PDGF stimulation induces phosphorylation of talin and cytoskeletal reorganization in skeletal muscle

Modifications in the interactions of the muscle cytoskeleton with the cell membrane occur during cell growth and adaptation, although the mechanisms regulating these interactions are unknown. We have observed that myotendinous junctions (MTJs), which are the primary sites of turnover of the thin filament-membrane associations in skeletal muscle, are greatly enriched in receptors for PDGF. The high concentration of PDGF receptors at MTJs suggested to us that receptor binding may initiate cytoskeletal remodeling in skeletal muscle. We tested this possibility by examining the organization and phosphorylation of cytoskeletal components of L6 myocytes after PDGF stimulation. We have found that 10 min after PDGF stimulation, L6 myoblasts exhibit no stress fibers discernible by phalloidin binding, and that vinculin relocates from focal contacts into a diffuse cytoplasmic distribution. After 60 min of incubation, these changes are largely reversed. Indirect immunofluorescence shows that at 10-min PDGF stimulation, there are no changes in the distribution of talin, the beta 1 subunit of integrin, pp125FAK or desmin. Phosphotyrosine distribution changes upon stimulation from focal contacts to being located both in focal contacts and granules concentrated in perinuclear regions. These granules also immunolabel with anti-PDGF receptor Immunoprecipitations with anti-phosphotyrosine show that polypeptides at 180 and 230 kD show the greatest increase in tyrosine phosphorylation after PDGF stimulation. Immunoblots of anti-phosphotyrosine precipitates show that these polypeptides are the PDGF receptor and talin. We also examined the possibility that the cytoskeletal reorganization observed may result from calpain activation caused by elevated intracellular calcium induced by PDGF stimulation. However, immunoblots of control and stimulated cells show no decrease in the inactive calpain proenzyme or increase in the proteolytic, autolyzed forms of calpain pursuant to stimulation. Furthermore, stimulation produces no increase in the proportion of the 190-kD talin fragment characteristic of calpain- mediated cleavage. The retention of talin and integrin at focal contacts after talin phosphorylation, while vinculin is redistributed, indicate that phosphorylation of talin in PDGF-stimulated cells leads to separation of talin-vinculin associations but not talin-integrin associations. We propose that PDGF binding to PDGF receptors at MTJs may provide one means of regulating myofibril associations with the muscle cell membrane.     

Localization of paxillin, a focal adhesion protein, to smooth muscle dense plaques, and the myotendinous and neuromuscular junctions of skeletal muscle

In this report we have demonstrated that paxillin, a cytoskeletal protein which is present in focal adhesions, localizes in vivo to regions of cell-extracellular matrix interaction which are believed to be analogous to focal adhesions. Specifically, it is enriched in the dense plaques of chicken gizzard smooth muscle tissue and in the myotendinous junctions formed in Xenopus laevis tadpole tail skeletal muscle. In addition, paxillin was identified at the rat diaphragm neuromuscular junction. The distribution of paxillin is thus comparable to that of other focal adhesion proteins, for example, talin and vinculin, in these structures.     

Molecular heterogeneity of adherens junctions

We describe here the subcellular distributions of three junctional proteins in different adherens-type contacts. The proteins examined include vinculin, talin, and a recently described 135-kD protein (Volk, T., and B. Geiger, 1984, EMBO (Eur. Mol. Biol. Organ.) J., 10:2249-2260). Immunofluorescent localization of the three proteins indicated that while vinculin was ubiquitously present in all adherens junctions, the other two showed selective and mutually exclusive association with either cell-substrate or cell-cell adhesions. Talin was abundant in focal contacts and in dense plaques of smooth muscle, but was essentially absent from intercellular junctions such as intercalated disks or adherens junctions of lens fibers. The 135-kD protein, on the other hand, was present in the latter two loci and was apparently absent from membrane-bound plaques of gizzard or from focal contacts. Radioimmunoassay of tissue extracts and immunolabeling of cultured chick lens cells indicated that the selective presence of talin and of the 135-kD protein in different cell contacts is spatially regulated within individual cells. On the basis of these findings it was concluded that adherens junctions are molecularly heterogeneous and consist of at least two major subgroups. Contacts with noncellular substrates contain talin and vinculin but not the 135-kD protein, whereas their intercellular counterparts contain the latter two proteins and are devoid of talin. The significance of these results and their possible relationships to contact-induced regulation of cell behavior are discussed.     

Desmin at myotendinous junctions

Myofibrils are linked to the cell membrane at myotendinous junctions located at the ends of muscle fibers, and at costameres, sites positioned periodically along lateral surfaces of muscle cells. Both of these sites are enriched in proteins that link active components of myofibrils to the cell membrane. Costameres are also enriched in desmin intermediate filaments that link passive components of myofibrils to the lateral surfaces of muscle cells. In this study, the possibility that desmin is also found between the terminal Z-disk of myofibrils and the myotendinous junction membrane is examined by immunocytochemistry and by KI-extraction procedures. Data presented show that desmin is located in the filamentous core of cellular processes at myotendinous junctions at sites 30 nm or more from the membrane. This core lies deep to subsarcolemmal material previously shown to contain talin, vinculin, and dystrophin. The distance from desmin to the membrane suggests desmin does not interact directly with membrane proteins at the junction. Immunoblots and indirect immunofluorescence of junctional regions of muscle compared to nonjunctional regions show no apparent enrichment of desmin at junctional sites, although vinculin, another costameric and junctional component, is significantly enriched at junctional regions. These findings show that passive elements of myofibrils may be continuous from myotendinous junctions of muscle origin to insertion via desmin filaments located between terminal Z-disks and the junctional membrane. This can provide a system in parallel to that involving thin filaments, vinculin, and talin for linking myofibrils to the cell membrane at myotendinous junctions.     

Progressive myopathy and defects in the maintenance of myotendinous junctions in mice that lack talin 1 in skeletal muscle

The development and function of skeletal muscle depend on molecules that connect the muscle fiber cytoskeleton to the extracellular matrix (ECM). beta1 integrins are ECM receptors in skeletal muscle, and mutations that affect the alpha7beta1 integrin cause myopathy in humans. In mice, beta1 integrins control myoblast fusion, the assembly of the muscle fiber cytoskeleton, and the maintenance of myotendinous junctions (MTJs). The effector molecules that mediate beta1 integrin functions in muscle are not known. Previous studies have shown that talin 1 controls the force-dependent assembly of integrin adhesion complexes and regulates the affinity of integrins for ligands. Here we show that talin 1 is essential in skeletal muscle for the maintenance of integrin attachment sites at MTJs. Mice with a skeletal muscle-specific ablation of the talin 1 gene suffer from a progressive myopathy. Surprisingly, myoblast fusion and the assembly of integrin-containing adhesion complexes at costameres and MTJs advance normally in the mutants. However, with progressive ageing, the muscle fiber cytoskeleton detaches from MTJs. Mechanical measurements on isolated muscles show defects in the ability of talin 1-deficient muscle to generate force. Collectively, our findings show that talin 1 is essential for providing mechanical stability to integrin-dependent adhesion complexes at MTJs, which is crucial for optimal force generation by skeletal muscle.     

The alpha 5 beta 1 integrin associates with a dystrophin-containing lattice during muscle development.

The organization of the alpha 5 beta 1 integrin on skeletal muscle was studied in culture and in sections from adult and embryonic tissue using monoclonal antibodies specific for the alpha 5 subunit. The alpha 5 beta 1 integrin showed changes in organization and in the molecules with which it colocalizes. On early myoblasts, possessing a fibroblast-like morphology, the alpha 5 integrin organization was indistinguishable from that on fibroblasts; it was expressed prominently and localized in numerous focal contacts around the cell periphery. In bipolar myoblasts and early myotubes, the alpha 5 integrin was expressed only weakly and localized in a small number of focal contact-like structures. As myogenesis proceeded there was an apparent increase in integrin expression and a change in organization. In addition to the focal contact-like structures that persist throughout myogenesis in vitro, a dense lattice-like structure of integrin appeared. Fibrillar fibronectin, talin, and non-muscle alpha-actinin did not colocalize with the alpha 5 beta 1 integrin in the lattice structure as they did in the focal contact-like structures. However, dystrophin, which displayed a diffuse distribution earlier, now colocalized with the alpha 5 beta 1 integrin in the punctate lattice. Coincident with the registration of myofibrils into visible sarcomeres, the prominent dense, lattice structure disappeared leaving the focal contact-like structures as the only regions of organized alpha 5 beta 1 integrin. Despite the presence of the beta 1 integrin in neuromuscular or myotendinous junctions in vivo and on myotubes in vitro, the alpha 5 beta 1 integrin was not present in either junction. These observations suggest that the alpha 5 beta 1 integrin is involved in the adhesion of muscle to the extracellular matrix, the organization of the dystrophin-containing lattice, and the organization of nascent myofibrils which emanate from the focal contact- and stress fiber-like structures in muscle. Other integrins appear to anchor myofibrils at the myotendinous and neuromuscular junctions.     

A protein homologous to the Torpedo postsynaptic 58K protein is present at the myotendinous junction

The 58K protein is a peripheral membrane protein enriched in the acetylcholine receptor (AChR)-rich postsynaptic membrane of Torpedo electric organ. Because of its coexistence with AChRs in the postsynaptic membrane in both electrocytes and skeletal muscle, it is thought to be involved in the formation and maintenance of AChR clusters. Using an mAb against the 58K protein of Torpedo electric organ, we have identified a single protein band in SDS-PAGE analysis of Xenopus myotomal muscle with an apparent molecular mass of 48 kD. With this antibody, the distribution of this protein was examined in the myotomal muscle fibers with immunofluorescence techniques. We found that the 48K protein is concentrated at the myotendinous junctions (MTJs) of these muscle fibers. The MTJ is also enriched in talin and vinculin. By double labeling muscle fibers with antibodies against talin and the 48K protein, these two proteins were found to colocalize at the membrane invaginations of the MTJ. In cultured myotomal muscle cells, the 48K protein and talin are also colocalized at sites of membrane-myofibril interaction. The 48K protein is, however, not found at focal adhesion sites in nonmuscle cells, which are enriched in talin. These data suggest that the 48K protein is specifically involved in the interaction of myofibrillar actin filaments with the plasma membrane at the MTJ. In addition to the MTJ localization, 48K protein is also present at AChR clusters both in vivo and in vitro. Thus, this protein is shared by both the MTJ and the neuromuscular junction.     

Filamin C plays an essential role in the maintenance of the structural integrity of cardiac and skeletal muscles, revealed by the medaka mutant zacro

Filamin C is an actin-crosslinking protein that is specifically expressed in cardiac and skeletal muscles. Although mutations in the filamin C gene cause human myopathy with cardiac involvement, the function of filamin C in vivo is not yet fully understood. Here we report a medaka mutant, zacro (zac), that displayed an enlarged heart, caused by rupture of the myocardiac wall, and progressive skeletal muscle degeneration in late embryonic stages. We identified zac to be a homozygous nonsense mutation in the filamin C (flnc) gene. The medaka filamin C protein was found to be localized at myotendinous junctions, sarcolemma, and Z-disks in skeletal muscle, and at intercalated disks in the heart. zac embryos showed prominent myofibrillar degeneration at myotendinous junctions, detachment of myofibrils from sarcolemma and intercalated disks, and focal Z-disk destruction. Importantly, the expression of γ-actin, which we observed to have a strong subcellular localization at myotendinous junctions, was specifically reduced in zac mutant myotomes. Inhibition of muscle contraction by anesthesia alleviated muscle degeneration in the zac mutant. These results suggest that filamin C plays an indispensable role in the maintenance of the structural integrity of cardiac and skeletal muscles for support against mechanical stress.     

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.     

Characterization of the column and autocellular junctions that define the vasculature of gill lamellae.

Novel adhesion junctions have been characterized that are formed at the interface between pillar cells and collagen columns, both of which are essential constituents of the gill lamellae in fish. We termed these junctions the "column junction" and "autocellular junction" and determined their molecular compositions by immunofluorescence microscopy using pufferfish. We visualized collagen columns by concanavalin A staining and found that the components of integrin-mediated cell-matrix adhesion, such as talin, vinculin, paxillin, and fibronectin, were concentrated on plasma membranes surrounding collagen columns (column membranes). This connection is analogous to the focal adhesion of cultured mammalian cells, dense plaque of smooth muscle cells, and myotendinous junction of skeletal muscle cells. We named this connection the "column junction." In the cytoplasm near the column, actin fibers, actinin, and a phosphorylated myosin light chain of 20 kDa are densely located, suggesting the contractile nature of pillar cells. The membrane infoldings surrounding the collagen columns were found to be connected by the autocellular junction, whose components are highly tyrosine-phosphorylated and contain the tight junction protein ZO-1. This study represents the first molecular characterization and fluorescence visualization of the column and autocellular junctions involved in both maintaining structural integrity and the hemodynamics of the branchial lamellae.     

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.     

New isoform-specific monoclonal antibodies reveal different sub-cellular localisations for talin1 and talin2

Talins are adaptor proteins that connect the integrin family of cell adhesion receptors to cytoskeletal actin. Vertebrates express two closely related talins encoded by separate genes, and while it is well established that talin1 plays a key role in cell adhesion and spreading, little is known about the role of talin2. To facilitate such studies, we report the characterisation of 4 new isoform-specific talin mouse monoclonal antibodies that work in Western blotting, immuno-precipitation, immuno-fluorescence and immuno-histochemistry. Using these antibodies, we show that talin1 and talin2 do not form heterodimers, and that they are differentially localised within the cell. Talin1 was concentrated in peripheral focal adhesions while talin2 was observed in both focal and fibrillar adhesions, and knock-down of talin2 compromised fibronectin fibrillogenesis. Although differentiated human macrophages express both isoforms, only talin1 showed discrete staining and was localised to the ring structure of podosomes. However, siRNA-mediated knock-down of macrophage talin2 led to a significant reduction in podosomal matrix degradation. We have also used the antibodies to localise each isoform in tissue sections using both cryostat and paraffin-embedded material. In skeletal muscle talin2 was localised to both myotendinous junctions and costameres while talin1 was restricted to the former structure. In contrast, both isoforms co-localised in kidney with staining of the glomerulus, and the tubular epithelial and interstitial cells of the cortex and medulla. We anticipate that these antibodies will form a valuable resource for future studies on the function of the two major talin isoforms.     

Localization of a 215-kDa tyrosine-phosphorylated protein that cross-reacts with tensin antibodies.

Tyrosine phosphorylation of cytoskeletal proteins at adhesive junctions has been speculated to play a role in the regulation of cell signaling at these sites. Previously, monoclonal antibodies were generated against phosphotyrosine-containing proteins from Rous sarcoma virus-transformed chick embryo fibroblasts, resulting in two antibodies which recognized antigens of 76 and 215 kDa that localized to focal contacts. We have now localized the 215-kDa antigen to a number of adhesive junctions in vivo, including the zonula adherens, intercalated discs, and myotendinous and neuromuscular junctions. In sections of skeletal muscle and in isolated myofibrils, the 215-kDa protein was localized to the I-band. By immunoprecipitation and immunoblot analysis, we determined that the 215-kDa antigen cross-reacts with a polyclonal anti-tensin antibody.     

Structure and protein composition of sites of papillary muscle attachment to chordae tendineae in avian hearts

Summary Most cardiac myocytes transmit force across fasciae adherentes, specialized sites of cell-cell adhesion. However, some cardiac myocytes in papillary muscle terminate on collagenous connective tissue in the chordae tendineae. These papillary myotendinous junctions (MTJs) are specialized for force transmission from myocytes to extracellular matrix. In the present study, we compared structural molecules at papillary MTJs to those at fasciae adherentes and skeletal MTJs. By using indirect immunofluorescence, we found that papillary MTJs more closely resemble skeletal MTJs in their molecular composition in that they are enriched in talin, vinculin, integrin, and fibronectin. Zeugmatin and -actinin, both components of fasciae adherentes, are absent from papillary MTJs. Although papillary MTJs and skeletal MTJs display strong similarities in structural protein composition, ultrastructural organization of the two junctions is different. Papillary MTJs display little folding of the junctional membrane and, according to morphological criteria, more closely resemble sites of thin filament-membrane association in smooth muscle than skeletal MTJs. Thus, papillary MTJs display a combination of structural characteristics described previously in skeletal and smooth muscles but exhibit few structural features observed previously in cardiac fasciae adherentes.     

Mechanical loading regulates expression of talin and its mRNA, which are concentrated at myotendinous junctions

The hypothesis that mechanical loading regulates talinexpression in developing and adult muscle was tested using in vitro andin vivo models. Talin was selected for study because it is a keystructural link between the cytoskeleton and cell membrane. In the invitro model,C₂C₁₂myotubes were subjected to cyclic strains for 48 h. In the in vivomodel, rat hindlimb muscles were unloaded for 10 days, then reloadedfor 2 days. Cyclic loading of myotubes resulted in significantincreases in the quantity of talin (68%) and its 190-kDa proteolyticfragment (70%), as well as talin mRNA (180%), relative to unloadedmyotube cultures. Similarly, talin concentration and its mRNA increasedby 68 and 136%, respectively, in soleus muscles reloaded for 2 daysrelative to ambulatory controls. Immunohistochemistry and in situRT-PCR showed that talin and its mRNA are concentrated and colocalized at myotendinous junctions. Thus these findings indicate that increased mechanical loading promotes talin synthesis, which occurs principally at myotendinous junctions, according to talin mRNA distribution.

     

The myotendinous junction of the smooth feather muscles (mm. pennati)

Summary Smooth feather muscles (mm. pennati) consist of bundles of smooth muscle cells which are attached to the feather follicles by short elastic tendons. In addition, some muscle bundles are interrupted by elastic tendons. The elastic tendon is composed of longitudinally arranged elastic fibers which branch and wavy bundles of collagen fibrils. Smooth muscle cells of the muscle bundles are attached to each other by desmosome-like junctions and by fusion of the basal laminae. The cytoplasm of the muscle cells is characterized by conspicuous thick filaments and abundant thin and intermediate filaments. These are attached to band-like dense patches (dense bands) at the plasma membrane which are particularly broad at the tapering end of the muscle cell. The contact surface between smooth muscle cells and their elastic tendon is considerably increased (i) by deep finger-like invaginations and indentations located at the tapering muscle end, and (ii) by branching of the coarse elastic fibers into slender processes, which are attached to the richly folded surface of the muscle cell endings by peripheral microfibrils. This intimate interlocking closely resembles the myotendinous junctions in skeletal muscle. In addition to fibroblasts and fibrocytes, the myotendinous junction of the young growing chicks contains numerous so-called myofibroblasts, which are suggested to represent smooth muscle cells differentiating into fibroblasts of the developing tendon.Dedicated to Professor Dr. Helmut Leonhardt on the occasion of his 60th birthdaySupported by a grant from the Deutsche Forschungsgemeinschaft (Dr. 91/1)     

Alpha-actinin is absent from the terminal segments of myofibrils and from subsarcolemmal densities in frog skeletal muscle

The presence and distribution of alpha-actinin, an actin-bundling protein, was investigated at sites where frog skeletal muscle forms junctions with tendon collagen fibers. These sites, called myotendinous junctions, are regions where myofibrils terminate and where the force of muscular contraction is transmitted from muscle cells to the substratum. An antibody manufactured to chicken smooth muscle alpha-actinin was used as a probe for alpha-actinin localization in this study. The cross-reactivity of this antibody with frog skeletal muscle alpha-actinin is demonstrated in immunoblots of one-dimensional (1D) electrophoretic separations of muscle proteins. Immunofluorescent localization of anti-alpha-actinin and electron microscopic immunolabelling confirms that the antibody binds to Z-discs with high affinity. However, in sections treated for electron microscopy with affinity-purified anti-alpha-actinin and a ferritin-conjugated, second antibody, there was no significant difference between experimental or control preparations in the number of ferritin grains overlying dense, subsarcolemmal material at junctional or non-junctional regions. Furthermore, Z-discs near myotendinous junctions displayed less binding of anti-alpha-actinin than Z-discs located several micrometers or more from the cells' termini. These findings indicate that thin filaments are not bundled by alpha-actinin near the sarcolemma. The results also provide evidence for molecular heterogeneity between Z-discs at the ends of muscle cells compared with other regions of the cell in that the terminal Z-discs of myofibrils contain very little or no alpha-actinin relative to non-terminal Z-discs.     

Early innervation of skeletal muscle during tail regeneration in urodele amphibians.

The innervation pattern of skeletal muscles was studied in the normal and regenerating tail of Notophthalmus viridescens. Silver staining for nerve endings and histochemical localization of acetylcholinesterase (AChE) were used for light microscopy. In In normal musculature, AChE positive reactions were localized at the ends of the muscle fibers where they are anchored on connective tissue septa by myotendinous junctions. At this level, silver staining shows nerve terminals forming endplates. During regeneration, positive reactions for AChE appear de novo as dense plates localized at the ends of the newly formed myotubes. The mechanisms involved in the localization of AChE on this surface seem to operate before previous local contacts by nerve terminals. From the ultrastructural data and immunohistochemical results with anti-laminin antibody, these observations suggest that regenerating muscle fibers determine a region of post-synaptic specialization in close relation with the organization of myotendinous regions and basement membrane formation. Nerve-muscle contacts appear at these levels at stage IV (15-20 days after amputation) in the stump and in the rostral part of the regenerate (transition zone). These nerve terminals are provided by the disorganized peripheral nervous system of the injured segment. In the regenerate a similar pattern of AChE reaction can be seen in every myotube, differentiating according to a rostro-caudal gradient. Innervation at the ends of the muscle fibers is in spatiotemporal relation with the exists of the ventral roots from the regenerating nerve cord as the regenerate continues to grow in length.     

Cell/substratum adhesions in RSV-transformed rat fibroblasts

Cell/substratum adhesions have been studied in rat fibroblasts transformed by a ts-mutant of Rous sarcoma virus (LA-29) using light and electron microscopy and a variety of preparative methods including immunolabeling. Cells were studied both during the process of transformation, i.e., shifting from 39 degrees to 35 degrees C, and in a fully transformed state (passaged at 35 degrees C continuously). The typical focal contacts observed at 39 degrees C (restrictive temperature) were replaced by "point-contacts" (100-200 per cell) which were classified by immunolabeling as podosome-like adhesions containing actin, beta 1 integrin subunit, vinculin, talin, alpha-actinin, and small membrane patches containing clathrin and integrin. Tyrosine-phosphorylated proteins and pp60src were found in association with groups of small particles on the protoplasmic surface of ventral membranes by gold immunolabeling. Both types of point-contacts were visualized by electron microscopy of ultrathin sections and shadowed replicas and characterized by gold immunolabeling wherever possible. The overall composition of podosome-like adhesions is similar to focal contacts but there are differences in the three-dimensional organization of the microfilaments and in the topography of vinculin which is associated more with actin filaments than with the plasma membrane. The presence of talin and extracellular matrix receptor in podosomes together with the adhesive properties of these actin-containing structures argues against the hypothesis that pp60src affects the interaction of actin with the plasma membrane by phosphorylating the fibronectin receptor and/or other associated proteins.