Rigidity of collagen fibrils controls collagen gel-induced down-regulation of focal adhesion complex proteins mediated by alpha2beta1 integrin

Previous studies have shown that collagen gel overlay induced selective proteolysis of focal adhesion complex proteins in Madin-Darby canine kidney (MDCK) cells. In this study, we examined whether morphological and biochemical changes were present in cells cultured on collagen gel. We found that focal adhesion complex proteins, including focal adhesion kinase (FAK), talin, paxillin, and p130cas, but not vinculin, were decreased within 1 h when MDCK cells were cultured on collagen gel. Collagen gel-induced selective decrease of focal adhesion proteins was observed in all lines of cells examined, including epithelial, fibroblastic, and cancer cells. Matrigel also induced selective down-regulation of focal adhesion proteins. However, cells cultured on collagen gel- or matrigel-coated dishes did not show any changes of focal adhesion proteins. These data suggest that the physical nature of the gel, i.e. the rigidity, is involved in the expression of focal adhesion proteins. The collagen gel-induced down-regulation of focal adhesion complex proteins was caused by reduction of protein synthesis and activation of proteases such as calpain. Overexpression of a dominant negative mutant of discoidin domain receptor 1 (DDR1) or FAK-related non-kinase (FRNK) did not prevent collagen gel-induced down-regulation of the focal adhesion complex protein, whereas an anti-alpha2beta1 integrin-neutralizing antibody completely blocked it. Taken together, our results indicate that the rigidity of collagen gel controls the expression of focal adhesion complex proteins, which is mediated by alpha2beta1 integrin but not DDR1.     

Vinculin regulates directionality and cell polarity in two- and three-dimensional matrix and three-dimensional microtrack migration

During metastasis, cells can use proteolytic activity to form tube-like “microtracks” within the extracellular matrix (ECM). Using these microtracks, cells can migrate unimpeded through the stroma. To investigate the molecular mechanisms of microtrack migration, we developed an in vitro three-dimensional (3D) micromolded collagen platform. When in microtracks, cells tend to migrate unidirectionally. Because focal adhesions are the primary mechanism by which cells interact with the ECM, we examined the roles of several focal adhesion molecules in driving unidirectional motion. Vinculin knockdown results in the repeated reversal of migration direction compared with control cells. Tracking the position of the Golgi centroid relative to the position of the nucleus centroid reveals that vinculin knockdown disrupts cell polarity in microtracks. Vinculin also directs migration on two-dimensional (2D) substrates and in 3D uniform collagen matrices, as indicated by reduced speed, shorter net displacement, and decreased directionality in vinculin-deficient cells. In addition, vinculin is necessary for focal adhesion kinase (FAK) activation in three dimensions, as vinculin knockdown results in reduced FAK activation in both 3D uniform collagen matrices and microtracks but not on 2D substrates, and, accordingly, FAK inhibition halts cell migration in 3D microtracks. Together these data indicate that vinculin plays a key role in polarization during migration.     

An in vitro model giving access to adhesion plaques

Summary A new approach was investigated to study the interaction between integrins and actin via intracytoplasmic proteins. Because intracellular processes are hampered by the limiting plasma membrane, we developed an in vitro model with cells perforated by a bacterial toxin, streptolysin O. The specific conditions for the use of permeabilized cells to study the intramolecular associations occurring at adhesion plaques are described. The two cell types used, HUVEC and CHO, showed that the choice of the perforation method is of great importance. After perforation of cells in a monolayer, 75±10% of the cells remained adherent to a fibronectin substrate; after perforation of cells in suspension, only 25±10% of the cells readhered. Specific conditions were required however to maintain these adhesive properties up to 4 h: the presence of 1 mM Mg⁺⁺ in the medium was crucial, and it was necessary to layer the cells on a specific coat rather than a substitute such as gelatin. Immunofluorescence investigations of actin, talin and vinculin, and Normarsky differential interference contrast microscopy showed retention of focal adhesion plaques in perforated cells. Moreover, in perforated cells antibodies directed against actin led to actin disorganization, showing that our model of perforated cells in a monolayer can give new insight to adhesion study.     

Comparing the mechanical influence of vinculin, focal adhesion kinase and p53 in mouse embryonic fibroblasts

Cytoskeletal reorganization is an ongoing process when cells adhere, move or invade extracellular substrates. The cellular force generation and transmission are determined by the intactness of the actomyosin-(focal adhesion complex)-integrin connection. We investigated the intracellular course of action in mouse embryonic fibroblasts deficient in the focal adhesion proteins vinculin and focal adhesion kinase (FAK) and the nuclear matrix protein p53 using magnetic tweezer and nanoparticle tracking techniques. Results show that the lack of these proteins decrease cellular stiffness and affect cell rheological behavior. The decrease in cellular binding strength was higher in FAK- to vinculin-deficient cells, whilst p53-deficient cells showed no effect compared to wildtype cells. The intracellular cytoskeletal activity was lowest in wildtype cells, but increased in the following order when cells lacked FAK+p53 > p53 > vinculin. In summary, cell mechanical processes are differently affected by the focal adhesion proteins vinculin and FAK than by the nuclear matrix protein, p53.     

Collagen gel overlay induces two phases of apoptosis in MDCK cells

We previously demonstrated that collagen gel overlayinduced cell remodeling to form lumen and apoptosis inMadin-Darby canine kidney cells. In the present study, we establishedthat collagen gel overlay-induced apoptosis was initiated atareas exclusive of cell remodeling within 24 h (first phase) andextended into areas of cell remodeling within 48 h (second phase).Collagen gel overlay-induced apoptosis was accompanied byselective proteolysis of focal adhesion kinase (FAK), talin,p130^cas, and c-src. Upon collagen geloverlay, FAK was initially degraded into a 90-kDa product during thefirst phase and subsequently into a 80-kDa product during the secondphase. Collagen gel overlay-induced apoptosis of focal adhesioncomplex proteins and apoptosis of the first phase could beblocked only by a protease inhibitor cocktail. In addition, we foundthat both DEVD-fmk and ZVAD-fmk inhibited secondary proteolysis of FAK,but only ZVAD-fmk blocked collagen gel overlay-inducedapoptosis of the second phase. Finally, collagen geloverlay-induced apoptosis and proteolysis of focal adhesioncomplex proteins were completely inhibited by the combination ofprotease inhibitor cocktail and ZVAD-fmk. Taken together, collagen geloverlay induces two phases of apoptosis; the first phase is dependent on proteolysis of focal adhesion complex proteins, and thesecond phase on activation of caspases.

     

Investigating lasp-2 in cell adhesion: new binding partners and roles in motility

Focal adhesions are intricate protein complexes that facilitate cell attachment, migration, and cellular communication. Lasp-2 (LIM-nebulette), a member of the nebulin family of actin-binding proteins, is a newly identified component of these complexes. To gain further insights into the functional role of lasp-2, we identified two additional binding partners of lasp-2: the integral focal adhesion proteins vinculin and paxillin. Of interest, the interaction of lasp-2 with its binding partners vinculin and paxillin is significantly reduced in the presence of lasp-1, another nebulin family member. The presence of lasp-2 appears to enhance the interaction of vinculin and paxillin with each other; however, as with the interaction of lasp-2 with vinculin or paxillin, this effect is greatly diminished in the presence of excess lasp-1. This suggests that the interplay between lasp-2 and lasp-1 could be an adhesion regulatory mechanism. Lasp-2’s potential role in metastasis is revealed, as overexpression of lasp-2 in either SW620 or PC-3B1 cells—metastatic cancer cell lines—increases cell migration but impedes cell invasion, suggesting that the enhanced interaction of vinculin and paxillin may functionally destabilize focal adhesion composition. Taken together, these data suggest that lasp-2 has an important role in coordinating and regulating the composition and dynamics of focal adhesions.     

Type XIII collagen: a novel cell adhesion component present in a range of cell–matrix adhesions and in the intercalated discs between cardiac muscle cells

Recent analysis of type XIII collagen surprisingly showed that it is anchored to the plasma membranes of cultured cells via a transmembrane segment near its amino terminus. Here we demonstrate that type XIII collagen is concentrated in cultured skin fibroblasts and several other human mesenchymal cell lines in the focal adhesions at the ends of actin stress fibers, co-localizing with the known focal adhesion components talin and vinculin. This co-occurrence was also observed in rapidly forming adhesive structures of spreading and moving fibroblasts and in disrupting focal adhesions following microinjection of the Rho-inhibitor C3 transferase into the cells, suggesting that type XIII collagen is an integral focal adhesion component. Moreover, it appears to have an adhesion-related function since cell-surface expression of type XIII collagen in cells with weak basic adhesiveness resulted in improved cell adhesion on selected culture substrata. In tissues type XIII collagen was found in a range of integrin-mediated adherens junctions including the myotendinous junctions and costameres of skeletal muscle as well as many cell–basement membrane interfaces. Some cell–cell adhesions were found to contain type XIII collagen, most notably the intercalated discs in the heart. Taken together, the results strongly suggest that type XIII collagen has a cell adhesion-associated function in a wide array of cell–matrix junctions.     

Effects of decorin and biglycan on human airway smooth muscle cell adhesion

Growth on a decorin matrix results in decreased human airway smooth muscle cell (HASMC) number, by decreasing proliferation and increasing apoptosis. We questioned whether these effects were related to abnormal extracellular matrix (ECM)-cell adhesion. HASMCs were seeded on decorin, biglycan, collagen type I or plastic. Actin organization and focal adhesion formation were assessed by staining for filamentous (F) and globular (G) actin, and vinculin, respectively. Gene expression for focal adhesion proteins, ECM molecules and HASMC receptors was measured. Protein levels for fibronectin, α(2), α(5), α(v) and β(3) integrin subunits and, focal adhesion kinase (FAK) were assessed. F-actin filaments were prominent in cells seeded on collagen I and plastic, less apparent in cells cultured on biglycan and faint in cells on decorin. Vinculin clustering was decreased in cells seeded on decorin and biglycan, as was vinculin gene expression. Compared to cells on plastic, cells on decorin had an increase in fibronectin gene expression. Seeding on decorin caused an increase in α(2) integrin subunit and platelet-derived growth factor receptor A gene expression. There was also an increase in α(2) and α(v) integrin subunit protein. Finally, FAK protein levels in cells seeded on decorin or biglycan were decreased compared to cells seeded on plastic or collagen I. Cells grown on proteoglycan matrices demonstrate evidence of abnormalities during many of the key processes involved in normal cell adhesion. Upregulation of cell surface receptor proteins, such as α(2) integrin subunit, may represent a compensatory mechanism to overcome poor adhesion induced by growth on these matrices.     

The Cytoskeleton Regulates Cell Attachment Strength

Quantitative information about adhesion strength is a fundamental part of our understanding of cell-extracellular matrix (ECM) interactions. Adhesion assays should measure integrin-ECM bond strength, but reports now suggest that cell components remain behind after exposure to acute force for radial shear assays in the presence of divalent cations that increase integrin-ECM affinity. Here, we show that focal adhesion proteins FAK, paxillin, and vinculin but not the cytoskeletal protein actin remain behind after shear-induced detachment of HT1080 fibrosarcoma cells. Cytoskeletal stabilization increased attachment strength by eightfold, whereas cross-linking integrins to the substrate only caused a 1.5-fold increase. Reducing temperature—only during shear application—also increased attachment strength eightfold, with detachment again occurring between focal adhesion proteins and actin. Detachment at the focal adhesion-cytoskeleton interface was also observed in mouse and human fibroblasts and was ligand-independent, highlighting the ubiquity of this mode of detachment in the presence of divalent cations. These data show that the cytoskeleton and its dynamic coupling to focal adhesions are critically important for cell adhesion in niche with divalent cations.     

Vinculin Is Indispensable for Repopulation by Hematopoietic Stem Cells,Independent of Integrin Function

Vinculin is a highly conserved actin-binding protein that is localized in integrin-mediated focal adhesion complexes. Although critical roles have been proposed for integrins in hematopoietic stem cell (HSC) function, little is known about the involvement of intracellular focal adhesion proteins in HSC functions. This study showed that the ability of c-Kit⁺Sca1⁺Lin⁻ HSCs to support reconstitution of hematopoiesis after competitive transplantation was severely impaired by lentiviral transduction with short hairpin RNA sequences for vinculin. The potential of these HSCs to differentiate into granulocytic and monocytic lineages, to migrate toward stromal cell-derived factor 1α, and to home to the bone marrow in vivo were not inhibited by the loss of vinculin. However, the capacities to form long term culture-initiating cells and cobblestone-like areas were abolished in vinculin-silenced c-Kit⁺Sca1⁺Lin⁻ HSCs. In contrast, adhesion to the extracellular matrix was inhibited by silencing of talin-1, but not of vinculin. Whole body in vivo luminescence analyses to detect transduced HSCs confirmed the role of vinculin in long term HSC reconstitution. Our results suggest that vinculin is an indispensable factor determining HSC repopulation capacity, independent of integrin functions.     

Leukocyte adhesion to vascular endothelium induces E-selectin linkage to the actin cytoskeleton

We have examined functions of the cytoplasmic domain of E-selectin, an inducible endothelial transmembrane protein, especially its ability to associate with the cytoskeleton during leukocyte adhesion. Confocal microscopy of interleukin-1 beta (IL-1 beta)-activated human umbilical vein endothelial cells (HUVEC) visualized clustering of E-selectin molecules in the vicinity of leukocyte-endothelial cell attachment sites. A detergent based extraction and Western blotting procedure demonstrated an association of E-selectin with the insoluble (cytoskeletal) fraction of endothelial monolayers that correlated with adhesion of leukocytes via an E-selectin-dependent mechanism. A mutant form of E-selectin lacking the cytoplasmic domain (tailless E-selectin) was expressed in COS-7 cell and supported leukocyte attachment (in a nonstatic adhesion assay) in a fashion similar to the native E-selectin molecule, but failed to become associated with the cytoskeletal fraction. To identify the cytoskeletal components that associate with the cytoplasmic domain of E-selectin, paramagnetic beads coated with the adhesion-blocking anti-E-selectin monoclonal antibody H18/7 were incubated with IL-1 beta-activated HUVEC, and then subjected to detergent extraction and magnetic separation. Certain actin-associated proteins, including alpha-actinin, vinculin, filamin, paxillin, as well as focal adhesion kinase (FAK), were copurified by this procedure, however talin was not. When a mechanical stress was applied to H18/7- coated ferromagnetic beads bound to the surface of IL-1 beta-activated HUVEC, using a magnetical twisting cytometer, the observed resistance to the applied stress was inhibited by cytochalasin D, thus demonstrating transmembrane cytoskeletal mechanical linkage. COS-7 cells transfected with the tailless E-selectin failed to show resistance to the twisting stress. Taken together, these data indicate that leukocyte adhesion to cytokine-activated HUVEC induces transmembrane cytoskeletal linkage of E-selectin through its cytoplasmic domain, a process which may have important implications for cell-cell signaling as well as mechanical anchoring during leukocyte- endothelial adhesive interactions.     

Maspin Regulates Endothelial Cell Adhesion and Migration through an Integrin Signaling Pathway

Maspin has been identified as a potent angiogenesis inhibitor. However, the molecular mechanism responsible for its anti-angiogenic property is unclear. In this study, we examined the effect of maspin on endothelial cell (EC) adhesion and migration in a cell culture system. We found that maspin was expressed in blood vessels ECs and human umbilical vein endothelial cells (HUVECs). Maspin significantly enhanced HUVEC cell adhesion to various matrix proteins. This effect was dependent on the activation of integrin β₁, which subsequently led to distribution pattern changes of vinculin and F-actin. These results indicated that maspin affects cell adhesion and cytoskeleton reorganization through an integrin signal transduction pathway. Analysis of HUVECs following maspin treatment revealed increased integrin-linked kinase activities and phosphorylated FAK levels, consistent with increased cell adhesion. Interestingly, when HUVECs were induced to migrate by migration stimulatory factor bFGF, active Rac1 and cdc42 small GTPase levels were decreased dramatically at 30 min following maspin treatment. Using phosphorylated FAK at Tyr³⁹⁷ as an indicator of focal adhesion disassembly, maspin-treated HUVECs had elevated FAK phosphorylation compared with the mock treated control. The results were a reduction in focal adhesion disassembly and the retardation in EC migration. This study uncovers a mechanism by which maspin exerts its effect on EC adhesion and migration through an integrin signal transduction pathway.     

Organization of the cytoskeleton and the focal contacts of bovine aortic endothelial cells cultured on type I and III collagen.

We investigated the organization of the cytoskeleton and the focal contacts of bovine aortic endothelial cells cultured on type I and III collagen. The influence of these collagens on cell morphology and the distribution pattern of actin, vimentin, talin, and vinculin was analyzed by light microscopy, conventional electron microscopy, immunofluorescence, and immunogold labeling after lysis-squirting. Whereas the morphology of the endothelial cells is not markedly influenced, the structure of the cytoskeleton and the focal contacts of the cells are altered by the different collagen types. Stress fibers are more distinct in cells grown on type I collagen; cells on type III collagen show a more diffuse distribution of actin molecules. Intermediate filaments seem not to be affected by the collagens. The areas of focal contacts are larger in cells on type I collagen. Additionally, the labeling pattern of talin and vinculin is denser in focal contacts of cells grown on type I collagen. These results suggest an important role of the type of collagen in mediation of the organization of the microfilament system and the adhesion structures of bovine aortic endothelial cells in culture.     

Synthetic peptide GRGDS induces dissociation of alpha-actinin and vinculin from the sites of focal contacts

The synthetic peptide Gly-Arg-Gly-Asp-Ser (GRGDS) mimics the cellular binding site of many adhesive proteins in the extracellular matrix and causes rounding and detachment of spread cells. We have studied whether its binding affects the associations of two major components, alpha-actinin and vinculin, at the adhesion plaque. Living 3T3 cells were microinjected with fluorescently labeled alpha-actinin and/or vinculin and observed using video microscopy before and after the addition of 50 micrograms/ml GRGDS. As soon as 5 min after treatment, fluorescent alpha-actinin and vinculin became dissociated simultaneously from the sites of many focal contacts. The proteins either moved away as discrete structures or dispersed from adhesion plaques. As a result, the enrichment of alpha-actinin and vinculin at these focal contacts was no longer detected. The focal contacts then faded away slowly without showing detectable movement. These data suggest that the binding state of integrin has a transmembrane effect on the distribution of cytoskeletal components. The dissociation of alpha-actinin and vinculin from adhesion plaques may in turn weaken the contacts and result in rounding and detachment of cells.     

Identification of LIM3 as the principal determinant of paxillin focal adhesion localization and characterization of a novel motif on paxillin directing vinculin and focal adhesion kinase binding

Paxillin is a 68-kD focal adhesion phosphoprotein that interacts with several proteins including members of the src family of tyrosine kinases, the transforming protein v-crk, and the cytoskeletal proteins vinculin and the tyrosine kinase, focal adhesion kinase (FAK). This suggests a function for paxillin as a molecular adaptor, responsible for the recruitment of structural and signaling molecules to focal adhesions. The current study defines the vinculin- and FAK-interaction domains on paxillin and identifies the principal paxillin focal adhesion targeting motif. Using truncation and deletion mutagenesis, we have localized the vinculin-binding site on paxillin to a contiguous stretch of 21 amino acids spanning residues 143-164. In contrast, maximal binding of FAK to paxillin requires, in addition to the region of paxillin spanning amino acids 143-164, a carboxyl-terminal domain encompassing residues 265-313. These data demonstrate the presence of a single binding site for vinculin, and at least two binding sites for FAK that are separated by an intervening stretch of 100 amino acids. Vinculin- and FAK-binding activities within amino acids 143-164 were separable since mutation of amino acid 151 from a negatively charged glutamic acid to the uncharged polar residue glutamine (E151Q) reduced binding of vinculin to paxillin by >90%, with no reduction in the binding capacity for FAK. The requirement for focal adhesion targeting of the vinculin- and FAK-binding regions within paxillin was determined by transfection into CHO.K1 fibroblasts. Significantly and surprisingly, paxillin constructs containing both deletion and point mutations that abrogate binding of FAK and/or vinculin were found to target effectively to focal adhesions. Additionally, expression of the amino-terminal 313 amino acids of paxillin containing intact vinculin- and FAK-binding domains failed to target to focal adhesions. This indicated other regions of paxillin were functioning as focal adhesion localization motifs. The carboxyl-terminal half of paxillin (amino acids 313-559) contains four contiguous double zinc finger LIM domains. Transfection analyses of sequential carboxyl-terminal truncations of the four individual LIM motifs and site-directed mutagenesis of LIM domains 1, 2, and 3, as well as deletion mutagenesis, revealed that the principal mechanism of targeting paxillin to focal adhesions is through LIM3. These data demonstrate that paxillin localizes to focal adhesions independent of interactions with vinculin and/or FAK, and represents the first definitive demonstration of LIM domains functioning as a primary determinant of protein subcellular localization to focal adhesions.     

Hepatic stellate cells (vitamin A-storing cells) change their cytoskeleton structure by extracellular matrix components through a signal transduction system

 When cultured on a polystyrene surface or aminoalkylsilane-coated cover glasses, rat and human hepatic stellate cells exhibit a flattened, fibroblast-like shape with well-developed stress fibers. However, culturing the cells on type I collagen gel results in the elongation of long, multipolar cellular processes, whereas cells cultured on Matrigel maintain their round shapes. Dual fluorescence staining of microtubules and fibrillar actin indicated that the processes extend together with collagen fibers and contained microtubules as the core, whereas the periphery contained fibrillar actin. Immunofluorescence staining of vinculin showed that the focal adhesions were distributed mainly in lamellipodia when cultured on aminoalkylsilane-coated cover glasses, whereas in the cells cultured on type I collagen gel they were localized to the tips of the processes and along their bottom surface contacting collagen fibers. Wortmannin, as well as staurosporin and herbimycin A, inhibited the elongation process and induced the retraction of elongated processes. The wortmannin treatment also resulted in an alteration in focal adhesion distribution from the processes to cell bodies. These results indicate that the cell surface integrin binding to interstitial collagen fibers induces the elongation of processes through signaling events and the subsequent cytoskeleton assembly in hepatic stellate cells. Accepted: 12 February 1998     

Tumorigenesis of rat mammary epithelial cells by N-nitroso-N-methylurea in anin vitro system: Characterization of the microtumors

Summary Chemical carcinogenesis is a lengthy process that involves the rather loosely defined stages of initiation, promotion, and progression. Several model systems of mammary carcinogenesis have been designed to elucidate the mechanisms of chemical carcinogenesis. Most of these systems have included animal models. While organ specific chemical carcinogenesis can be initiated in these systems, the subsequent stages of promotion and progression are difficult to study in detail. Investigations onin vitro carcinogenesis have shown transformation of mammalian cells in culture; the transformational event, however, is difficult to discern within the monolayer culture. We have recently reported the development of anin vitro carcinogenesis system that allows both the initiation as well as the progression of mammary cells in a collagen gel matrix culture system. The cells transformed by a chemical carcinogen develop into discernible microtumors with the three dimensions of a collagen gel culture. Isolation of these microtumors from the collagen gel an subsequent culture in monolayer has produced cells capable of colony formation in soft agar. The present study further characterizes these microtumors originatedin vitro by analysis of cell growth kinetics versus parallel control cells. In addition, flow cytometric and cytogenetic studies have been performed to investigate the chromosomal stability of these cells. It was also observed that the microtumors, producedin vitro from mammary epithelial cells of an inbred strain of rats, show the ability to form tumors upon transplantation into the fat pad of syngeneic hosts.     

Tyrosine kinase activity, cytoskeletal organization, and motility in human vascular endothelial cells.

Tyrosine phosphorylation of cytoskeletal proteins occurs during integrin-mediated cell adhesion to extracellular matrix proteins. We have investigated the role of tyrosine phosphorylation in the migration and initial spreading of human umbilical vein endothelial cells (HUVEC). Elevated phosphotyrosine concentrations were noted in the focal adhesions of HUVEC migrating into wounds. Anti-phosphotyrosine Western blots of extracts of wounded HUVEC monolayers demonstrated increased phosphorylation at 120-130 kDa when compared with extracts of intact monolayers. The pp125FAK immunoprecipitated from wounded monolayers exhibited increased kinase activity as compared to pp125FAK from intact monolayers. The time to wound closure in HUVEC monolayers was doubled by tyrphostin AG 213 treatment. The same concentration of AG 213 interfered with HUVEC focal adhesion and stress fiber formation. AG 213 inhibited adhesion-associated tyrosine phosphorylation of pp125FAK in HUVEC. Tyrphostins AG 213 and AG 808 inhibited pp125FAK activity in in vitro kinase assays. pp125FAK immunoprecipitates from HUVEC treated with both of these inhibitors also had kinase activity in vitro that was below levels seen in untreated HUVEC. These findings suggest that tyrosine phosphorylation of cytoskeletal proteins may be important in HUVEC spreading and migration and that pp125FAK may mediate phosphotyrosine formation during these processes.     

Kinetic determination of focal adhesion protein formation

I examined the binding kinetics between integrin (alpha(IIb)beta(3)) and purified focal adhesion proteins, including alpha-actinin, filamin, vinculin, talin, and F-actin. Using static light-scatter technique, I observed affinities of the order talin > filamin > F-actin > alpha-actinin > (talin when bound to vinculin) which were lower when integrin was complexed with fibronectin. No binding between integrin and vinculin was detected. The calculated dissociation constants (K(d)) ranged between 0.4 microM and 5 microM. These results in part confirm previously published data using different methods. The modest affinity with which the focal adhesion proteins interact in vitro might be indicative of how cells, e.g., thrombocytes, gain a high degree of versatility and velocity.