Mapping in vivo associations of cytoplasmic proteins with integrin beta 1 cytoplasmic domain mutants.

Integrins promote formation of focal adhesions and trigger intracellular signaling pathways through cytoplasmic proteins such as talin, alpha-actinin, and focal adhesion kinase (FAK). The beta 1 integrin subunit has been shown to bind talin and alpha-actinin in in vitro assays, and these proteins may link integrin to the actin cytoskeleton either directly or through linkages to other proteins such as vinculin. However, it is unknown which of these associations are necessary in vivo for formation of focal contacts, or which regions of beta 1 integrin bind to specific cytoskeletal proteins in vivo. We have developed an in vivo assay to address these questions. Microbeads were coated with anti-chicken beta 1 antibodies to selectively cluster chicken beta 1 integrins expressed in cultured mouse fibroblasts. The ability of cytoplasmic domain mutant beta 1 integrins to induce co-localization of proteins was assessed by immunofluorescence and compared with that of wild-type integrin. As expected, mutant beta 1 lacking the entire cytoplasmic domain had a reduced ability to induce co-localization of talin, alpha-actinin, F-actin, vinculin, and FAK. The ability of beta 1 integrin to co-localize talin and FAK was found to require a sequence near the C-terminus of beta 1. The region of beta 1 required to co-localize alpha-actinin was found to reside in a different sequence, several amino acids further from the C-terminus of beta 1. Deletion of 13 residues from the C-terminus blocked co-localization of talin, FAK, and actin, but not alpha-actinin. Association of alpha-actinin with clustered integrin is therefore not sufficient to induce the co-localization of F-actin.     

The vinculin binding sites of talin and alpha-actinin are sufficient to activate vinculin

Vinculin regulates both cell-cell and cell-matrix junctions and anchors adhesion complexes to the actin cytoskeleton through its interactions with the vinculin binding sites of alpha-actinin or talin. Activation of vinculin requires a severing of the intramolecular interactions between its N- and C-terminal domains, which is necessary for vinculin to bind to F-actin; yet how this occurs in cells is not resolved. We tested the hypothesis that talin and alpha-actinin activate vinculin through their vinculin binding sites. Indeed, we show that these vinculin binding sites have a high affinity for full-length vinculin, are sufficient to sever the head-tail interactions of vinculin, and they induce conformational changes that allow vinculin to bind to F-actin. Finally, microinjection of these vinculin binding sites specifically targets vinculin in cells, disrupting its interactions with talin and alpha-actinin and disassembling focal adhesions. In their native (inactive) states the vinculin binding sites of talin and alpha-actinin are buried within helical bundles present in their central rod domains. Collectively, these results support a model where the engagement of adhesion receptors first activates talin or alpha-actinin, by provoking structural changes that allow their vinculin binding sites to swing out, which are then sufficient to bind to and activate vinculin.     

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.     

The cytoplasmic domain of L-selectin interacts with cytoskeletal proteins via alpha-actinin: receptor positioning in microvilli does not require interaction with alpha-actinin

The leukocyte adhesion molecule L-selectin mediates binding to lymph node high endothelial venules (HEV) and contributes to leukocyte rolling on endothelium at sites of inflammation. Previously, it was shown that truncation of the L-selectin cytoplasmic tail by 11 amino acids abolished binding to lymph node HEV and leukocyte rolling in vivo, but the molecular basis for that observation was not determined. This study examined potential interactions between L-selectin and cytoskeletal proteins. We found that the cytoplasmic domain of L- selectin interacts directly with the cytoplasmic actin-binding protein alpha-actinin and forms a complex with vinculin and possibly talin. Solid phase binding assays using the full-length L-selectin cytoplasmic domain bound to microtiter wells demonstrated direct, specific, and saturable binding of purified alpha-actinin to L-selectin (Kd = 550 nM), but no direct binding of purified talin or vinculin. Interestingly, talin potentiated binding of alpha-actinin to the L- selectin cytoplasmic domain peptide despite the fact that direct binding of talin to L-selectin could not be measured. Vinculin binding to the L-selectin cytoplasmic domain peptide was detectable only in the presence of alpha-actinin. L-selectin coprecipitated with a complex of cytoskeletal proteins including alpha-actinin and vinculin from cells transfected with L-selectin, consistent with the possibility that alpha- actinin binds directly to L-selectin and that vinculin associates by binding to alpha-actinin in vivo to link actin filaments to the L- selectin cytoplasmic domain. In contrast, a deletion mutant of L- selectin lacking the COOH-terminal 11 amino acids of the cytoplasmic domain failed to coprecipitate with alpha-actinin or vinculin. Surprisingly, this mutant L-selectin localized normally to the microvillar projections on the cell surface. These data suggest that the COOH-terminal 11 amino acids of the L-selectin cytoplasmic domain are required for mediating interactions with the actin cytoskeleton via a complex of alpha-actinin and vinculin, but that this portion of the cytoplasmic domain is not necessary for proper localization of L- selectin on the cell surface. Correct L-selectin receptor positioning is therefore insufficient for leukocyte adhesion mediated by L- selectin, suggesting that this adhesion may also require direct interactions with the cytoskeleton.     

Aggregatibacter actinomycetemcomitans lipopolysaccharide affects human gingival fibroblast cytoskeletal organization

The cytoskeleton is a dynamic structure that plays a key role in maintaining cell morphology and function. This study investigates the effect of bacterial wall lipopolysaccharide (LPS), a strong inflammatory agent, on the dynamics and organization of actin, tubulin, vimentin, and vinculin proteins in human gingival fibroblasts (HGF). A time-dependent study showed a noticeable change in actin architecture after 1.5 h of incubation with LPS (1 microg/ml) with the formation of orthogonal fibers and further accumulation of actin filament at the cell periphery by 24 h. When 0.01-10 microg/ml of LPS was added to human gingival fibroblast cultures, cells acquired a round, flat shape and gradually developed cytoplasmic ruffling. Lipopolysaccharides extracted from Aggregatibacter actinomycetemcomitans periodontopathogenic bacteria promoted alterations in F-actin stress fibres of human gingival cells. Normally, human gingival cells have F-actin fibres that are organized in linear distribution throughout the cells, extending along the cell's length. LPS-treated cells exhibited changes in cytoskeletal protein organization, and F-actin was reorganized by the formation of bundles underneath and parallel to the cell membrane. We also found the reorganization of the vimentin network into vimentin bundling after 1.5 h of treatment. HGF cells exhibited diffuse and granular gamma-tubulin stain. There was no change in LPS-treated HGF. However, vinculin plaques distributed in the cell body diminished after LPS treatment. We conclude that the dynamic and structured organization of cytoskeletal filaments and actin assembly in human gingival fibroblasts is altered by LPS treatment and is accompanied by a decrease in F-actin pools.     

Specific interaction of vinculin with alpha-actinin

Vinculin and alpha-actinin are cytoskeletal proteins present at focal contacts of the ventral surface of cultured fibroblasts. We labelled alpha-actinin with an acceptor fluorophore and vinculin with a donor. A mixture of vinculin and alpha-actinin showed a 28% quench, due to energy transfer, suggesting an interaction. Quench of vinculin was dependent on the concentration of alpha-actinin; Scatchard analysis gives a dissociation constant in the microM range. Quench was inhibited by excess unlabelled alpha-actinin, and by reaction of the acceptor protein with p-chloromercuribenzoate. We found that vinculin had a slightly greater elution volume in a gel filtration column equilibrated with alpha-actinin, indicating a higher effective Stokes radius due to the interaction of the two proteins.     

F-actin aggregates in transformed cells contain alpha-actinin and fimbrin but apparently lack tropomyosin

Transformation-specific F-actin structures are examined in tumor cells after in vitro tumor cell growth alone or on an untransformed cell monolayer. In transformed cells F-actin aggregates near the ventral plasma membrane in close substrate adhesion areas contain the cytoskeletal proteins alpha-actinin and fimbrin but, unlike microfilament bundles, are not labeled with antibody against tropomyosin. By electron microscopy the dense ventral aggregates in transformed cells resemble stress fiber termini found at the membrane in normal cells. These transformed-cell cytoskeletal structures are not limited solely to substrate adhesion areas; they are also expressed at cell-cell contacts about 48 h after transformed cells are plated on untransformed cells. These specialized F-actin aggregates appear to be implicated in the processes of penetration of these transformed cells between adjoining untransformed cells in vitro.     

Architectural dynamics of F-actin in eupodia suggests their role in invasive locomotion in Dictyostelium.

Eupodia are F-actin-containing cortical structures similar to vertebrate podosomes or invadopodia found in metastatic cells. Eupodia are rich in alpha-actinin and myosin IB/D, but not a Dictyostelium homologue of talin. In the present study, we localized other actin-binding proteins, ABP120, cofilin, coronin, and fimbrin, in the eupodia and examined the three-dimensional organization of their F-actin system by confocal microscopy and transmission electron microscopy. To examine their function, we analyzed the assembly and disassembly dynamics of the F-actin system in eupodia and its relation to lamellipodial protrusion. Actin dynamics was examined by monitoring S65T-GFP-coronin and rhodamine-actin using a real-time confocal unit and a digital microscope system. Fluorescence morphometric analysis demonstrates the presence of a precise spatiotemporal coupling between F-actin assembly in eupodia and lamellipodial protrusion. When a lamellipodium advances to invade a tight space, additional rows of eupodia are sequentially formed at the base of that lamellipodium. These results indicate that mechanical stress at the leading edge modulates the structural integrity of actin and its binding proteins, such that eupodia are formed when anchorage is needed to boost for invasive protrusion of the leading edge.     

Talin requires beta-integrin, but not vinculin, for its assembly into focal adhesion-like structures in the nematode Caenorhabditis elegans.

In cultured cells, the 230-kDa protein talin is found at discrete plasma membrane foci known as focal adhesions, sites that anchor the intracellular actin cytoskeleton to the extracellular matrix. The regulated assembly of focal adhesions influences the direction of cell migrations or the reorientation of cell shapes. Biochemical studies of talin have shown that it binds to the proteins integrin, vinculin, and actin in vitro. To understand the function of talin in vivo and to correlate its in vitro and in vivo biochemical properties, various genetic approaches have been adopted. With the intention of using genetics in the study of talin, we identified a homologue to mouse talin in a genetic model system, the nematode Caenorhabditis elegans. C. elegans talin is 39% identical and 59% similar to mouse talin. In wild-type adult C. elegans, talin colocalizes with integrin, vinculin, and alpha-actinin in the focal adhesion-like structures found in the body-wall muscle. By examining the organization of talin in two different C. elegans mutant strains that do not make either beta-integrin or vinculin, we were able to determine that talin does not require vinculin for its initial organization at the membrane, but that it depends critically on the presence of integrin for its initial assembly at membrane foci.     

In vitro regulation of granulosa cell differentiation. Involvement of cytoskeletal protein expression

The link between the biochemical and morphological differentiation of granulosa cells was studied by investigating the organization and the expression of cytoskeletal proteins which determine cell shape and contacts. In cells treated with follicle-stimulating hormone (FSH), in a serum- and growth factor-free medium, or with other compounds which elevate cellular cAMP levels, the synthesis of the adherens junction proteins, vinculin, alpha-actinin, and actin was reduced significantly when compared to unstimulated cells (7-fold for vinculin, 5-fold for alpha-actinin, and 3-fold for actin). The in vitro translatability of the mRNAs coding for these proteins and the level of actin mRNA determined by RNA blot hybridization were generally reduced in differentiating cells. The synthesis and the organization of vimentin and tubulin was unaffected during this process, whereas the organization of actin and vinculin was dramatically affected, with FSH-treated cells displaying a diffuse pattern of actin and vinculin, with very little vinculin in adhesion plaques. Gonadotropin-releasing hormone agonist and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate which are known to antagonize the cAMP-mediated biochemical differentiation of granulosa cells by reducing cAMP levels or by activating protein kinase C and phospholipid turnover, blocked to a large extent the FSH-induced effect on the adherens junction proteins. Epidermal growth factor, which blocked the FSH-induced cAMP increase, but not the FSH-induced progesterone production, failed to block the synthesis of vinculin, alpha-actinin, and actin. Cytochalasin B could induce steroidogenesis and similar changes in the synthesis of these cytoskeletal proteins, whereas fibronectin, which causes cell spreading, blocked in part the FSH-induced effect on the expression of cytoskeletal proteins. The modulation of cytoskeletal proteins may therefore be an essential feature of programmed differentiation events leading to the final phenotype of granulosa cells.     

Inositol 1,4,5-trisphosphate receptor associated with focal contact cytoskeletal proteins

The linkage between inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)Rs) and cytoskeletal proteins is considered to be important in cell function. In the present study, the association of IP(3)R subtypes with cytoskeletal proteins was examined using monoclonal antibodies specific to each IP(3)R subtype. We found that IP(3)R type 2 colocalized with talin, a focal contact cytoskeletal protein. IP(3)R type 2 exhibited a patchy distribution in the peripheral cytoplasm differently from type 1 and type 3 IP(3)R. Furthermore, IP(3)R subtypes co-immunoprecipitated with talin, vinculin and alpha-actin, but not alpha-actinin or paxillin.     

Accumulation of talin in nodes at the edge of the lamellipodium and separate incorporation into adhesion plaques at focal contacts in fibroblasts

The focal contact forms beneath F-actin-rich ribs, or cytoplasmic precursors, present in the lamellipodia of fibroblasts. The basal part of the precursor is retained at the contact as the initial adhesion plaque. We have examined the distribution of talin in the lamellipodia and adhesion plaques of chicken embryo fibroblasts relative to the process of focal contact formation. Motility of single cells was recorded with differential interference contrast or interference reflection microscopy before fixation and fluorescent staining for talin, F-actin, and vinculin. Talin is present along the extreme edge of the lamellipodium, where it is further concentrated into a series of nodes. The nodes of talin are present at the tips of both larger and finer F-actin-rich ribs and at small structural nodes at the edge of the lamellipodium. We suggest that the talin in the nodes functions, via a cross-linking activity, in the convergence of actin filaments at the membrane during development of the ribs. Talin accumulates de novo in the adhesion plaque, independent of that at the tip of the precursor, in response to contact with the substrate. This second accumulation of talin at the focal contact starts before vinculin, consistent with a sequential binding of talin at the membrane and of vinculin to talin. The results imply that talin functions independently at two steps during formation of the focal contact: the development of the F-actin-rich precursor of the contact; and development of the contact-associated adhesion plaque, both involving organization of F-actin at the membrane.     

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.     

Reduced cell migration and disruption of the actin cytoskeleton in calpain-deficient embryonic fibroblasts

The physiological functions and substrates of the calcium-dependent protease calpain remain only partly understood. The mu- and m-calpains consist of a mu- or m-80-kDa large subunit (genes Capn1 and Capn2), and a common 28-kDa small subunit (Capn4). To assess the role of calpain in migration, we used fibroblasts obtained from Capn4(-/-) mouse embryos. The cells lacked calpain activity on casein zymography and did not generate the characteristic calpain-generated spectrin breakdown product that is observed in wild-type cells. Capn4(-/-) cells had decreased migration rates and abnormal organization of the actin cytoskeleton with a loss of central stress fibers. Interestingly, these cells extended numerous thin projections and displayed delayed retraction of membrane protrusions and filopodia. The number of focal adhesions was decreased in Capn4(-/-) cells, but the cells had prominent vinculin-containing focal complexes at the cell periphery. The levels of the focal adhesion proteins, alpha-actinin, focal adhesion kinase (FAK), spectrin, talin, and vinculin, were the same in Capn4(+/+) and Capn4(-/-) cells. FAK, alpha-actinin, and vinculin were not cleaved in either cell type plated on fibronectin. However, proteolysis of the focal complex component, talin, was detected in the wild-type cells but not in the Capn4(-/-) cells, suggesting that calpain cleavage of talin is important during cell migration. Moreover, talin cleavage was again observed when calpain activity was partially restored in Capn4(-/-) embryonic fibroblasts by stable transfection with a vector expressing the rat 28-kDa calpain small subunit. The results demonstrate unequivocally that calpain is a critical regulator of cell migration and of the organization of the actin cytoskeleton and focal adhesions.     

Localization of brush border cytoskeletal proteins in gastric oxynticopeptic cells from the bullfrog Rana catesbeiana

The contribution of brush border cytoskeletal proteins (actin, villin, fimbrin, and brush border myosin-1) to organization of the cytoskeletal network underlying apical plications of oxynticopeptic cells was examined by immunohistochemical techniques in frozen sections of gastric mucosa from the bullfrog, Rana catesbeiana. Apical localization of F-actin with phalloidin in oxynticopeptic cells inhibited with cimetidine revealed small, punctate domains within the apical cytoplasm that were consistent with the presence of short microvilli revealed by electron microscopy. Localization of F-actin in cells stimulated with forskolin was limited to a wide continuous band of cytoplasm corresponding to the location of numerous long surface folds. Inhibition of protein synthesis with cycloheximide did not prevent acid secretion or formation of actin filaments within surface folds in stimulated oxynticopeptic cells, suggesting that the formation of filaments does not require actin synthesis. Staining of gastric mucosae with fluorescent DNase-1 demonstrated that oxynticopeptic cells possess an unusually large pool of non-filamentous actin. Taken together, these results suggest that actin-filament formation in stimulated cells occurs by polymerization of an existing pool of non-filamentous actin. Localization of antibodies specific for villin and fimbrin revealed that these proteins were present within intestinal absorptive cells and gastric surface and neck cells but were not present within inhibited or stimulated oxynticopeptic cells. Brush border myosin-1, present in intestinal absorptive cells, was not present in gastric epithelium. Thus, we propose that actin-containing projections in oxynticopeptic cells are not organized like intestinal microvilli and that filament formation occurs after stimulation by modulating intracellular pools of filamentous and non-filamentous actin.     

Phorbol myristate acetate induces changes on F-actin and vinculin content in immature rat Sertoli cells

Actin and vinculin are two of the most abundant cytoskeletal proteins, widely expressed in nearly all types of eukaryotic cells. It has been well established that long-term exposure to the tumor promoter phorbol myristate acetate (PMA) affects Sertoli cell morphology, as well as F-actin and vinculin organization in vitro. To analyze in a quantitative manner the F-actin and vinculin content of rat immature Sertoli cells in vitro in response to PMA exposure, cytoskeletal fractions were prepared following extraction with Triton X-100. Analysis of the isolated cytoskeletal fractions by immunoblotting showed that exposure of immature rat Sertoli cells to PMA for 8h has an appreciable effect on the cellular level of both the actin and vinculin. Interestingly, as revealed by using calphostin C, a specific protein kinase C inhibitor, the observed F-actin and vinculin changes are most probably mediated by a mechanism that depends on protein kinase activity. A discussion is made concerning PKC modulation by PMA and the subsequent actin and vinculin quantitative changes and reorganization, phenomena that have been closely related to cell transformation.     

Protein kinase C regulates alpha v beta 5-dependent cytoskeletal associations and focal adhesion kinase phosphorylation

Integrins alpha v beta 3 and alpha v beta 5 both mediate cell adhesion to vitronectin yet trigger different postligand binding events. Integrin alpha v beta 3 is able to induce cell spreading, migration, angiogenesis, and tumor metastasis without additional stimulators, whereas alpha v beta 5 requires exogenous activation of protein kinase C (PKC) to mediate these processes. To investigate this difference, the ability of beta 3 or beta 5 to induce colocalization of intracellular proteins was assessed by immunofluorescence in hamster CS-1 melanoma cells. We found that alpha v beta 5 induced colocalization of talin, alpha-actinin, tensin, and actin very weakly relative to alpha v beta 3. alpha v beta 5 was able to efficiently induce colocalization of focal adhesion kinase (FAK); however, it was unable to increase phosphorylation of FAK on tyrosine. Activation of PKC by adding phorbol ester to alpha v beta 5-expressing cells induced spreading, increased colocalization of alpha-actinin, tensin, vinculin, p130cas and actin, and triggered tyrosine phosphorylation of FAK. Unexpectedly, talin colocalization remained low even after activation of PKC. Treatment of cells with the PKC inhibitor calphostin C inhibited spreading and the colocalization of talin, alpha-actinin, tensin, and actin for both alpha v beta 3 and alpha v beta 5. We conclude that PKC regulates localization of cytoskeletal proteins and phosphorylation of FAK induced by alpha v beta 5. Our results also show that FAK can be localized independent of its phosphorylation and that cells can spread and induce localization of other focal adhesion proteins in the absence of detectable talin.     

Comparison of the regional distribution of calspectin (nonerythroid spectrin or fodrin), alpha-actinin, vinculin nonerythroid protein 4.1, and calpactin in normal and avian sarcoma virus- or Rous sarcoma virus-induced transformed cells

With fluorescence and interference reflection microscopy (IRM), we compared the regional distribution of calspectin, its interacting proteins (nonerythroid protein 4.1 and calpactin), alpha-actinin, and vinculin in NRK cells and their avian sarcoma virus (ASV)- or temperature-sensitive (ts) Rous sarcoma virus (RSV)-transformed cells. The localization of these cytoskeletal proteins was determined with the specific antibodies. In NRK cells, alpha-actinin and vinculin were concentrated at adhesion plaques. By contrast, calspectin was distributed throughout the cytoplasm, but not concentrated at adhesion plaques. In ASV- and ts RSV-transformed cells, all three cytoskeletal proteins were concentrated at dot structures representing cellular feet. Nonerythroid protein 4.1 and calpactin were diffusely distributed throughout the cytoplasm of NRK cells and their transformed counterparts. In the case of calpactin, a part of this protein was excluded near regions of the terminal ends of stress fibers. These two proteins did not show the restricted location at the dot structures of transformed cells. From these findings, it is apparent that the accumulation of calspectin into dot structures is a specific event for cell transformation induced by the src protein.     

Organization of pp60src and selected cytoskeletal proteins within adhesion plaques and junctions of Rous sarcoma virus-transformed rat cells

The localization of pp60src within adhesion structures of epithelioid rat kidney cells transformed by the Schmidt-Ruppin strain of Rous sarcoma virus was compared to the organization of actin, alpha-actinin, vinculin (a 130,000-dalton protein), tubulin, and the 58,000-dalton intermediate filament protein. The adhesion structures included both adhesion plaques and previously uncharacterized adhesive regions formed at cell-cell junctions. We have termed these latter structures "adhesion junctions." Both adhesion plaques and adhesion junctions were identified by interference-reflection microscopy and compared to the location of pp60src and the various cytoskeletal proteins by double fluorescence. The results demonstrated that the src gene product was found within both adhesion plaques and the adhesion junctions. In addition, actin, alpha-actinin, and vinculin were also localized within the same pp60src-containing adhesion structures. In contrast, tubulin and the 58,000-dalton intermediate filament protein were not associated with either adhesion plaques or adhesion junctions. Both adhesion plaques and adhesion junctions were isolated as substratum-bound structures and characterized by scanning electron microscopy. Immunofluorescence revealed that pp60src, actin, alpha-actinin, and vinculin were organized within specific regions of the adhesion junctions. Heavy accumulations of actin and alpha-actinin were found on both sides of the junctions with a narrow gap of unstained material at the midline, whereas pp60src stain was more intense in this central region. Antibody to vinculin stained double narrow lines defining the periphery of the junctional complexes but was excluded from the intervening region. In addition, the distribution of vinculin relative to pp60src within adhesion plaques suggested an inverse relationship between the presence of these two proteins. Overall, these results establish a close link between the src gene product and components of the cytoskeleton and implicate the adhesion plaques and adhesion junctions in the mechanism of Rous sarcoma virus-induced transformation.