to autophosphory-lated SH2 domains of receptor tryosine kinases (29).Additionally, studies using either deletion constructsof the PDGF receptor and the p85-regulatory sub-unit or chemical inhibitors of PI 3-kinase (such aswortmannin) indicate that in addition to mediatingmitogenic signaling (27), PI 3-kinase plays a regu-latory role in growth factor-induced membrane ruf-fling (59,87,174).
How does PI 3-kinase facilitate actin reorganiza-tion? This remains a controversial and active area ofstudy. There are reports to suggest that PI 3-kinasemediates ruffling through the activation of proteinkinase C (42). Phosphoinositide 3-kinase activity
may be a downstream effector of actin-binding pro-teins. Profilin has been shown to bind the p85 sub-unit of PI3-kinase, and profilin and gelsolin stimu-late PI 3-kinase lipid kinase activity (152). Whetherthis is an effect of direct interactions between PI3-kinase and these proteins or through the interac-tion of gelsolin and profilin with phosphatidylinositi-des remains to be seen. Studies of PDGF-inducedruffling in endothelial cells indicate that PI 3-kinaseis required for the activation of a ras-related smallGTPase, p21 rac, that, in turn, mediates actin poly-merization and membrane ruffling (59). This studysuggested that the lipid products of PI 3-kinase(phosphatidylinositol 3,4,5 triphosphate) may affectthe binding of the rho-binding protein rhoGDI,which binds rho-family members and preventsnucleotide exchange (and activation) (59). However,there is compelling evidence to suggest that the lipidproducts of PI 3-kinase stimulate the activity of gua-nosine nucleotide exchange Factors (GEFs) such asSos and Vav, which, in turn, stimulate the actions ofrho-family GTPases (36,122). Thus, given the im-portance of PI 3-kinase in mitogenic responses insmooth muscle and endothelial cells, the role of PI3-kinase in regulating vascular cell–cytoskeletal re-modeling certainly merits further examination.
An interesting area that is attracting considerableattention is the potential role of a family of smallras-related GTP-binding proteins in controlling ac-tin polymerization and organization. It had beenknown for some time that bacterial ribonucleotidetransferases such as the C3 toxin from Clostridiumbotulinum had marked effects on the actin cytoskel-eton (32). The targets of these and other toxins havebeen characterized as a family of small ubiquitousGTPases related to the ras oncogene, which includerho, rac, and cdc42 (55,138). Studies using consti-tutively active or dominant negative mutants ofthese proteins reveal that their putative roles in regu-lating cytoskeletal remodeling include membraneruffling, filopodia extension, stress fiber and focaladhesion formation (123,135,136). Indeed, there isgrowing evidence to suggest that these proteins alsopromote endothelial-junctional integrity (69),growth factor-induced membrane ruffling (59), aswell as activation on myosin contractility in vascularsmooth muscle cells (7,90). Although these regula-tory proteins are capable of activating each other,these proteins appear to regulate the actin cytoskel-eton by distinct mechanisms. Thus, by the selectiveactivation of these GTPases, different signaling
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pathways may give rise to a certain subset of remod-eling events. Although the molecular details regard-ing these potential interactions is lengthy, one shouldconsider that many of the experiments demonstrat-ing cytoskeletal alterations by these molecules havebeen performed on serum-starved Swiss 3T3 cells,expressing extrapharmacological doses of the rho-related GTPases. Thus, it is uncertain whether theseras-related peptides function similarly under physi-ologic conditions.
p21rac
As mentioned above, experiments using either con-stitutively active or dominant negative forms of therho-family member, p21rac, suggest that this GT-Pase plays a role in growth factor-induced mem-brane ruffling in 3T3 cells (135). Activated rac canstimulate the activity of several protein kinases, in-cluding those of the jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) family (163).However, deletion analysis of rac reveals that rac-mediated membrane ruffling is independent of theseprotein kinases (91), but is instead dependent on theactivation of lipid kinases. This was demonstrated ina series of experiments using permeabilized platelets,where the dramatic increase in actin polymerizationfollowing thrombin stimulation could be attributedto a PIP2-dependent uncapping of existing filamentends, which was shown to be under the control ofrac, but not rho (57). Presumably, rac mediates thisuncapping through the stimulation of phosphatidyli-nositol 4-phosphate 5-kinase (PI 5-kinase), and bystimulating PI 5-kinase activity to produce PIP2, racGTPase is capable of stimulating a rapid burst ofactin polymerization in response to an externalstimulus. It will be of great interest to see if rac isalso activated in other instances of vascular cell mo-tility, such as during endothelial monolayer injury,where the rates of forward protrusion and motilityare slower and more sustained than the rapid re-sponses seen during platelet activation. Recently, re-constitution experiments of digitonin-extractedSwiss 3T3 cells suggest that rac-mediated ruffling isdependent on an indirect interaction with membersof the ezrin-radixin-moesin (ERM) family (104).These results are intriguing in light of our findingsdemonstrating that ezrin and the b–actin specific-binding protein, bcap73, are recruited to the leadingedge of crawling endothelial cells and pericytes dur-ing protrusion formation and can be coprecipitatedfrom pericyte extracts (150, 151; Lin et al., inpreparation). Further investigation will determinewhether rac actually plays a role in regulating thiscomplex as well other nucleating/capping factors
such as VASP and the Arp2/3 complex, whose lo-calization to motile cytoplasmic domains stronglyimplicates them as important factors mediating vas-cular cell motility.
p21 rho
p21 rho has been implicated in the regulation of awide variety of actin-based functions, includingstress fiber formation, focal adhesion assembly andcontractile ring integrity (163). However, themechanisms by which rho mediates these processesare not well understood. Like p21 rac, rho stimulatesboth the activity of lipid kinases such as PI-5 kinase(134) as well as several protein kinases (163). Ad-ditionally, it has been recently shown that activatedrho stimulates the activity of rho-associated kinase(Rho-K or ROCK), which, in turn, may directlyphosphorylate myosin regulatory light chain on resi-dues previously shown to stimulate myosin ATPaseand motor activity (7). In permeabilized smoothmuscle cells, addition of a constitutively activatedform of rho kinase results in phosphorylation ofmyosin light chain and muscle contraction (90).Similarly, the stimulation of rho in fibroblasts resultsin myosin light chain phosphorylation, concomitantwith FA and SF formation (33). This notion that rhocontrols myosin-based contractility through the ac-tion of ROCK has been recently supported by phar-macological studies in smooth muscle cells. A potentdrug named Y-27632 has been developed that in-hibits agonist-stimulated, Ca++-mediated contrac-tion in vascular smooth muscle, and is capable oflowering blood pressure in spontaneously hyperten-sive rats (170). Photoaffinity labeling studies revealthat the target of this drug is, in fact, a rho-associated kinase. From these results, it has beenproposed that rho mediates smooth muscle contrac-tility in vivo and SF formation in vitro, through theactivation of myosin heavy chain (163). As for FAassembly, the stimulation of PI-5 kinase by rho mayactivate the FA protein, vinculin, which exists in aninactive conformation that can be ‘‘opened’’ to re-veal talin and actin-binding domains in the presenceof PIP2 (51,78). Thus, by the selective activation ofboth lipid and protein kinases, rho may affect a sub-set of actin-associated proteins or motors to promotethe assembly of metastable structures such as actinbundles and FA.
Cdc42
Cdc42 has been implicated in filopodia formation incultured cells as well as polarization during T-cell
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activation and bud-site selection in Saccharomycescereviseae (55), and like the other rho-family pro-teins, has been the subject of a great deal of attentionover the past three years. From these efforts therehas emerged a multitude of candidate effector mol-ecules, any one of which may be responsible for theeffects of Cdc42 on the actin cytoskeleton. Like theother rho-family GTPases, Cdc42 is capable of ac-tivating the jun kinase pathway (91) as well a myo-tonic dystrophy kinase-related kinase (96) and aDrosophila serine/threonine kinase (98), both ofwhich have been implicated in cytoskeletal alter-ations. One of the first effector molecules to be iden-tified for Cdc42 was the Wiskott–Aldrich syndromeprotein (WASP) (86,158). The affected gene prod-uct in an X-linked immune disorder that affectsplatelet function as well as T-cell–B-cell interactions,WASP contains pleckstrin homology domains, a co-filin-homogous domain, and contains several poly-proline-rich domains capable of binding SH3-containing proteins such as the adapter protein Nck(84). The multiple binding domains suggest thatWASP may associate with acidic phospholipids aswell as components of receptor tyrosine kinase-signaling cascades (84). Wiskott–Aldrich syndromeprotein specifically associates with the rho-familyGTPase Cdc42, and to a lesser extent rac (10,86,158). Wiskott–Aldrich syndrome protein colocal-izes with actin filaments, and it is possible thatWASP interacts not only via the cofilin-homologydomain but also through the C-terminal 59 aminoacids (158). Additionally, a poly-proline-richWASP-interacting protein (WIP) has been isolatedthat promotes actin polymerization through actin-and proline-binding domains (132). Still, other re-ports suggest that a related protein, N-WASP (butnot WASP itself), promotes filopodia in a Cdc42-dependent manner (112). N-WASP constructs con-taining point mutations in the actin-binding domainor the PIP2-binding PH domain inhibit EGF-induced actin polymerization in cultured cells (111),and further studies of N-WASP suggest that it is thecofilin-like domain that promotes filopodia forma-tion (112). Thus, while the exact role that WASPplays in Cdc42-mediated cytoskeletal rearrange-ments remains controversial, it is clear that WASPrepresents an important effector molecule that maythen go on to participate in as many as three signal-ing- or second-messenger pathways (acidic phos-pholipids, tyrosine kinases, and rho-family GT-Pases).
Another important effector molecule of Cdc42 isIQGAP1. IQGAP1, a 190-kd calmodulin- and
Cdc42-binding protein, directly binds and crosslinksactin filaments in a calcium/calmodulin-sensitivemanner (15). Growth factor stimulation of cells pro-motes the formation of Cdc42/IQGAP1 complexes(46), and the GTP-bound form of Cdc42 promotesIQGAP1 oligomerization that, in turn, enhances fila-ment crosslinking activity (50). Genetic analyses infission yeast and Dictyostelium suggest that thisbundling activity is required for proper contractilering formation during cytokinesis (5,45). Thus,IQGAP1 represents a potential direct link betweenrho-family GTPases and the actin-based cytoskele-ton. This interaction may explain how Cdc42 pro-motes the elaboration of actin-based structures dis-tinct from those stimulated by the other rho-familyGTPases, even when the all three GTPases are acti-vated by a common stimulus.
The Cytoskeleton As a Reflection of Cell Function
Cytoskeletal proteins that are subject to regulationby multiple signaling pathways may not only shedlight on the complex regulation of the cytoskeleton ingeneral, but may also lend insight into vascular cellfunction in vivo. The pericyte has long been thoughtof as the ‘‘smooth muscle’’ equivalent of the micro-vasculature, and also plays a regulatory role in con-trolling endothelial cell proliferation (36). Indeed,the ability of pericytes to contract silica gels in vitro(82) and the expression of both muscle and non-muscle actin protein isoforms (67) implicate a con-tractile role for pericytes in the microvasculature.Pericytes express an abundance of both muscle andnonmuscle isoactins (67), and like smooth muscle,this contractile phenotype may be modulated bygrowth factors and extracellular matrices (121). Inlight of the plasticity of the pericyte contractile phe-notype and in the absence of any solid informationregarding the developmental lineage of this cell type,there remains much to be learned regarding the roleof pericytes in regulating blood flow in the micro-vasculature.
The regulation of smooth muscle cell contraction haslong been an intensive field of study, and the acti-vation of myosin heavy chain has been firmly estab-lished (147). Unlike skeletal muscle, smooth muscleand nonmuscle contractility is primarily regulated atthe level of the myosin thick filament, through thecalcium-activated phosphorylation of the myosinregulatory light chain (6). However, several obser-vations suggest that there lies an additional, thinfilament-based, level of myosin control. This regu-lation appears to be mediated by two proteins, cal-desmon and calponin. Caldesmon is found in two
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forms, a high-molecular-weight isoform (h; 120–150,000) that is exclusively expressed in smoothmuscle cells, and a lower molecular-weight form (l;70–80,000), found in most non-muscle cell types(147). Calponin is a 32-kd protein isolated fromchicken gizzard smooth muscle (160) by virtue of itsability to bind calmodulin and its crossreactivitywith anti-troponin antibodies. There are two iso-forms of calponin (162) whose expression is re-stricted to smooth muscle cells, although there arereports of the existence of nonmuscle homologues(44). Although caldesmon and calponin share verylittle sequence homology, they have similar bindingproperties: Both proteins bind actin, calmodulin,myosin, and tropomyosin (44,147,179), and mayeven associate with each other (53). Additionally,each molecule is capable of inhibiting actin-activated ATPase activity of myosin heavy chain andare phosphorylated by several kinases, includingprotein kinase C, Ca++-calmodulin-dependent ki-nase and, cAMP-dependent kinase. Phosphorylationby these kinases reduces the affinity of caldesmonand calponin for actin and thus lowers the ability ofthese molecules to inhibit myosin ATPase activity(157,179). Whether these molecules perform thesefunctions in vivo has yet to be clearly established.The stoichiometries of caldesmon and calponin insmooth muscle cells suggest that the amounts of cal-desmon found in cells is not consistent with its roleas a thin-filament regulatory protein (108). How-ever, experiments in permeabilized model systemssuggest that both molecules may play importantroles in the maintenance of smooth muscle relax-ation (80,106).
Regarding pericytes, both calponin and caldesmonare expressed. By Western blotting, calponin anti-bodies recognize a single immunoreactive band,whereas both the h and l isoforms of caldesmon canbe identified (C Shuster, unpublished observations).In pericytes, caldesmon appears to localizedthroughout the actin cytoskeleton (Shuster and Her-man, unpublished results). But due to antibodycrossreactivity between the h and l forms, which aresimultaneously expressed in pericytes, it is impos-sible to say whether caldesmon localization reflectswhat is seen in smooth muscle cells (21). Calponinlocalization is, however, restricted to a actin- andmyosin-containing SF (Fig. 4). The isoform of cal-ponin seen in pericytes appears to be a truly smoothmuscle specific isoform, because no such localizationcan be seen in endothelial cells using anti-smoothmuscle calponin antibodies (C. Shuster, unpublishedobservations). This restricted localization of calpo-
nin to ‘‘contractile’’ domains of pericyte cytoplasm isin contrast to localization studies in gizzard smoothmuscle, where calponin is localized to both cytoskel-etal and contractile domains (100,124).Defining ex-actly which isoforms are expressed in pericytes, andthe generation of isoform-specific antibodies may re-solve these issues, and may lend insight into the spe-cific functions that calponin isoforms play in vivo.
The expression of calponin and the h isoform of cal-desmon reflects the notion that pericytes perform acontractile as well as growth-regulatory role in themicrovasculature. Because these molecules are sub-ject to regulation by multiple pathways, it is a dis-tinct possibility that molecules such as calponinserve as a focal point for the regulation of contrac-tility by multiple and divergent signaling pathways.Revealing how these proteins, as well as the rho
Figure 4. Calponin associates with a actin-containingstress fibers in retinal pericytes. Bovine retinal pericyteswere plated onto glass coverslips, then fixed in a buffercontaining 4% formaldehyde, briefly permeablized with0.1% Triton X-100, and processed for actin localizationwith NBD-phallicidin (A) and calponin localization withanti-smooth muscle calponin IgG (B).
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GT-Pases, are regulated under conditions of vasodi-lation or constriction will ultimately yield a greaterunderstanding of how the cytoskeleton participatesin vascular function in vivo.
Much of our understanding of the molecular mecha-nisms of cell motility have come from a wide varietyof experimental approaches using different modelsystems. As to vascular cell motility, there are littledata to suggest that endothelial and smooth muscleresponses to chemotactic stimuli are fundamentallydifferent. However, in the case of vascular cells,there is the added dimension of contractile proteinisoform switching from a contractile (muscle) to aproliferative (nonmuscle) phenotype when pericytesand smooth muscle cells are exposed to certain ex-tracellular matrix proteins and growth factors(60,61). The multiple (and in some cases, simulta-neous) expression of muscle and non-muscle con-tractile protein isoforms, and their intracellular com-partmentalization to distinct functional cytoplasmicdomains have provided an ideal model system toexamine questions regarding the role of these iso-forms in the molecular basis of cell motility. Withour recent discoveries regarding novel binding pro-teins such as bcap73, nucleating factors such asVASP and the ARP2/3 complex, it will soon be pos-sible to directly address how these proteins functionin coordination with the different actin isoforms toinitiate forward protrusion and other ultrastructuralalterations in response to extracellular stimuli.
Sorting the exact pathways by which the engage-ment of cell-matrix interactions or receptor–ligandcomplexes results in actin-based cytoskeletal reorga-nization will not be so straightforward. Becausecomponents of the ECM as well as growth factors arecapable of eliciting very different responses depend-ing on the cell type, a healthy degree of skepticism isin order when considering how these molecules elicitactin-based reorganizations in vascular cells. Giventhat cells of the vessel wall are in constant contactwith the extracellular matrix, the physiological sig-nificance of the activation of multiple mitogenicpathways upon integrin-matrix binding must betested with more stringency. Likewise, the notionthat rho-family GTPases act as master regulators ofthe actomyosin-based cytoskeleton also merits fur-ther consideration. There are multiple reports tosuggest that membrane ruffling can occur either inthe presence of PI 3-kinase inhibitors (14) or dom-inant negative forms of rac (79). That growth factor-
independent membrane ruffling can occur even inthe presence of dominant negative forms of rho-family GTPases (79) suggests that other regulatorycascades are present. Regarding the endothelium,rho GTPases may play a more critical role in themaintenance of cadherin-based junctions, where ithas been recently shown in MDCK cells and kera-tinocytes that rho and rac play crucial roles not onlyin recruiting actin to the adherens junction, but alsoin cadherin clustering (18, 161). Revealing the sig-naling pathways that mobilize some of the more re-cently characterized capping and nucleation factorsduring vascular cell motility will help workers re-solve many of these issues, therein identifying thecritical cascades required to launch motility in re-sponse to an extracellular stimulus.
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