Role Of Vinculin Research Articles

Mechanical regulation of vinculin conformations and interactions.

Vinculin, as an essential mechanosensitive protein, transmits intracellular tension initiated from the cell-cell and the cell-matrix interfaces, to modulate cellular and tissue functions. However, the tension-dependent conformations and interactions of vinculin are largely unknown. Vinculin provides the mechanical linkages between talin and actin at Focal adhesions, or alpha-catenin and actin at Adherens junctions. In drosophila, vinculin also functions at the muscle attachment sites with Cbl-associated proteins (CAPs) to regulate the cytoskeletal organization and membrane morphology. In this work, we combined single-molecule manipulation, AlphaFold2-based structural prediction, molecular dynamic (MD) simulation and theoretical modelling to understand the tension-dependent conformational changes of drosophila vinculin domains and its interaction with drosophila CAP isoform E (dCAP-E). We reveal that vinculin domains unfold in discrete steps, within 5-15 pN at a physiologically relevant tension loading rate. Based on the structural conformations predicted by AlphaFold2 and confirmed by MD simulation, a theoretical model is developed which predicts a tension-dependent binding affinity between the drosophila vinculin linker and dCAP-E within the physiologically relevant tension range. Together, the results suggest two roles of vinculin, as a tension buffering structure via tension-dependent unfolding and refolding of domains and as a mechanosensor via tension-dependent binding with its binding partners.

Adhesion strength and anti-tumor agents regulate vinculin of breast cancer cells.

The onset and progression of cancer are strongly associated with the dissipation of adhesion forces between cancer cells, thus facilitating their incessant attachment and detachment from the extracellular matrix (ECM) to move toward metastasis. During this process, cancer cells undergo mechanical stresses and respond to these stresses with membrane deformation while inducing protrusions to invade the surrounding tissues. Cellular response to mechanical forces is inherently related to the reorganization of the cytoskeleton, the dissipation of cell–cell junctions, and the adhesion to the surrounding ECM. Moreover, the role of focal adhesion proteins, and particularly the role of vinculin in cell attachment and detachment during migration, is critical, indicating the tight cell–ECM junctions, which favor or inhibit the metastatic cascade. The biomechanical analysis of these sequences of events may elucidate the tumor progression and the potential of cancer cells for migration and metastasis. In this work, we focused on the evaluation of the spreading rate and the estimation of the adhesion strength between breast cancer cells and ECM prior to and post-treatment with anti-tumor agents. Specifically, different tamoxifen concentrations were used for ER+ breast cancer cells, while even concentrations of trastuzumab and pertuzumab were used for HER2+ cells. Analysis of cell stiffness indicated an increased elastic Young’s modulus post-treatment in both MCF-7 and SKBR-3 cells. The results showed that the post-treatment spreading rate was significantly decreased in both types of breast cancer, suggesting a lower metastatic potential. Additionally, treated cells required greater adhesion forces to detach from the ECM, thus preventing detachment events of cancer cells from the ECM, and therefore, the probability of cell motility, migration, and metastasis was confined. Furthermore, post-detachment and post-treatment vinculin levels were increased, indicating tighter cell–ECM junctions, hence limiting the probability of cell detachment and, therefore, cell motility and migration.

Vinculin-mediated axon growth requires interaction with actin but not talin in mouse neocortical neurons.

The actin-binding protein vinculin is a major constituent of focal adhesion, but its role in neuronal development is poorly understood. We found that vinculin deletion in mouse neocortical neurons attenuated axon growth both in vitro and in vivo. Using functional mutants, we found that expression of a constitutively active vinculin significantly enhanced axon growth while the head-neck domain had an inhibitory effect. Interestingly, we found that vinculin-talin interaction was dispensable for axon growth and neuronal migration. Strikingly, expression of the tail domain delayed migration, increased branching, and stunted axon. Inhibition of the Arp2/3 complex or abolishing the tail domain interaction with actin completely reversed the branching phenotype caused by tail domain expression without affecting axon length. Super-resolution microscopy showed increased mobility of actin in tail domain expressing neurons. Our results provide novel insights into the role of vinculin and its functional domains in regulating neuronal migration and axon growth.

Context-Dependent Role of Vinculin in Neutrophil Adhesion, Motility and Trafficking

Neutrophils are innate immune effector cells that traffic from the circulation to extravascular sites of inflammation. β2 integrins are important mediators of the processes involved in neutrophil recruitment. Although neutrophils express the cytoskeletal protein vinculin, they do not form mature focal adhesions. Here, we characterize the role of vinculin in β2 integrin-dependent neutrophil adhesion, migration, mechanosensing, and recruitment. We observe that knockout of vinculin attenuates, but does not completely abrogate, neutrophil adhesion, spreading, and crawling under static conditions. However, we also found that vinculin deficiency does not affect these behaviors in the presence of forces from fluid flow. In addition, we identify a role for vinculin in mechanosensing, as vinculin-deficient neutrophils exhibit attenuated spreading on stiff, but not soft, substrates. Consistent with these findings, we observe that in vivo neutrophil recruitment into the inflamed peritoneum of mice remains intact in the absence of vinculin. Together, these data suggest that while vinculin regulates some aspects of neutrophil adhesion and spreading, it may be dispensable for β2 integrin-dependent neutrophil recruitment in vivo.

Vinculins interaction with talin is essential for mammary epithelial differentiation

Vinculin is an essential component of cell adhesion complexes, where it regulates the strength and stability of adhesions. Whilst the role of vinculin in cell motility is well established, it remains unclear how vinculin contributes to other aspects of tissue function. Here we examine the role of vinculin in mammary epithelial cell phenotype. In these cells, correct adhesion to the extracellular matrix is essential for both the formation of polarised secretory acini and for the transcription of tissue-specific milk protein genes. We show that vinculin, through its interaction with talin, controls milk protein gene expression. However, vinculin is not required for the formation of polarised acini. This work reveals new roles for vinculin that are central to cellular differentiation, and for the ability of cells to interpret their extracellular microenvironment.

Abstract 77: The Role of Vinculin in Cardiomyocyte Adhesion and Mechanical Continuity

Cardiomyocytes are joined end-to-end by a complex adhesive structure known as the intercalated disc (ICD). The ICD is composed of mechanical and electrical junctional complexes including the adherens junction (AJ). The core of the AJ is the cadherin/catenin complex, which links the actin cytoskeletons of neighboring cells to provide mechanical continuity. Myofibrils – the contractile machinery of the heart – are coupled to the ICD, presumably through the AJ; however, this mechanical linkage is poorly defined. Tension applied to the AJ is sensed in part through alpha-catenin, which connects the cadherin/catenin complex to actin. Force alters the conformation of alpha-catenin to reveal binding sites for actin-binding ligands, including vinculin and afadin, which are both found at the ICD. We investigated the role of alpha-catenin and its ligands in linking the AJ to actin in cardiomyocytes. Using a Förester resonance energy transfer tension-sensing module inserted into N-cadherin, we found that N-cadherin is under tension at the ICD. We then measured protein mobility of EGFP-tagged N-cadherin and found that N-cadherin dynamics were reduced at sites of myofibril integration, suggesting that myofibrils stabilize N-cadherin. To examine the composition of AJs in cardiomyocytes, we used immunofluorescence and found that vinculin and afadin were preferentially recruited to myofibril integration sites at the ICD. To further examine the linkage between AJs and myofibrils, we built a series of EGFP-tagged N-cadherin:alpha-catenin fusion constructs to delineate the individual and combined roles of alpha-catenin, vinculin and afadin. A constitutively active alpha-catenin increased vinculin recruitment and decreased the mobility of the N-cadherin fusion, suggesting that vinculin recruitment stabilizes the AJ. In contrast, restricting the AJ-actin interface to afadin reduced stability, indicating afadin alone cannot replace vinculin in linking AJs to the myofibril network. Together, our data support a model in which alpha-catenin, vinculin and afadin cooperate to anchor myofibrils at the ICD to provide mechanical continuity between cells.

Role of vinculin in cellular mechanotransduction.

Cell-matrix adhesion and cell-cell contacts are essential for the metabolism, protein synthesis, survival, and cancer metastasis of cells. Major transmembrane receptors are the integrins, which are responsible for cell-matrix adhesions, and the cadherins, which are important for cell-cell adhesions. Adherent cells anchor via focal adhesion proteins to the extracellular matrix, whereas cell-cell contacts connect via focal adherens junction proteins. The temporal formation of these connections is greatly strengthened either through externally applied stresses on the cell or by myosin-driven cell contractility. The mechanism by which protein(s) within these connections sense, transmit, and respond to mechanochemical signaling is currently strongly debated and various candidates have been named. Vinculin has been described as one of the key players in cell-matrix and cell-cell adhesions that build a strong physical connection for transmitting forces between the cytoskeleton, the extracellular matrix, and cell-cell connections.

Novel Role for Vinculin in Ventricular Myocyte Mechanics and Dysfunction

Vinculin (Vcl) plays a key structural role in ventricular myocytes that, when disrupted, can lead to contractile dysfunction and dilated cardiomyopathy. To investigate the role of Vcl in myocyte and myocardial function, cardiomyocyte-specific Vcl knockout mice (cVclKO) and littermate control wild-type mice were studied with transmission electron microscopy (TEM) and in vivo magnetic resonance imaging (MRI) tagging before the onset of global ventricular dysfunction. MRI revealed significantly decreased systolic strains transverse to the myofiber axis in vivo, but no changes along the muscle fibers or in fiber tension in papillary muscles from heterozygous global Vcl null mice. Myofilament lattice spacing from TEM was significantly greater in cVclKO versus wild-type hearts fixed in the unloaded state. AFM in Vcl heterozygous null mouse myocytes showed a significant decrease in membrane cortical stiffness. A multiscale computational model of ventricular mechanics incorporating cross-bridge geometry and lattice mechanics showed that increased transverse systolic stiffness due to increased lattice spacing may explain the systolic wall strains associated with Vcl deficiency, before the onset of ventricular dysfunction. Loss of cardiac myocyte Vcl may decrease systolic transverse strains in vivo by decreasing membrane cortical tension, which decreases transverse compression of the lattice thereby increasing interfilament spacing and stress transverse to the myofibers.

Role of Vinculin in Ventricular Wall Function

Vinculin (Vcl) is a ubiquitously expressed cytoskeletal protein that localizes at cell adhesion sites in cardiomyocytes. It is a key component in the costamere, which anchors the sarcomeric cytoskeleton to the extracellular matrix via integrins. It has been previously shown that it plays a significant role in mechanotransduction and may affect myocardial mechanical function in a directional-dependent manner. To determine the role of Vcl in regional wall mechanics, cardiac MRI tagging was performed in a cardiac-myocyte-specific vinculin knockout (VclKO) mouse model revealing significant decreases in systolic sheet-normal shear strain (P<0.05) and systolic sheet strain (P<0.05) in KO mice compared with littermate wildtype (WT) controls. In addition, measurements in isolated papillary muscles in heterozygous global vinculin knockout mice showed no difference in isometric fiber tension development consistent with no change in systolic fiber strain in vivo. A finite element model of ventricular mechanics suggested an increase in transverse systolic stress development may explain these observations. We hypothesized that this may be due to an increase in myofilament lattice spacing in the VclKO. Lattice spacing and sarcomere length were measured from optical diffraction patterns generated from FFTs of electron micrographs of VclKO and control hearts. The center-to-center spacing between myosin filaments was 37.6±2.55 nm in barium-contracted VclKO hearts (n=3) compared with 32.9±2.37 nm in WT controls (n=3). In hearts fixed at zero load, this spacing averaged 33.5±1.35 nm in VclKO (n=3) vs. 30.8±0.68 nm in WT (n=3). These results along with sarcomere length measurements suggest Vcl can mediate myofilament architecture and hence directional-dependent systolic force generation. We hypothesize that increasing lattice spacing alters the crossbridge binding angle of myosin heads, increasing the transverse force that they generate. The resulting increase in transverse myofiber stiffness during systole decreases systolic sheet strains.

The Role of Vinculin in the Regulation of the Mechanical Properties of Cells

Vinculin couples as a focal adhesion protein the extracellular matrix (ECM) through integrins to the actomyosin cytoskeleton. During the last years vinculin has become the focus of cell mechanical measurements and a key protein regulating the transmission of contractile forces. In earlier reports vinculin has been described as an inhibitor of cell migration on planar substrates, because knock-out of vinculin in F9 mouse embryonic carcinoma cells and mouse embryonic fibroblasts showed increased cell motility on 2D substrates. The role of vinculin in cell invasion through a 3D extracellular matrix is still fragmentarily investigated. This review presents vinculin in its role as a regulator of cellular mechanical functions. Contractile force generation is reduced when vinculin is absent, or enhanced when vinculin is present. Moreover, the generation of contractile forces is a prerequisite for cell invasion through a dense 3D ECM, where the pore-size is smaller than the diameter of the cell nucleus (<2 microm). Measurements of cell's biophysical properties will be presented. In summary, vinculin's leading role among focal adhesion proteins in regulating the mechanical properties of cells will be discussed.

Role of vinculin in regulating focal adhesion turnover

Although vinculin (−/−) mouse embryo fibroblasts assemble focal adhesions (FAs), they spread more slowly, less extensively, and close a wound more rapidly than vinculin (+/+) cells. To investigate the structure and dynamics of FAs in these cells, we used real-time interference reflection microscopy (IRM) thus avoiding the need to express exogenous GFP-tagged FA proteins which may be misregulated. This showed that the FAs were smaller, less abundant and turned over more rapidly in vinculin null compared to wild-type cells. Expression of vinculin rescued the spreading defect and resulted in larger and more stable FAs. Phosphatidylinositol 4,5-bisphosphate (PIP2) is thought to play a role in vinculin activation by relieving an intramolecular association between the vinculin head (Vh) and tail (Vt) that masks the ligand binding sites in Vh and Vt. To investigate the role of the vinculin/PIP2 interaction in FA dynamics, we used a vinculin mutant lacking the C-terminal arm (residues 1053–1066) and referred to as the ΔC mutation. This mutation reduced PIP2 binding to a VtΔC polypeptide by >90% compared to wild type without affecting binding to Vh or F-actin. Interestingly, cells expressing the vinculinΔC mutant assembled remarkably stable FAs. The results suggest that vinculin inhibits cell migration by stabilising FAs, and that binding of inositol phospholipids to Vt plays an important role in FA turnover.

Shigella interactions with the actin cytoskeleton in the absence of Ena/VASP family proteins.

Shigella move through the cytosol of infected cells by assembly of a propulsive actin tail at one end of the bacterium. Vasodilator-stimulated phosphoprotein (VASP), a member of the Ena/VASP family of proteins, is important in cellular actin dynamics and is present on intracellular Shigella. VASP binds both profilin, an actin monomer-binding protein, and vinculin, a component of intercellular contacts that also binds the Shigella actin assembly protein IcsA. It has been postulated that VASP might serve as a linker between vinculin and profilin on intracellular Shigella, thereby delivering profilin to the Shigella actin assembly machinery. We show that Shigella actin-based motility is unaltered in cells that are deficient for the Ena/VASP family of proteins. In these cells, Shigella form normal-appearing actin tails and move at rates that are comparable to the rates of bacterial movement in Ena/VASP-deficient cells complemented with the Ena/VASP family member Mena. Finally, whereas vinculin can bind the Arp2/3 complex, we show that Arp2/3 recruitment to Shigella is not correlated with vinculin recruitment, indicating that the role of vinculin in Shigella motility is not recruitment of Arp2/3. Thus, although VASP is recruited to the surface of intracellular Shigella, it is not essential for Shigella actin-based motility.

Role of vinculin in the maintenance of cell-cell contacts in kidney epithelial MDBK cells

Microinjection of fluorophore-tagged cytoskeletal proteins has been a useful tool in studies of formation of focal adhesions (FA). We used this method to study the maintenance of adherens junctions (AJ) and tight junctions (TJ) of epithelial Madin-Darby bovine kidney cells. We chose alpha-actinin and vinculin as markers, because they are present both at adherens junctions and focal adhesions and their binding partners have been well characterized. Isolated FITC-labelled chicken alpha-actinin and vinculin were injected into confluent cells where they were rapidly incorporated both in FAs and AJs. The FAs remained unchanged, whereas cell-cell contacts began to fade within an hour after injection and the cells were joined to polykaryons having 5 to 13 nuclei. Short fragments of cell membranes containing injected proteins, actin, beta-catenin, cadherin, claudin, occludin and ZO-1 were visible inside the polykaryons indicating that both AJs and TJs were disintegrated as a single complex. Microinjected FITC-labelled vinculin head domain was also incorporated to both AJs and FAs, but instead of fusions it rapidly induced the detachment of the cells from the substratum probably due to high affinity of vinculin head to talin. Vinculin tail domain had no apparent effect on the cell morphology. Since small GTPases are involved in the building up of AJs, we injected active and inactive forms of cdc42 and rac proteins together with vinculin to see their effect. Active forms reduced the formation of polykaryons presumably by strengthening AJs, whereas inactive forms had no apparent effect. We suggest that excess alpha-actinin and vinculin uncouple the cell-cell adhesion junctions from the intracellular cytoskeleton which leads to fragmentation of junctional complexes and subsequent cell fusion. The results show that cell-cell adhesion sites are more dynamic and more sensitive than FAs to an imbalance in the amount of free alpha-actinin and intact vinculin.

Vinculin and cell-cell adhesion.

Vinculin, a 117-kDa protein, is a constituent of adhesion plaques and adherence junctions in non-muscle cells. We investigated the role of vinculin on the physical strength of cell-cell adhesion by conducting disaggregation assays on aggregates of parental wild-type F9 mouse embryonal carcinoma cells (clone BIM), two vinculin-depleted F9 cell lines, gamma 227 and gamma 229, and a reconstituted gamma 229 cell line (R3) that re-express vinculin. Immunoblotting demonstrated that the four cell lines used in the study had similar expressions of the cell-cell adhesion molecule E-cadherin and associated membrane proteins alpha- and beta-catenin. Double immunofluorescence analysis showed that, in contrast to the vinculin-null cell lines. BIM and R3 cells expressed abundant vinculin at the cell margins in adhesion plaques and in cell-cell margins that also contained actin. Laminar flow assays showed that both the vinculin-positive and vinculin-negative cell aggregates that were formed in culture in the course of 24 to 48 hours largely remained intact despite the imposition of shear flow at high shear rates. Since laminar flow imposed on cell aggregates act to separate cells from each other, our data indicate that F9 cells that were adherent to a substrate formed strong cell-cell adhesion bonds independent of vinculin expression. On the other hand, aggregates of vinculin-depleted gamma 229 and gamma 227 cells that were formed in suspension during a two-hour static incubation at 37 degrees C were desegregated more easily with the imposition of shear flow than the BIM and R3 cell aggregates formed under identical conditions. Loss of vinculin was associated with a reduction in cell-cell adhesion strength only among those cells lacking contact to a substrate. Overall, the results indicate that vinculin is not needed for forming strong cell-cell adhesion bonds between neighboring carcinoma cells which are adherent to the basal lamina.

Characterization of two F-actin-binding and oligomerization sites in the cell-contact protein vinculin.

Vinculin, a structural protein of animal cells, is critically involved in the assembly of microfilament/plasma membrane junctions at cell contacts. To understand its role in organizing the distal portions of microfilaments into specific, morphologically distinct structures at these sites in more detail, we characterized its interaction with filamentous actin and with itself by means of in vitro assays. Using recombinant proteins comprising different parts of the vinculin tail fused to the maltose-binding protein of Escherichia coli, we show in sedimentation assays that this part of vinculin harbors two discrete sites that can bind to actin independently. They reside within amino acid residues 893-985 and 1016-1066 of the 1066-residue polypeptide chain. However, both sites are necessary to cross-link or bundle actin filaments, as demonstrated by low shear viscometry. Crosslinking and bundling are alternatives determined by the molar ratio of fusion protein to F-actin. Both actin-binding sequences are capable of oligomer formation, as shown in chemical-cross-linking and dot-overlay assays. These data allow us to propose a possible role for vinculin in organizing the distal ends of microfilaments at the plasma membrane into the point-like structure characteristic for cell-matrix contacts.

No mechanical role for vinculin in strain transduction in primary bovine osteoblasts

Vinculin is thought to play a central role in linking the actin cytoskeleton to the integrin adhesion proteins of focal contacts and also in cell-cell adhesion sites. We have investigated aspects of attachment and the transduction of mechanical stimulation on the function of vinculin and its assembly in primary bovine osteoblasts. The dynamic attachment process was concomitant with the reassembly of vinculin from a soluble cytoplasmic pool into Triton-insoluble focal adhesions. Immunoblotting experiments demonstrated a slight increase in the amount of vinculin concentrated in focal contacts that was paralleled by a discrete decrease in the Triton-soluble fraction of cytoplasmic vinculin. Sixty cycles of 1 Hz deformations at 0.02% (hypophysiological, no change in cell division rate), 0.2% (within the normal physiological range), and 1% (hyperphysiological) resulted in a rapid and reversible disassembly of vinculin from focal contacts to a homogeneous cytoplasmic localization, although alterations in the shape and morphology of cells could not be detected. Although all mechanical loading protocols dramatically depleted vinculin from focal contacts, its initial distribution in adhesion plaques was fully restored within the next 60 min, demonstrating the highly dynamic and reversible shift of this protein from a membrane-associated pool to the cytoplasm. By first depleting vinculin from the focal adhesion sites by applying 0.02% strain, waiting 60 s, and then applying 0.2% strain, we show that the transduction of mechanical deformation was identical with controls that had not been pretreated. This indicates that vinculin does not play a role in mechanical transduction, that significant mechanical forces are not transmitted through vinculin, and also that loss of vinculin at the focal adhesion site does not have significant effects, in the short term, on cell adhesion.

Functional Analysis of Shigella VirG Domains Essential for Interaction with Vinculin and Actin-based Motility

The VirG (IcsA) protein of Shigella is required for recruitment of host actin filament (F-actin) by intracellularly motile bacteria. An N-terminal 80-kDa VirG portion (alpha-domain) is exposed on the bacterial surface, while the following C-terminal 37-kDa portion (beta-core) is embedded in the outer membrane. Here, we report that the surface exposed alpha-domain of VirG possesses two distinct functional domains; one is the N-terminal two-thirds portion of the alpha-domain which is required for eliciting F-actin assembly on the bacteria in infected cells, and the other one is the rest of the C-terminal portion of the VirG alpha-domain, which is essential for the asymmetric distribution of VirG on the bacterial surface. Furthermore, we found that vinculin, an actin-binding cytoskeletal protein, accumulates on the surface of bacteria expressing VirG in infected cells, and that the distribution of vinculin coincided with the distribution of VirG and assembled F-actin. The vinculin accumulation depended on the expression of the alpha-domain VirG portion required for F-actin assembly, but the recruitment of vinculin on Shigella appeared prior to the appearance of F-actin in the infected cells. Analysis of proteins interacting with VirG using Xenopus laevis eggs extracts revealed that vinculin was a protein that bound to the alpha-domain portion. This was further confirmed using purified chicken gizzard vinculin, in that the 95-kDa vinculin head part, but not the 30-kDa tail part, directly bound to the alpha-domain portion. These results suggest a possible role for vinculin in recruitment of F-actin to the VirG moiety exposed on Shigella in infected mammalian cells.

Presence and Localization of Vinculin in Giardia

A requisite element of pathogenicity in Giardia infections is the parasites' ability to adhere to the intestinal epithelial brush border. The presence of vinculin in Giardia was studied because this protein is known to link the cytoskeleton to the plasma membrane and is localized at adhesion foci in many cell-cell and cell-substrate contact sites. Actin, alpha-actinin, and vinculin were identified in Giardia by western blot analysis. Giardia trophozoites attached to glass substrates were examined by interference reflection microscopy (IRM) and immunofluorescence. The IRM defined the lateral crest, bare area, and overlap region of the ventral disk, as well as the ventrolateral flange and lateral shields as close contact areas between parasite and substrate. These close contact regions were then correlated with immunofluorescence localization of actin, alpha-actinin, and vinculin. Actin was seen in the lateral crest, while alpha-actinin was observed in the ventral disc periphery and lateral shields. Vinculin was viewed at the bare and overlap areas of the ventral disc and portions of the lateral crest, as well as the ventrolateral flange and lateral shields. The correspondence of close contact sites with vinculin localization suggests a role for vinculin in Giardia attachment and adherence.

The role of vinculin in irreversible myocardial cell injury induced by neutrophils

The role of vinculin in irreversible myocardial cell injury induced by neutrophils

Evidence for an actin-containing cytoplasmic precursor of the focal contact and the timing of incorporation of vinculin at the focal contact.

The distribution of F-actin and vinculin in chicken embryo fibroblasts has been examined by nitrobenzoxadiazol (NBD)-phallacidin and indirect immunofluorescent staining, respectively, and related to the process of focal contact formation by recording the motility of the cell with differential interference contrast (DIC) or interference reflection microscopy (IRM) before fixation for staining. Linear cytoplasmic precursors of the focal contact, present within unattached lamellipodia, stained intensely with NBD-phallacidin. Without exception new focal contacts, 8 s and older at fixation, were associated with either a longer F-actin rib in the lamellipodium or, in older contacts, an F-actin structure of similar dimensions to the contact. This change in distribution of F-actin over the new contacts was accounted for by the segregation of the structural precursor into an attached part over the focal contact and a separate motile part. These results show that F-actin accumulates in the precursor adjacent to areas of the membrane competent to form the focal contact, and are consistent with the interpretation that this F-actin contributes to the initial adhesion plaque associated with the new contact. Vinculin was essentially absent from motile lamellipodia, showed no preferential association with F-actin rich precursors or very young focal contacts, but accumulated over new contacts during a 90-s period. Therefore, the association of F-actin with the membrane that precedes and persists in the initial focal contact is independent of vinculin, and the role of vinculin in development of the focal contact remains unclear.