Assembly and Reorientation of Stress Fibers Drives Morphological Changes to Endothelial Cells Exposed to Shear Stress
2004; Elsevier BV; Volume: 164; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)63209-9
ISSN1525-2191
AutoresSabrena Noria, Xu Feng, Shannon McCue, Mara Jones, Avrum I. Gotlieb, B. Lowell Langille,
Tópico(s)Angiogenesis and VEGF in Cancer
ResumoFluid shear stress greatly influences the biology of vascular endothelial cells and the pathogenesis of atherosclerosis. Endothelial cells undergo profound shape change and reorientation in response to physiological levels of fluid shear stress. These morphological changes influence cell function; however, the processes that produce them are poorly understood. We have examined how actin assembly is related to shear-induced endothelial cell shape change. To do so, we imposed physiological levels of shear stress on cultured endothelium for up to 96 hours and then permeabilized the cells and exposed them briefly to fluorescently labeled monomeric actin at various time points to assess actin assembly. Alternatively, monomeric actin was microinjected into cells to allow continuous monitoring of actin distribution. Actin assembly occurred primarily at the ends of stress fibers, which simultaneously reoriented to the shear axis, frequently fused with neighboring stress fibers, and ultimately drove the poles of the cells in the upstream and/or downstream directions. Actin polymerization occurred where stress fibers inserted into focal adhesion complexes, but usually only at one end of the stress fiber. Neither the upstream nor downstream focal adhesion complex was preferred. Changes in actin organization were accompanied by translocation and remodeling of cell-substrate adhesion complexes and transient formation of punctate cell-cell adherens junctions. These findings indicate that stress fiber assembly and realignment provide a novel mode by which cell morphology is altered by mechanical signals. Fluid shear stress greatly influences the biology of vascular endothelial cells and the pathogenesis of atherosclerosis. Endothelial cells undergo profound shape change and reorientation in response to physiological levels of fluid shear stress. These morphological changes influence cell function; however, the processes that produce them are poorly understood. We have examined how actin assembly is related to shear-induced endothelial cell shape change. To do so, we imposed physiological levels of shear stress on cultured endothelium for up to 96 hours and then permeabilized the cells and exposed them briefly to fluorescently labeled monomeric actin at various time points to assess actin assembly. Alternatively, monomeric actin was microinjected into cells to allow continuous monitoring of actin distribution. Actin assembly occurred primarily at the ends of stress fibers, which simultaneously reoriented to the shear axis, frequently fused with neighboring stress fibers, and ultimately drove the poles of the cells in the upstream and/or downstream directions. Actin polymerization occurred where stress fibers inserted into focal adhesion complexes, but usually only at one end of the stress fiber. Neither the upstream nor downstream focal adhesion complex was preferred. Changes in actin organization were accompanied by translocation and remodeling of cell-substrate adhesion complexes and transient formation of punctate cell-cell adherens junctions. These findings indicate that stress fiber assembly and realignment provide a novel mode by which cell morphology is altered by mechanical signals. There is compelling evidence that early atherosclerotic lesions most often originate near arterial bends and branch sites, where complex blood flow patterns occur. This finding, together with observations that experimental perturbations in flow can initiate or redistribute lesions,1Roach MR Fletcher J Effect of unilateral nephrectomy of the localization of aortic sudanophilic lesions in cholesterol-fed rabbits.Atherosclerosis. 1977; 24: 327-333Abstract Full Text PDF Scopus (10) Google Scholar, 2Zand T Hoffman AH Savilonis BJ Underwood JM Nunnari JJ Majno G Joris IS Lipid deposition in rat aortas with intraluminal hemispherical plug stenosis.Am J Pathol. 1999; 155: 85-92Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar strongly implicates local hemodynamics in atherogenesis. More specifically, exhaustive study of selected arterial sites, eg, the carotid bifurcation, has provided evidence that low and/or fluctuating shear stresses are proatherogenic.3Ku DN Giddens DP Zarins CK Glagov S Pulsatile flow and atherosclerosis in the human carotid bifurcation. Positive correlation between plaque location and low and oscillating shear stress.Arteriosclerosis. 1985; 5: 293-302Crossref PubMed Google Scholar Shear stresses are very small forces that typically cause bulk deformations (strains) of artery walls that are much less than 1%;4Langille BL O'Donnell F Reductions in arterial diameter produced by chronic decreases in blood flow are endothelium-dependent.Science. 1986; 231: 405-407Crossref PubMed Scopus (941) Google Scholar however, the endothelium of arteries is directly exposed to flowing blood and these cells have proven to be exquisitely sensitive to shear. This sensitivity may impact on atherogenesis through both modulation of classic functions of endothelium, ie, its role as a nonthrombogenic transport barrier that regulates inflammatory responses, and through regulation of growth and contractile functions of smooth muscle.5Ross R Fuster V The pathogenesis of atherosclerosis.in: Fuster V Ross R Topol EJ Atherosclerosis and Coronary Artery Disease. Lippincott-Raven Publishers, Philadelphia1996: 441-460Google Scholar, 6Ross R Mechanisms of disease—atherosclerosis—an inflammatory disease.N Engl J Med. 1999; 340: 115-126Crossref PubMed Scopus (19570) Google Scholar Consequently, much effort has been expended to explore endothelial sensitivity to shear stress. Examination of candidate genes has revealed that many potentially atherosclerosis-related gene products display shear-sensitive expression by endothelium, including VCAM-1, MCP-1, ICAM-1, eNOS, platelet-derived growth factors, transforming growth factor-β1, and endothelin.7Ando J Tsuboi H Korenaga R Takada Y Toyama-Sorimachi N Miyasaka M Kamiya A Shear stress inhibits adhesion of cultured mouse endothelial cells to lymphocytes by downregulating VCAM-1 expression.Am J Physiol. 1994; 267: C679-C687PubMed Google Scholar, 8Ranjan V Diamond SL Fluid shear stress induces synthesis and nuclear localization of c-fos in cultured human endothelial cells.Biochem Biophys Res Commun. 1993; 196: 79-84Crossref PubMed Scopus (57) Google Scholar, 9Walpola PL Gotlieb AI Cybulsky MI Langille BL Expression of ICAM-1 and VCAM-1 and monocyte adherence in arteries exposed to altered shear stress.Arterioscler Thromb Vasc Biol. 1995; 15: 2-10Crossref PubMed Scopus (337) Google Scholar, 10Ohno M Cooke JP Dzau VJ Gibbons GH Fluid shear stress induces endothelial transforming growth factor beta-1 transcription and production. Modulation by potassium channel blockade.J Clin Invest. 1995; 95: 1363-1369Crossref PubMed Scopus (307) Google Scholar, 11Nagel T Resnick N Atkinson WJ Dewey Jr, CF Gimbrone Jr, MA Shear stress selectively upregulates intercellular adhesion molecule-1 expression in cultured human vascular endothelial cells.J Clin Invest. 1994; 94: 885-891Crossref PubMed Google Scholar More recently, microarray-based studies have demonstrated that expression of hundreds of genes display sensitivity to both the duration and nature (eg, laminar versus turbulent) of shear imposed on endothelial cells.12Garcia-Cardena G Comander J Anderson KR Blackman BR Gimbrone Jr, MA Biomechanical activation of vascular endothelium as a determinant of its functional phenotype.Proc Natl Acad Sci USA. 2001; 98: 4478-4485Crossref PubMed Scopus (462) Google Scholar The sensitivity of endothelium to shear forces is most obviously manifest through changes in cell morphology: the cells elongate, the cell outline and its cytoskeleton becomes highly oriented in the direction of shear stress, and the profile of the cells becomes substantially more flattened when they are exposed to shear stress.13Noria S Cowan DB Gotlieb AI Langille BL Transient and steady-state effects of shear stress on endothelial cell adherens junctions.Circ Res. 1999; 85: 504-514Crossref PubMed Scopus (203) Google Scholar Morphological responses also include partial disassembly and then reassembly of adherens junctions, the primary sites of mechanical coupling between endothelial cells.13Noria S Cowan DB Gotlieb AI Langille BL Transient and steady-state effects of shear stress on endothelial cell adherens junctions.Circ Res. 1999; 85: 504-514Crossref PubMed Scopus (203) Google Scholar Consequently, long-term alterations in secondary flow patterns near branch sites, eg, because of circadian fluctuations in tissue perfusion, may contribute to the high endothelial permeability and low-density lipoprotein uptake at atherosclerosis-prone regions near arterial bends and branch sites14Caplan BA Schwartz CJ Increased endothelial cell turnover in areas of Evans blue uptake in the pig aorta.Atherosclerosis. 1973; 17: 401-417Abstract Full Text PDF PubMed Scopus (277) Google Scholar and thereby contribute to lesion formation. Finally, shear-stressed endothelial cells provide one of the few cases in which all cells in a monolayer undergo a common morphological response to a synchronized stimulus. Sheared endothelial cells therefore provide a unique model system with which to examine control of cell motility and morphology. Given the importance of shear-induced morphological changes, it is surprising that little is known concerning how reorganization of cellular structures drives these changes. The actin cytoskeleton initiates most changes in cell morphology. Contractile function of actin-myosin complexes is responsible in some cases;15Costa M Raich W Agbunag C Leung B Hardin J Priess JR A putative catenin-cadherin system mediates morphogenesis of the Caenorhabditis elegans embryo.J Cell Biol. 1998; 141: 297-308Crossref PubMed Scopus (313) Google Scholar however, actin polymerization is most commonly exploited.16Borisy GG Svitkina TM Actin machinery: pushing the envelope.Curr Opin Cell Biol. 2000; 12: 104-112Crossref PubMed Scopus (404) Google Scholar Usually, cell shape change follows from nucleation and assembly of thin parallel bundles of actin microfilaments at the cell edge that produce filopodia, ie, finger-like projections of the plasma membrane, or from assembly of dendritic arrays of actin that protrude foot-like processes referred to as lamellipodia. Coordinate remodeling of cell-cell and cell-substrate adhesion complexes then allows cell shape change to proceed. Actin also assembles into basal stress fibers but these are generally associated with adhesion to, or reorganization of, extracellular matrix.17Pankov R Cukierman E Katz B-Z Matsumoto K Lin DC Lin S Hahn CS Yamada KM Integrin dynamics and matrix assembly: tension-dependent translocation of α5β1 integrins promotes early fibronectin fibrillogenesis.J Cell Biol. 2000; 148: 1075-1090Crossref PubMed Scopus (387) Google Scholar We have investigated the role of actin assembly, and associated changes in cell adhesion complexes, in endothelial morphological responses to shear stress. The onset and rate of morphological changes to the cells depended greatly on the state and duration of confluence of the cells before initiation of shear stress: much slower morphological responses were observed in cells that were long confluent. As cells changed shape, formation of filopodia or lamellipodia did not occur; instead, a novel mode of cell shape change was driven by coincident extension and reorientation of stress fibers at the basal aspect of the cell, fusion of adjacent stress fibers, and translocation of the focal adhesion complexes into which these stress fibers insert. Ultimately, stress fiber assembly drove protrusion of the cell membrane in the direction of the shear axis to elongate the cells. This process was independent of the zyxin/vasodilator-stimulated phosphoprotein (VASP) complex, which associates with focal adhesions and can assemble G-actin into microfilament bundles, because introduction of peptide sequences that disrupted this complex do not suppress shear-induced actin assembly. Focal adhesion complexes that were associated with stress fibers also translocated with stress fibers and elongated. Simultaneously, cell-cell adherens junctions transiently assumed a punctate pattern and then re-established a linear distribution, in association with sporadic junctional actin assembly. Porcine aortic endothelial cells, at passages four to six, were maintained at 37°C in medium 199 plus 5% fetal bovine serum (Gibco, Invitrogen, Barlington, Canada), 1% amphotericin B (Fungizone), and 2% penicillin/streptomycin, and equilibrated with humidified 95% air and 5% CO2. At 2 days after confluence, cells were subjected to laminar shear stress of 15 dynes/cm2 in a parallel plate flow chamber.13Noria S Cowan DB Gotlieb AI Langille BL Transient and steady-state effects of shear stress on endothelial cell adherens junctions.Circ Res. 1999; 85: 504-514Crossref PubMed Scopus (203) Google Scholar, 18Frangos JA Eskin SG McIntire LV Ives CL Flow effects on prostacyclin production by cultured human endothelial cells.Science. 1985; 227: 1477-1479Crossref PubMed Scopus (1009) Google Scholar In some cases, subconfluent or just confluent cultures were exposed to shear stress to assess the effects of monolayer stabilization on responses to shear. Cells maintained in static culture served as controls (n ≥ 3 for each experiment). Cells were fixed with 3% paraformaldehyde as described,13Noria S Cowan DB Gotlieb AI Langille BL Transient and steady-state effects of shear stress on endothelial cell adherens junctions.Circ Res. 1999; 85: 504-514Crossref PubMed Scopus (203) Google Scholar permeabilized with 0.2% Triton X-100 and washed 3 times for 5 minutes with phosphate-buffered saline (PBS). Cells were then incubated with primary antibody for 1 hour, and then washed with PBS followed by incubation with secondary antibody. Primary antibodies included mouse monoclonal antibodies to paxillin (1:200; Transduction Laboratories, Lexington, KY), vinculin (1:50; Sigma BioSciences, Oakville, Canada), tensin or VASP (1:50; Transduction Laboratories). Primary antibodies were detected using Alexa Fluor 488 goat anti-mouse antibody (1:50; Molecular Probes, Eugene, OR). Staining frequently was done in the presence of rhodamine phalloidin (1:20; Molecular Probes), which labels F-actin. Animal experiments were conducted in accord with the guidelines of the Canadian Council of Animal Care and were approved by the Animal Care Committee of the Toronto General Hospital. Rabbits were killed by intravenous infusion of 1.0 ml of euthanasia solution (T-61; Hoechst, Montreal, Canada).9Walpola PL Gotlieb AI Cybulsky MI Langille BL Expression of ICAM-1 and VCAM-1 and monocyte adherence in arteries exposed to altered shear stress.Arterioscler Thromb Vasc Biol. 1995; 15: 2-10Crossref PubMed Scopus (337) Google Scholar The carotid arteries were fixed and immunostained9Walpola PL Gotlieb AI Cybulsky MI Langille BL Expression of ICAM-1 and VCAM-1 and monocyte adherence in arteries exposed to altered shear stress.Arterioscler Thromb Vasc Biol. 1995; 15: 2-10Crossref PubMed Scopus (337) Google Scholar using a goat polyclonal antibody to β-catenin (1:800; Jackson ImmunoResearch Laboratories, West Grove, PA) and a fluorescein isothiocyanate-conjugated donkey anti-goat secondary antibody (1:50, Jackson ImmunoResearch Laboratories). The arteries were viewed en face by confocal microscopy.9Walpola PL Gotlieb AI Cybulsky MI Langille BL Expression of ICAM-1 and VCAM-1 and monocyte adherence in arteries exposed to altered shear stress.Arterioscler Thromb Vasc Biol. 1995; 15: 2-10Crossref PubMed Scopus (337) Google Scholar The protocol for visualizing actin assembly was based on studies by Symons and Mitchison,19Symons MH Mitchison TJ Control of actin polymerization in live and permeabilized fibroblasts.J Cell Biol. 1991; 114: 503-513Crossref PubMed Scopus (230) Google Scholar with modifications.20Vasioukhin V Bauer C Yin M Fuchs E Directed actin polymerization is the driving force for epithelial cell-cell adhesion.Cell. 2000; 100: 209-219Abstract Full Text Full Text PDF PubMed Scopus (960) Google Scholar Cultures were gently washed with rinsing buffer (20 mmol/L Hepes, pH 7.5, 138 mmol/L KCl; 4 mmol/L MgCl2, 3 mmol/L EGTA) and then incubated for up to 10 minutes with fluorescently labeled G-actin in permeabilization buffer [20 mmol/L Hepes, pH 7.5, 138 mmol/L KCl, 4 mmol/L MgCl2, 3 mmol/L EGTA, 0.01% saponin, 0.5 μmol/L Alexa 488-conjugated G-actin from rabbit muscle (Molecular Probes, Eugene, OR)]. After washing with buffer, cells were fixed for 20 minutes with 3% paraformaldehyde followed by a 3 × 5-minute wash with PBS. To detect endogenous F-actin, cells were incubated with rhodamine-labeled phalloidin for 30 minutes. In separate experiments, endothelial cells were triple stained for newly polymerized F-actin, endogenous F-actin, and with monoclonal anti-vinculin antibody (1:50; Sigma BioSciences) and a CY5-conjugated donkey anti-mouse secondary antibody, in the presence of rhodamine-labeled phalloidin. To visualize the cellular actin pool in live cells, subconfluent cells grown on coverslips were microinjected with Alexa Fluor 488-conjugated actin (6 mg/ml, Molecular Probes) using an Eppendorf transjector 5246. Cells were exposed to shear stress 24 hours after injection. Time-lapse images were collected using a fluorescence microscope (TE300; Nikon, Melville, NY) equipped with a cooled charge-coupled device camera (ORCA ER; Hamamatsu). To label cell membrane, cells were incubated for 10 minutes with 20 mmol/L DiI (1.1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate; Molecular Probes). Cells were fixed as described above. Fixed endothelial cells were examined using a Bio-Rad MRC 1024 laser scanning confocal microscope (Nikon ×60 oil-immersion objective; NA = 1.4). The peak excitation/emission wavelengths for the fluorescent probes, and the detection filters used were: fluorescein isothiocyanate = 492/520 nm and detected at 522 nm; CY5 = 650/680 nm and detected at 680 nm; rhodamine = 554/573 nm and detected at 605 nm. For each experiment, at least10 fields per slide were collected. Peptide sequences comprising a domain of zyxin that mediates binding to VASP (PPEDFPLPPPPLAGD),21Drees B Friederich E Fradelizi J Louvard D Beckerle MC Golysteyn RM Characterization of the interaction between zyxin and members of the Ena/VASP (vasodilator stimulated phosphoprotein) family of proteins.J Biol Chem. 2000; 275: 22503-22511Crossref PubMed Scopus (137) Google Scholar or a scrambled version of this peptide (control, PPEDAPLPPPPLAGD), in tandem with a Trojan peptide sequence (a string of nine arginines)22Wender PA Mitchell DJ Pattabiraman K Pelkey ET Steinman L Rothbard JB The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: peptoid molecular transporters.Proc Natl Acad Sci USA. 2000; 97: 13003-13008Crossref PubMed Scopus (1445) Google Scholar to facilitate cell entry, were synthesized by the Biotechnology Service Centre (Hospital for Sick Children, Toronto, Canada). The peptide (300 μmol/L) was introduced into the flow loop at 15.5 hours of shear stress and then incorporation of fluorescently labeled monomeric actin into microfilaments was assessed 30 minutes later, as described above. Four replicate experiments were performed. Actin most often assembled at one end of stress fibers. To assess bias in the direction of actin assembly, cells were classified according to whether actin assembly was predominantly in the upstream, downstream, or both ends of stress fibers (bipolar), or directed away from the nucleus (anti-podal). At least 30 cells per time point were scored by an unbiased (blinded) observer and the cells to be counted were defined from a predefined grid using the Vernier scales on the stage drives, so that cell selection was unbiased. Statistical analysis was analysis of variance followed by a Tukey's post hoc test. Endothelial cells in healthy arteries are permanently confluent; therefore, cells generally were not exposed to shear until 2 days after confluence was achieved. Throughout these 2 days, stress fibers became less prominent and a junctional dense peripheral band of F-actin was established (Figure 1), then no further changes in actin distribution were detectable. This stabilization proved important because subconfluent or just confluent cells initiate reorganization of actin very quickly after shear stress is imposed (not shown), whereas only very modest shear-related F-actin reorganization occurred in cells that were 2 days postconfluent until 8 hours of shear (Figure 1).13Noria S Cowan DB Gotlieb AI Langille BL Transient and steady-state effects of shear stress on endothelial cell adherens junctions.Circ Res. 1999; 85: 504-514Crossref PubMed Scopus (203) Google Scholar Partial loss of the dense peripheral band of actin was then followed by elongation of cells and alignment of actin microfilaments with shear throughout 8 to 24 hours. No further changes in cell shape or actin distribution were observed. To define shear-induced changes to cell shape and cell junctions, we labeled membrane phospholipid with DiI.13Noria S Cowan DB Gotlieb AI Langille BL Transient and steady-state effects of shear stress on endothelial cell adherens junctions.Circ Res. 1999; 85: 504-514Crossref PubMed Scopus (203) Google Scholar Under static conditions, the cells displayed a typical cobblestone morphology with multiple regions of membrane overlap that were manifest as areas of enhanced DiI fluorescence, because of fluorescence from multiple layers of membrane. These regions were separated by linear cell-cell junctions (Figure 2a). While elongating, cell margins did not display classical lamellipodia; instead, pointed projections were commonplace (Figure 2b). These structures that appeared to initiate shape change were often quite broadly based, but could not be definitely differentiated from filopodia, finger-like cellular projections that result from actin assembly at the cell edge.20Vasioukhin V Bauer C Yin M Fuchs E Directed actin polymerization is the driving force for epithelial cell-cell adhesion.Cell. 2000; 100: 209-219Abstract Full Text Full Text PDF PubMed Scopus (960) Google Scholar To assess actin assembly during changes in cell shape, we permeabilized endothelial cell membranes after varying periods of shear stress and then incubated the cells with Alexa 488-labeled monomeric actin (G-actin) briefly (up to 10 minutes) to define sites of actin assembly, then cells were fixed and stained for total F-actin. When it occurred, actin incorporation into microfilaments was detectable at 3 minutes of incubation, but optimal labeling was seen with 8 to 10 minutes of incubation. Uptake of G-actin into microfilaments was readily detected in subconfluent endothelial cells (not shown), which are highly motile, but little or no incorporation of labeled G-actin was detected in cultures 2 days after confluence that were not exposed to shear stress (Figure 3A). Approximately 50% of cells displayed incorporation of exogenous G-actin at ends of randomly oriented stress fibers after 8 hours of shear (Figure 3B, green). Most frequently, actin assembly occurred at only one end of the stress fibers (see below). Less prominent, punctate staining of exogenous actin was also observed along the lengths of microfilaments. Most cells displayed actin assembly at the ends of stress fibers by 16 hours, when many cells were elongate and aligned with shear (Figure 3C). In shear-aligned cells, basal stress fibers and actin assembly were parallel to shear. Many other cells were elongated but not yet parallel to shear, in which case basal stress fibers tended to align with the long axis of the cell (not shown). Cell elongation and alignment with shear stress was completed at 24 hours; nonetheless, actin assembly continued for as long as we examined the cells (up to 96 hours, Figure 3D). Optical sectioning with confocal microscopy and double staining for actin assembly and the focal adhesion proteins, vinculin and paxillin, demonstrated that actin assembly occurred at the basal aspect of the cells, where microfilament bundles insert into focal adhesion complexes (Figure 4). Notably, both ends of stress fibers terminated in adhesion complexes, but actin assembly most often occurred at only one of these sites (Figure 4, arrows). Importantly, actin assembly was constrained to subdomains of focal adhesion complexes, as indicated by only partial overlap of G-actin fluorescence with immunostaining for vinculin (Figure 4, insets). This finding probably reflects remodeling and translocation of the focal adhesion/stress fiber complex during adaptation to shear stress, as described below. Within individual cells, actin polymerized predominantly in the upstream direction, predominantly in the downstream direction, or predominantly directed away from the nucleus (anti-podal) with approximately equal frequencies (Figure 5). Assembly at both ends of the stress fiber (bipolar assembly) was also observed occasionally, but random distribution of actin polymerization to one or the other end of stress fibers within individual cells was seen in only 10 to 20% of cells and never at times later than 24 hours. The only other statistically significant time dependence in direction of actin assembly was the elevation in bipolar assembly at 48 hours. These findings indicate that shear stress determined the orientation but not direction of actin polymerization. Subtle intracellular or cell-cell signaling may provide cues that define the latter. The scaffolding protein, zyxin, can link VASP to the actin-bundling protein, α-actinin, which concentrates at focal adhesions.23Rottner K Krause M Gimona M Small JV Wehland J Zyxin is not colocalized with vasodilator-stimulated phosphoprotein (VASP) at lamellipodial tips and exhibits different dynamics to vinculin, paxillin, and VASP in focal adhesions.Mol Biol Cell. 2001; 12: 3103-3113Crossref PubMed Scopus (96) Google Scholar VASP interaction with cytoplasmic G-actin-profilin complexes can then incorporate actin monomer into microfilaments. VASP localized to focal adhesions in endothelium (Figure 6, A and B) but it did not mediate shear-induced actin assembly at these sites. Accordingly, a peptide sequence that was designed to interfere with zyxin-VASP interaction successfully dislodged VASP from focal adhesions (Figure 6, A and B) but had no impact on shear-induced actin assembly (Figure 6, C and D). VASP was not dislodged from focal adhesions by control peptide (not shown). By 16 to 24 hours, actin assembly in all cells became oriented with the shear axis and frequently extended to the upstream and/or downstream poles of the cell (Figure 3, Figure 4). We therefore hypothesized that stress fiber reorientation and assembly protruded the cell membrane along the shear axis to achieve elongation. To test this hypothesis, we microinjected endothelial cells with fluorescently labeled G-actin and then exposed the cells to shear stress 24 hours later, when the labeled G-actin was distributed throughout the cellular pool, so that microfilaments in live cells could be visualized. Individual stress fibers extended and changed their orientation, most often toward the shear axis, but excursion away from this direction also occurred. During and after alignment of stress fibers in the direction of shear, assembling stress fibers extended to the cell periphery and further actin assembly drove protrusion of the cell membrane in the direction of cell elongation (Figure 7). Stress fibers originated proximal to the cell periphery then extended to the cell-cell junction and advanced the adjacent cell membrane. In no cells did we see evidence of actin nucleation at the cell edge that would characterize filopodia. Interestingly, when assembling and reorienting stress fibers encountered adjacent stress fibers, they often merged to produce larger microfilament bundles (Figure 7, arrows). Reorganization of cell-substrate adhesion complexes accompanied realignment of stress fibers. In static cultures, focal adhesions were large, ovoid, or arrowhead complexes, whereas shear stress caused most of these complexes to assume an extended, linear morphology (Figure 8, Figure 9). These changes may reflect conversion from classical focal adhesions (focal contacts),24Zamir E Katz B-Z Aota S Yamada KM Geiger B Kam Z Molecular diversity of cell-matrix adhesions.J Cell Sci. 1999; 112: 1655-1669Crossref PubMed Google Scholar seen in many stable cultures, to the fibrillar adhesions that characterize motile cells;24Zamir E Katz B-Z Aota S Yamada KM Geiger B Kam Z Molecular diversity of cell-matrix adhesions.J Cell Sci. 1999; 112: 1655-1669Crossref PubMed Google Scholar however, not all of our data were consistent with this interpretation. We immunostained adhesion complexes with antibodies to vinculin, paxillin, and tensin, because the former two proteins preferentially localize to focal contacts whereas tensin preferentially concentrates at fibrillar adhesions.25Zamir E Katz M Posen Y Erez N Yamada KM Katz B-Z Lin S Lin DC Bershadsky A Kam Z Geiger B Dynamics and segregation of cell-matrix adhesions in cultured fibroblasts.Nat Cell Biol. 2000; 2: 191-196Crossref PubMed Scopus (470) Google Scholar, 26Geiger B Cell biology: encounters in space.Science. 2001; 294: 1661-1663Crossref PubMed Scopus (56) Google Scholar Some depletion of vinculin and paxillin at focal adhesions was seen with shear but there was also a dramatic loss of tensin that persisted until after shape change had gone to completion (Figure 9).Figure 9Focal adhesion complexes become depleted of tensin under the influence of shear stress. Endothelium in postconfluent cultures was immunostained for tensin and viewe
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