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  • Vinculin promotes cell spreading by mechanically coupling integrins to the cytoskeleton. 9056408

    Mouse F9 embryonic carcinoma 5.51 cells that lack the cytoskeletal protein vinculin spread poorly on extracellular matrix compared with wild-type F9 cells or two vinculin-transfected clones (5.51Vin3 and Vin4; Samuels et al., 1993, J. Cell Biol. 121, 909-921). In the present study, we used this model system to determine how the presence of vinculin promotes cytoskeletal alterations and associated changes in cell shape. Microscopic analysis of cell spreading at early times, revealed that 5.51 cells retained the ability to form filopodia; however, they could not form lamellipodia, assemble stress fibers, or efficiently spread over the culture substrate. Detergent (Triton X-100) studies revealed that these major differences in cell morphology and cytoskeletal organization did not result from differences in levels of total polymerized or cross-linked actin. Biochemical studies showed that 5.51 cells, in addition to lacking vinculin, exhibited slightly reduced levels of alpha-actinin and paxillin in their detergent-insoluble cytoskeleton. The absence of vinculin correlated with a decrease in the mechanical stiffness of the integrin-cytoskeleton linkage, as measured using cell magnetometry. Furthermore, when vinculin was replaced by transfection in 5.51Vin3 and 5.51Vin4 cells, the levels of cytoskeletal-associated alpha-actinin and paxillin, the efficiency of transmembrane mechanical coupling, and the formation of actin stress fibers were all restored to near wild-type levels. These findings suggest that vinculin may promote cell spreading by stabilizing focal adhesions and transferring mechanical stresses that drive cytoskeletal remodeling, rather than by altering the total level of actin polymerization or cross-linking.
    Document Type:
    Reference
    Product Catalog Number:
    MAB3060
    Product Catalog Name:
    Anti-Paxillin Antibody, clone 349

  • Requirement of the integrin beta 3 subunit for carcinoma cell spreading or migration on vitronectin and fibrinogen. 1374415

    FG human pancreatic carcinoma cells use integrin alpha v beta 5 as their primary vitronectin receptor since they fail to express integrin alpha v beta 3. These cells are unable to form focal contacts, spread, or migrate on vitronectin but readily do so on collagen in a beta 1 integrin-dependent manner. Transfection of FG cells with a cDNA encoding the integrin beta 3 subunit results in the surface expression of a functional integrin alpha v beta 3 heterodimer providing these cells with novel adhesive and biological properties. Specifically, FG cells expressing beta 3 acquire the capacity to attach and spread on vitronectin as well as fibrinogen with beta 3 localization to focal contacts. Moreover, these cells gain the capacity to migrate through a porous membrane in response to either vitronectin or fibrinogen. These results demonstrate that the beta 3 and beta 5 integrin subunits when associated with alpha v, promote distinct cellular responses to a vitronectin extracellular environment.
    Document Type:
    Reference
    Product Catalog Number:
    AB1926
    Product Catalog Name:
    Anti-Integrin β5 Antibody, CT/cytosolic

  • Rap1 can bypass the FAK-Src-Paxillin cascade to induce cell spreading and focal adhesion formation. 23209645

    We developed new image analysis tools to analyse quantitatively the extracellular-matrix-dependent cell spreading process imaged by live-cell epifluorescence microscopy. Using these tools, we investigated cell spreading induced by activation of the small GTPase, Rap1. After replating and initial adhesion, unstimulated cells exhibited extensive protrusion and retraction as their spread area increased, and displayed an angular shape that was remodelled over time. In contrast, activation of endogenous Rap1, via 007-mediated stimulation of Epac1, induced protrusion along the entire cell periphery, resulting in a rounder spread surface, an accelerated spreading rate and an increased spread area compared to control cells. Whereas basal, anisotropic, spreading was completely dependent on Src activity, Rap1-induced spreading was refractory to Src inhibition. Under Src inhibited conditions, the characteristic Src-induced tyrosine phosphorylations of FAK and paxillin did not occur, but Rap1 could induce the formation of actomyosin-connected adhesions, which contained vinculin at levels comparable to that found in unperturbed focal adhesions. From these results, we conclude that Rap1 can induce cell adhesion and stimulate an accelerated rate of cell spreading through mechanisms that bypass the canonical FAK-Src-Paxillin signalling cascade.
    Document Type:
    Reference
    Product Catalog Number:
    MAB374
    Product Catalog Name:
    Anti-Glyceraldehyde-3-Phosphate Dehydrogenase Antibody, clone 6C5

  • Use of an Immobilized Monoclonal Antibody to Examine Integrin alpha5beta1 Signaling Independent of Cell Spreading. 12734565

    Cell attachment to the extracellular matrix (ECM) engages integrin signaling into the cell, but part of the signaling response also stem from cell spreading (3). To analyze specific integrin signaling-mediated responses independent of cell spreading, we developed a method engaging integrin signaling by use of an immobilized anti-integrin monoclonal antibody (mab) directed against the fibronectin (FN) receptor integrin alpha5beta1. ECV 304 cells were plated onto FN or immobilized mab JBS5 (anti-integrin alpha5beta1) or onto poly-L-lysin (P-L-L), which mediates integrin-independent attachment. Cells attached and spread on FN, while cells on JBS5 or P-L-L attached but did not spread. Importantly, plating onto FN or mab JBS5 gave rise to identical integrin-induced responses, including a down-regulation of the cyclin-dependent kinase (Cdk2) inhibitors p21(CIP1) and p27(KIP1), while attachment to P-L-L did not. We conclude that engagement of the FN-receptor integrin alpha5beta1 induces integrin signaling regulating the Cdk2-inhibitors independent of cell spreading and present a method for how integrin signaling can be analyzed separate from the effects of cell spreading.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Filamin links cell shape and cytoskeletal structure to Rho regulation by controlling accumulation of p190RhoGAP in lipid rafts. 17227794

    Cytoskeleton-dependent changes in the activity of the small GTPase Rho mediate the effects of cell shape on cell function; however, little is known about how cell spreading and related distortion of the cytoskeleton regulate Rho activity. Here we show that rearrangements of the actin cytoskeleton associated with early phases of cell spreading in human microvascular endothelial (HMVE) cells suppress Rho activity by promoting accumulation of p190RhoGAP in lipid rafts where it exerts its Rho inhibitory activity. p190RhoGAP is excluded from lipid rafts and Rho activity increases when cell rounding is induced or the actin cytoskeleton is disrupted, and p190RhoGAP knockdown using siRNA prevents Rho inactivation by cell spreading. Importantly, cell rounding fails to prevent accumulation of p190RhoGAP in lipid rafts and to increase Rho activity in cells that lack the cytoskeletal protein filamin. Moreover, filamin is degraded in spread cells and cells that express a calpain-resistant form of filamin exhibit high Rho activity even when spread. Filamin may therefore represent the missing link that connects cytoskeleton-dependent changes of cell shape to Rho inactivation during the earliest phases of cell spreading by virtue of its ability to promote accumulation of p190RhoGAP in lipid rafts.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Low Mr phosphotyrosine protein phosphatase associates and dephosphorylates p125 focal adhesion kinase, interfering with cell motility and spreading. 12055185

    Low M(r) phosphotyrosine protein phosphatase interferes in vivo with the activation of several growth factor receptors and is transiently redistributed, following cell stimulation with platelet-derived growth factor, from the cytosol to the cytoskeleton. We demonstrate here that this phosphatase also participates in the regulation of cell spreading and migration, pointing to its involvement in cytoskeleton organization. Low M(r) phosphotyrosine protein phosphatase-overexpressing fibroblasts are, indeed, less spread than controls and display a significantly decreased number of focal adhesions and increased cell motility. Furthermore, p125 focal adhesion kinase is associated to, and dephosphorylated by, low M(r) phosphotyrosine protein phosphatase both in vitro and in vivo. This event is consistent with an altered association of pp60(src) with focal adhesion kinase. The activation of extracellular signal-regulated kinase, another well known event downstream of the focal adhesion kinase, is also affected. On the other hand, cells overexpressing the dominant-negative form of low M(r) phosphotyrosine protein phosphatase exhibit hyperphosphorylated focal adhesion kinase, reduced motility, and an increased number of focal adhesions, which are distributed all over the ventral cell surface. Taken together, the results reported here are in keeping with low M(r) phosphotyrosine protein phosphatase participation in FAK-mediated focal adhesion remodeling.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple
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