Interaction of iodinated vinculin, metavinculin and α-actinin with cytoskeletal proteins

Iodinated vinculin, metavinculin and α-actinin were used to probe the interaction of these proteins with electrophoretically separated cytoskeletal proteins. Using the gel overlay technique, we detected strong binding of ¹²⁵I-vinculin and ¹²⁵I-metavinculin to α-actinin, 175 kDa polypeptide, talin, vinculin and metavinculin themselves, and moderate binding to actin.¹²⁵I-α-actinin was capable of interacting with vinculin and metavinculin. The specific binding of ¹²⁵-I-α-actinin to vinculin and metavinculin immobilized on a polysterene surface was also demonstrated. We suggest that the ability of vinculin and α-actinin to form a complex may be realized in microfilament-membrane linkages.     

Serine phosphorylation on position 1033 of vinculin impacts cellular mechanics

This study evaluates the influence of S1033 vinculin phosphorylation on the mechanical properties of cells. We demonstrate that MEFvcl KO cells transfected with the non-phosphorylatable eGFP-vinculin mutant S1033A are of lower stiffness compared to MEFvcl Rescue and phospho-mimicking mutant S1033D cells, which were of similar stiffness. Analogous, 2D traction microscopy indicates that MEFvcl Rescue and MEF mutant S1033D cells generate similar strain energy, but mutant S1033A cells display ∼50% less strain energy. Fluorescence recovery after photobleaching demonstrates that the recovery time for mutant S1033A was significantly lower compared to MEFvcl Rescue and mutant S1033D and that the mobile fraction was smaller for MEFvcl Rescue and mutant S1033D than for mutant S1033A cells. This indicates that serine phosphorylation is required for the activation of vinculin and force transmission in focal adhesions.     

A theoretical model for F-actin remodeling in vascular smooth muscle cells subjected to cyclic stretch

A constrained mixture theory model was developed and used to estimate remodeling of F-actin in vascular smooth muscle cells that were subjected to 10% equibiaxial stretching for up to 30min. The model was based on a synthesis of data on time-dependent changes in atomic force microscopy measured cell stiffness and immunofluorescence measured focal adhesion associated vinculin as well as data on stress fiber stiffness and pre-stretch. Results suggest that an observed acute (after 2min of stretching) increase in cell stiffness is consistent with an increased stretch of the originally present F-actin plus an assembly of new F-actin having nearly homeostatic values of stretch. Moreover, the subsequent (after 30min of stretching) decrease in cell stiffness back towards the baseline value is consistent with a replacement of the overstretched original filaments with the new (reassembled), less stretched filaments. That is, overall cell response is consistent with a recently proposed concept of "tensional homeostasis" whereby cells seek to maintain constant certain mechanical factors via a remodeling of intracellular and transmembrane proteins. Although there is a need to refine the model based on more comprehensive data sets, using multiple experimental approaches, the present results suggest that a constrained mixture theory can capture salient features of the dynamics of F-actin remodeling and that it offers some advantages over many past methods of modeling, particularly those based on classical linearized viscoelasticity.     

Myocilin binding to Hep II domain of fibronectin inhibits cell spreading and incorporation of paxillin into focal adhesions

Myocilin, a novel matricellular protein found in the human eye, can modify signaling events mediated by the Heparin II domain of fibronectin. Using myocilin produced in sf9 insect cells, myocilin inhibited spreading of cycloheximide-treated human skin fibroblasts plated on substrates co-coated with myocilin and either fibronectin or its Heparin II domain. Cell spreading could be rescued by adding back either substrate adsorbed or soluble Heparin II domains. Myocilin did not inhibit cell attachment to fibronectin even in the presence of a 2400 M excess of myocilin. Myocilin impaired focal adhesion formation and specifically blocked the incorporation of paxillin, but not vinculin, into focal adhesions. The Heparin II domain mediated the incorporation of paxillin into focal adhesions, since paxillin was not assembled into focal adhesions unless the Heparin II domain was present. The effect of myocilin on focal adhesions could be overcome by treating cells with either phorbol 12-myristate (PMA) or oleoyl-L-alpha-lysophosphatidic acid (LPA). Myocilin bound to the fibroblast cell surface, but its binding could not be competed with excess fibronectin, suggesting that myocilin does not compete for cell surface binding sites of fibronectin. Myocilin therefore appears to specifically block functions mediated by the Heparin II domain possibly through direct interactions with it.     

Central Region of Talin Has a Unique Fold That Binds Vinculin and Actin

Talin is an adaptor protein that couples integrins to F-actin. Structural studies show that the N-terminal talin head contains an atypical FERM domain, whereas the N- and C-terminal parts of the talin rod include a series of α-helical bundles. However, determining the structure of the central part of the rod has proved problematic. Residues 1359–1659 are homologous to the MESDc1 gene product, and we therefore expressed this region of talin in Escherichia coli. The crystal structure shows a unique fold comprised of a 5- and 4-helix bundle. The 5-helix bundle is composed of nonsequential helices due to insertion of the 4-helix bundle into the loop at the C terminus of helix α3. The linker connecting the bundles forms a two-stranded anti-parallel β-sheet likely limiting the relative movement of the two bundles. Because the 5-helix bundle contains the N and C termini of this module, we propose that it is linked by short loops to adjacent bundles, whereas the 4-helix bundle protrudes from the rod. This suggests the 4-helix bundle has a unique role, and its pI (7.8) is higher than other rod domains. Both helical bundles contain vinculin-binding sites but that in the isolated 5-helix bundle is cryptic, whereas that in the isolated 4-helix bundle is constitutively active. In contrast, both bundles are required for actin binding. Finally, we show that the MESDc1 protein, which is predicted to have a similar fold, is a novel actin-binding protein.     

Backbone 1H, 13C, and 15N NMR assignments of the tail domain of vinculin

Vinculin is an essential protein involved in linking the actin cytoskeleton to sites of cell-cell and cell-matrix adhesion. Here we report the majority of the backbone ¹H_N, ¹⁵N, ¹³C_α, ¹³CO, and side chain ¹³C_β NMR resonance assignments of the actin binding tail domain of vinculin (Vt).     

Early stages of endothelial wound repair: conversion of quiescent to migrating endothelial cells involves tyrosine phosphorylation and actin microfilament reorganization

Endothelial repair to reestablish structural integrity following wounding is a complex process. Since the actin cytoskeleton undergoes specific changes in distribution as quiescent endothelial cells switch to activated migrating cells over a 6-h period following wounding (Lee et al. 1996), we studied tyrosine phosphorylation in association with actin microfilaments and adhesion proteins using double immunofluorescent confocal microscopy. We showed that in a confluent monolayer phosphotyrosine localized at the periphery of the cell at vinculin cell-cell adhesion sites within the actin-dense peripheral band (DPB) and centrally at talin/vinculin cell-substratum adhesion sites at the ends of central microfilaments. Over a period of 6 h following in vitro wounding there was a reduction of peripheral phosphotyrosine associated with the loss of both cell-cell adhesion sites and the DPB (stage I). Concomitantly, an increase in central phosphotyrosine was associated with an increase in cell-substratum adhesion sites and central microfilaments parallel to the wound edge (stage II), which subsequently redistributed perpendicular to the wound edge (stage III). We also localized FAK and paxillin at the ends of parallel and perpendicular central microfilaments. Immunoprecipitation of paxillin showed increased phosphotyrosine and protein levels when prominent central microfilaments were present and underwent remodeling. Inhibition of tyrosine kinases by genistein and tyrosine phosphatases by sodium orthovanadate resulted in reduced endothelial repair associated with disruption of adhesion site formation and central microfilament formation/redistribution in each stage of repair. We suggest that tyrosine phosphorylation of adhesion proteins, such as paxillin, may be important in regulating the early stages of endothelial wound repair. Received: 22 March 1999 / Accepted: 24 March 1999     

Relationship between the organization of actin bundles and vinculin plaques

Summary The temporal pattern of the formation and dissolution of vinculin patches during experimental manipulation of the state of actin within the cell was studied. Cytochalasin D-induced retraction and disappearance of stress fibers is followed, with a brief delay, by the dissolution of vinculin-containing patches and the coordinated redistribution of both actin and vinculin into newly formed amorphous aggregates or foci. Recovery from cytochalasin treatment begins with a transformation of these foci into doughnut-shaped assemblies in which actin and vinculin are precisely co-localized. The emergence and growth of filament bundles is paralleled by the appearance of faint vinculin patches that gradually increase in size in parallel with the stress fibers. If stress fibers are stabilized by microinjected rhodamine-phalloidin against stimuli that normally induce a coordinated redistribution of actin and vinculin, also the vinculin patches persist. These observations indicate that treatments influencing the state of actin in the cell have corresponding effects on the stability of vinculin patches and suggest a strong interdependency of actin and vinculin organization.     

Identification and isolation of vinculin from platelets

A vinculin-like protein was identified in chicken as well as in bovine platelets by ELISA competitive binding assay using antibodies against vinculin from chicken gizzard. By a modified procedure (J. Biol. Chem. (1980) 255, 1194–1199) we succeeded in isolating bovine platelet vinculin to apparent homogeneity. The structural identity of platelet and chicken gizzard vinculin was demonstrated by circular dichroism analysis. It was also shown that platelet vinculin induces a significant decrease in the low shear viscosity of F-actin. Vinculin, in all probability, plays an important role in the organization of actin filaments in platelets, especially in the linkages of microfilaments to the membrane.