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At least 19 recordsLinked to original sources
PubMed1992

A unique talin antigenic determinant and anomalous megakaryocyte talin distribution associated with abnormal platelet formation in the Wistar Furth rat.

Rats of the Wistar Furth (WF) strain have hereditary macrothrombocytopenia with decreased platelet alpha-granule proteins. The autosomal recessive pattern of inheritance of the large mean platelet volume (MPV) phenotype and platelet alpha-granule protein deficiencies suggest that a component common to both formation of platelet alpha-granules and subdivision of megakaryocyte cytoplasm into platelets is quantitatively or qualitatively abnormal in WF megakaryocytes and platelets. We examined WF platelets for such an abnormality using electrophoretic and immunologic analyses. Rabbit antiserum prepared against WF rat platelets and absorbed with Wistar rat platelets recognized a major 235-Kd band, and minor bands of WF rat platelets ranging from 200 to 130 Kd, not present in immunoblots of Wistar, Sprague-Dawley, or Long-Evans rat platelets. The minor bands were labeled with affinity-isolated antibody to the 235-Kd band, indicating that all bands contained the same unique antigenic site. The 235-Kd antigen had the same mobility as rat platelet talin identified with a platelet antitalin antibody. Activation of calcium-dependent proteases during Triton X-100 extraction caused conversion of the 235-Kd antigen into a major fragment of 200 Kd and minor fragments ranging to 115 Kd, identical in mobility to fragments of rat platelet talin produced in the same samples. The absorbed anti-WF platelet antiserum also detected a 235-Kd antigen in WF lung, kidney, and small intestine by immunoblotting. Finally, the 235-Kd antigen unique to WF rats was immunoprecipitated from Triton X-100 supernatants of WF platelets with an antitalin monoclonal antibody (MoAb). These data indicate that the unique antigenic site is on WF talin. Examination of talin distribution in Wistar megakaryocytes showed localization beneath the plasma membrane, on the cytosolic face of demarcation membranes, associated with alpha-granule membranes, and diffusely throughout the cytoplasm. Although WF megakaryocytes showed the same general distribution pattern, some differences were apparent. In contrast to membrane systems of the Wistar rat, the large membrane complexes in WF megakaryocytes contained little or no talin. In addition, approximately half of WF megakaryocytes showed an increased peripheral localization of talin, often associated with membrane blebs, with decreased talin in the cytoplasmic interior. The association of the unique talin antigenic determinant and anomalous megakaryocyte talin distribution with abnormal platelet formation in WF rats suggests that talin is abnormal in this rat strain and that talin plays an important role in subdivision of megakaryocyte cytoplasm into platelets.

Animals↗
PubMed1992

Probing actin and liposome interaction of talin and talin-vinculin complexes: a kinetic, thermodynamic and lipid labeling study.

Talin purified from human platelets and chicken gizzard smooth muscle is an actin and lipid binding protein. Here, we have investigated the effect of vinculin on (a) talin-nucleated actin polymerization and (b) insertion of talin into lipid bilayers. Calorimetric data show ternary complex formation between talin, vinculin, and actin. Actin-talin, actin-vinculin and actin-(talin-vinculin) binding and rate constants as well as actin polymerization rates for all three protein species have been determined by steady state titration, stopped-flow, and fluorescence assay. In contrast to an increase of the polymerization rate by a factor of less than 2 for actin-talin and actin-(talin-vinculin) when lowering the temperature, we measured a decrease in rates for actin alone and actin-vinculin. The overall equilibrium constants (Keq) in the van't Hoff plot proved linear and were of one-step reactions. Thermodynamic data exhibited signs of van der Waal's binding forces. Using the photoactivatable lipid analogue [3H]PTPC/11, which selectively labels membrane-embedded hydrophobic domains of proteins, we also show that talin partially inserts into the hydrophobic bilayer of liposomes. This insertion occurs in a similar manner irrespective of preincubation with vinculin.

Actins↗
PubMed1986

Demonstration of a relationship between talin and P235, a major substrate of the calcium-dependent protease in platelets.

Talin is a 225,000-Dalton protein we have purified from smooth muscle. In chick embryo fibroblasts talin is found in adhesion plaques (focal contacts), areas where the cell is closely apposed to the substratum. In comparison with other cytoskeletal proteins, we found talin to be unusually susceptible to proteolysis and have identified a 190,000-Dalton proteolytic fragment of talin in the immunoblots of many tissues. These observations raised the possibility that the cleavage of talin to this fragment has physiological relevance. One system that we have investigated in which significant proteolysis occurs is platelets. During platelet activation several high-molecular-weight proteins are cleaved to lower-molecular-weight forms. Here we demonstrate that talin is closely related to one of these platelet high-molecular-weight proteins, P235. The purification of talin is comparable to that developed for P235, and the two proteins have similar biophysical properties. In addition, antibodies raised against chicken gizzard talin recognize P235 in purified form as well as in crude platelet extracts. The platelet protein also resembles smooth-muscle talin in its susceptibility to endogenous proteolysis: P235 is rapidly cleaved to a 190-200 kD polypeptide by a calcium-activated protease found in platelet extracts. Moreover, partial proteolysis of P235 and talin with chymotrypsin, elastase, or trypsin also generates remarkably similar one-dimensional peptide maps. Because of their similar biophysical properties, immunological crossreactivity, and similar one-dimensional partial peptide maps, we conclude that P235 is the platelet form of talin.

Animals↗
PubMed1990

Functional studies of the domains of talin.

The protein talin has two domains of approximately 200 and 47 kD, which can be cleaved apart by a variety of proteases. To examine the function of these two structural domains of talin, we have digested purified talin with a calcium-dependent protease and separated the resulting fragments chromatographically. Both fragments were radioiodinated and used to probe Western blots of whole fibroblasts and chicken gizzard extracts. The large talin fragment bound to vinculin and metavinculin. The small fragment did not demonstrate any binding in this assay. The fragments were labeled fluorescently and microinjected into fibroblasts in tissue culture. The large talin fragment incorporated quickly into focal adhesions where it remained stable for at least 14 h. The small fragment associated with focal adhesions of fibroblasts but was also distributed diffusely in the cytoplasm and the nucleus. These experiments suggest that talin has at least two sites that contribute to its localization in focal adhesions. Intact talin microinjected into Madin-Darby bovine kidney epithelial cells localized to the focal adhesions but was excluded from the zonulae adherentes, despite the localization of vinculin to both of these sites. In contrast, the large talin fragment, when microinjected into these epithelial cells, incorporated into both focal adhesions and zonulae adherentes. The difference in localization between the large talin fragment and intact talin seems to be due to the removal of the small domain. This difference in localization suggests that talin binding sites in zonulae adherentes have limited accessibility.

Actins↗
PubMed1990

The adhesion plaque protein, talin, is phosphorylated in vivo in chicken embryo fibroblasts exposed to a tumor-promoting phorbol ester.

Talin is a high molecular weight phosphoprotein that is localized at adhesion plaques. We have found that talin phosphorylation increases 3.0-fold upon exposure of chicken embryo fibroblasts to the tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate. Talin isolated from tumor promoter-treated cells is phosphorylated on serine and threonine residues. Vinculin, a 130 kDa talin-binding protein, also exhibits increased phosphorylation in vivo in response to tumor promoter, but to a lesser degree than does talin. Because tumor-promoting phorbol esters augment protein kinase C activity, we have compared the ability of purified protein kinase C to phosphorylate talin and vinculin in vitro. Both talin and vinculin were found to be substrates for protein kinase C; however, talin was phosphorylated to a greater extent than was vinculin. Cleavage of protein kinase C-phosphorylated talin by the calcium-dependent protease (Type II) revealed that while both the resulting 190-200 and 46 kDa proteolytic peptides were phosphorylated, the majority of label was contained within the 46-kDa fragment. Although incubation of chicken embryo fibroblasts with tumor-promoting phorbol ester induces a dramatic increase in talin phosphorylation, we detected no change in the organization of stress fibers and focal contacts in these cells. Exposure of the cells to tumor promoter did, however, result in a loss of actin and talin-rich cell surface elaborations that resemble focal contact precursor structures.

Animals↗
PubMed1992

Vinculin binding site mapped on talin with an anti-idiotypic antibody.

Vinculin and talin are major adhesion plaque components which interact in vitro and presumably in vivo. The amino acid sequence of talin is now known so details of its domain structure can be mapped. We localized vinculin binding sites in the talin sequence by overlaying peptide maps of talin with an anti-idiotypic vinculin antibody that recognizes talin and with 125I-vinculin. A rabbit injected only twice with vinculin and producing anti-vinculin antibodies spontaneously generated a second antibody that recognizes talin. Vinculin and anti-vinculin antibodies specifically compete with this second antibody for binding to talin as determined by solid-phase binding and overlay assays. The antibody is thus most likely an anti-idiotypic antibody which mimics a region of vinculin that interacts with talin. The binding site of the anti-idiotypic antibody on talin was mapped to the 196 amino acids spanning residues 1653 to 1848. A second vinculin binding site identified with an 125I-vinculin blot overlay technique was located between residues 483 and 1652. The observation that talin has two immunologically distinct vinculin binding sites suggests that vinculin may have two different talin binding sites or one "complex" site with two interacting regions.

Amino Acid Sequence↗
PubMed1989

Talin dynamics in living microinjected nonmuscle cells.

To investigate the role of talin in the anchoring of actin-containing stress fibers to the cell membrane of nonmuscle cells, a fluorescent analog of the adhesion plaque protein talin was developed, characterized, and microinjected into living cells. Purified chicken gizzard talin was covalently labeled with the fluorescent dye lissamine rhodamine B sulfonyl chloride. The fluorescently labeled protein was then chromatographed on Sephadex G-25 and DEAE-cellulose in order to remove free dye and denatured protein. The fluorescent talin was able to bind purified vinculin and was localized in adhesion plaques, membrane ruffles, microspikes, and polygonal networks in acetone-permeabilized nonmuscle cells. In cells that were double-stained with fluorescent talin and an affinity-purified anti-talin antibody, a one-to-one correspondence of adhesion plaque staining was seen. Living epithelial cells (PtK2) were microinjected during interphase with fluorescent talin. Computer-enhanced video microscopy was used to document adhesion plaque dynamics such as 1) changes in plaque shape, 2) alterations in plaque positions, and 3) the appearance, growth, and dissolution of plaques. In cells that were followed during mitosis, the adhesion plaques disappeared during cell rounding and then subsequently reappeared upon spreading of the two daughter cells. Treatment of microinjected cells with DMSO in order to disassemble stress fibers resulted in an altered localization of the fluorescent talin. Upon recovery of the cell from the drug, the talin was visualized in its characteristic submembraneous position. These results are the first to document the role and distribution of talin in dynamic processes occurring in living microinjected nonmuscle cells.

Animals↗
PubMed1990

Direct interactions between talin and actin.

Talin was purified from chicken gizzard by a modification of the method of L. Molony et al. [J. Biol. Chem.(1987) 262, 7790-7795]. Unlike the talin purified by the previous method, the talin purified by the new method was found to bind to both F- and G-actin: Talin cosedimented with F-actin. On gel filtration of a mixture of talin and G-actin, a complex of talin and action was obtained. Talin stimulated the polymerization rate of G-actin. A major proteolytic fragment of talin that retained the binding ability to F-actin was also identified. These results indicate that talin can bind directly to actin and suggest that talin plays a key role in the organization of actin filaments at the actin-membrane attachment sites in vivo also.

Actins↗
PubMed1991

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.

Actins↗
PubMed1992

Microinjection of antibodies against talin inhibits the spreading and migration of fibroblasts.

Talin is believed to be one of the key proteins involved in linking actin filaments to extracellular matrix receptors in focal adhesions. Our strategy for studying the function of talin has been to inactivate talin in living fibroblasts in tissue culture through the microinjection of affinity-purified, polyclonal anti-talin antibodies. The effect of the injected anti-talin antibodies on cell spreading was found to depend on how recently the cells had been plated. Cells that were in the process of spreading on a fibronectin substratum, and which had newly developed focal adhesions, were induced to round up and to disassemble many of the adhesions. However, if fibroblasts were allowed to spread completely before they were microinjected with the anti-talin antibody, focal adhesions remained intact and the flat morphology of the cells was unaffected. The percentage of cells that were able to maintain a spread morphology despite the injection of anti-talin antibodies increased during the first few hours after plating on fibronectin substrata. Fibroblasts that were allowed to spread completely before microinjection with the anti-talin antibody retained both intact focal adhesions and a flat, well-spread morphology, but failed to migrate effectively. Our experiments do not directly address the role of talin in mature focal adhesions, but they indicate that talin is essential for the spreading and migration of fibroblasts on fibronectin as well as for the development and initial maintenance of focal adhesions on this substratum.

Animals↗
PubMed1990

Purification and characterization of an 85 kDa talin-binding fragment of vinculin.

Vinculin and talin are adhesion plaque proteins which have been shown to interact with each other in vitro. In order to begin to investigate where the talin-binding domain is in vinculin, vinculin was digested with Staphylococcus aureus V8 protease to generate two major fragments of 85 and 30 kDa, and these fragments were purified. Nitrocellulose overlays with 125I-talin and the 125I-85 kDa vinculin fragment and sucrose density gradient centrifugation demonstrated that the talin-binding domain was localized to the 85 kDa vinculin fragment. Quantification of 125I-talin binding in the overlays showed that four times more talin bound to the 85 kDa fragment as compared to intact vinculin. Competitive immunoprecipitation experiments demonstrated that unlabeled 85 kDa fragment was about three-fold more effective at competing for 125I-85 kDa binding to talin than was unlabeled vinculin. These results suggest that the 30 kDa fragment inhibits the vinculin-talin interaction even though the talin-binding domain is localized in the 85 kDa fragment.

Binding Sites↗
PubMed1990

Identification and localization of talin in chick retinal pigment epithelial cells.

Retinal pigmented epithelial cells are adherent at their basal surface to Bruch's membrane and at their apical surface to the neural retina. We examined the expression and distribution of two proteins that are found in regions of cell-matrix interaction, talin and integrin. Talin is a 235-kDa cytoplasmic protein that has been localized to regions of cell-substrate adhesion. It binds to both integrin, a transmembrane glycoprotein complex, and to vinculin, a cytoskeletal protein. In the present study, we produced a polyclonal antibody to chicken gizzard talin. Using this antibody we showed by western blot analysis that talin is expressed by RPE cells and is found in the triton-soluble fraction. Talin was shown to co-localize with integrin and vinculin in the basal region of chick RPE cells isolated from 18-day-old chick embryos. Neither talin nor integrin was found in the apical processes or in the zonula adherens. Antibodies to vinculin showed staining both in the apical and basal regions of the RPE cells. The localization of integrin, talin and vinculin along the basal membrane suggests that this complex is important in the attachment of the RPE cells to the basement membrane. The distribution of integrin and talin was examined in primary cultures of RPE cells grown on permeable filters. In these cells, a polarized distribution of integrin and talin was not observed. This may suggest that the neural retina may be important for maintaining the differentiated state of the RPE cells.

Animals↗
PubMed1990

Sequence and domain structure of talin.

Talin is a high-molecular-weight cytoskeletal protein concentrated at regions of cell-substratum contact and, in lymphocytes, at cell-cell contacts. Integrin receptors are involved in the attachment of adherent cells to extracellular matrices and of lymphocytes to other cells. In these situations, talin codistributes with concentrations of integrins in the cell surface membrane. Furthermore, in vitro binding studies suggest that integrins bind to talin, although with low affinity. Talin also binds with high affinity to vinculin, another cytoskeletal protein concentrated at points of cell adhesion. Finally, talin is a substrate for the Ca2(+)-activated protease, calpain II, which is also concentrated at points of cell-substratum contact. To learn more about the structure of talin and its involvement in transmembrane connections between extracellular adhesions and the cytoskeleton, we have cloned and sequenced murine talin. We describe a model for the structure of talin based on this sequence and other data. Homologies between talin and other proteins define a novel family of submembranous cytoskeleton-associated proteins all apparently involved in connections to the plasma membrane.

Amino Acid Sequence↗
PubMed1989

Activation-dependent redistribution of the adhesion plaque protein, talin, in intact human platelets.

Talin is a high molecular weight protein localized at adhesion plaques in fibroblasts. It binds vinculin and integrin and appears to participate in generating a transmembrane connection between the extracellular matrix and the cytoskeleton. We have recently shown that talin is an abundant protein in platelets, cells highly specialized for regulated adhesion. Although talin constitutes greater than 3% of the total protein in intact human platelets, its location within the cells had not been defined. In the work reported here, we have investigated the distribution of talin in resting and activated human platelets by immunofluorescence and immunoelectron microscopy. We have found that talin undergoes an activation-dependent change in its subcellular location. In resting platelets, which are nonadhesive, talin is uniformly distributed throughout the cytoplasm. In contrast, in thrombin- and glass-activated, substratum-adherent platelets, talin is concentrated at the cytoplasmic face of the plasma membrane. This dramatic, regulated redistribution of talin raises the possibility that talin plays a role in the controlled development of platelet adhesion.

Blood Platelets↗
PubMed1986

Talin is a post-synaptic component of the rat neuromuscular junction.

Talin is a protein, recently discovered in chicken gizzard, which occurs at sites of actin-plasma membrane interaction in several cell types. Vinculin also occurs at many of these sites, possibly in association with talin. In this study, three antisera against talin were used to probe the neuromuscular junction of rat skeletal muscle, which is also a site of vinculin accumulation. By immunofluorescence, all three sera stained the junction strongly in frozen sections of rat diaphragm. The extrajunctional periphery was lightly and irregularly stained in some muscle cells; others seemed not to be stained outside the junction. Staining remained at junctions and increased in extrajunctional regions of muscle denervated 6 weeks before sacrifice. The staining in all cases was abolished by competition with purified talin. One serum tested by immunoblotting recognized one protein at Mr 215 000 (identical with the value for chicken gizzard talin) and traces of a second at Mr 190 000 (corresponding to a known proteolytic fragment of talin). We conclude that rat muscle talin is similar in its general protein structure to chicken gizzard talin, and is a post-synaptic component of the neuromuscular junction.

Animals↗
PubMed1986

Purification of a 190 kDa protein from smooth muscle: relationship to talin.

Several studies of vinculin-binding proteins have described a 190 kDa protein in chicken gizzard smooth muscle which binds radioiodinated vinculin. We have purified and studied the 190 kDa protein from chicken gizzard smooth muscle. By indirect immunofluorescence, an antiserum raised against the 190 kDa protein stains adhesion plaques (focal contacts), ruffling membranes, and fibrillar streaks on the dorsal and ventral surfaces of fibroblasts. Both the binding to vinculin and the location of the protein in fibroblasts are properties shared with talin, a 215 kDa protein in smooth muscle and fibroblasts. Because antisera against talin and the 190 kDa cross-react the relationship of these two proteins has been investigated further. Upon prolonged storage at 4 degrees C, purified talin degrades into a 190 kDa fragment. A 190 kDa fragment is also generated from talin by the Staphylococcus aureus V-8 proteinase and by trypsin. Comparison of partial peptide maps of talin and the 190 kDa protein reveal that the proteins are very similar and when the 190 kDa fragment of talin is compared with the purified 190 kDa protein by partial proteolytic digestion no differences are found in the pattern of peptides generated. In addition, the amount of 190 kDa protein detected in muscle tissues excised from chick embryos can be drastically reduced if proteinase inhibitors are added to the tissue homogenates. We conclude that the purified 190 kDa dalton protein is a proteolytic fragment of talin. Although markedly reduced by proteinase inhibitors, detection of the 190 kDa protein is not completely abolished, suggesting that some talin may already be cleaved within living cells.

Animals↗