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PubMed1992

Augmentation of alpha-actinin-induced gelation of actin by talin.

Interactions among the three major constituents of focal adhesions, talin, actin, and alpha-actinin, were studied. No evidence was obtained for the direct interaction between talin and alpha-actinin. Both talin and alpha-actinin increased the rate and extent of polymerization of actin, and their effects were additive. Whereas talin alone exhibited very little actin-gelating activity, it potentiated markedly the gelation in the presence of alpha-actinin and lowered the concentration of alpha-actinin necessary for the gel formation. Its gelation-potentiating activity on prepolymerized actin was much smaller than observed on G-actin. Treatment of talin with a cross-linking reagent, 1-ethyl-3[3-(dimethylamino)propyl]carbodiimide or dimethyl suberimidate, resulted in the formation of its oligomeric polypeptides. The complexes of talin and G-actin were also demonstrated with the cross-linking reagents and fluorescence-labeled actin. These results indicate that talin is able to cross-link some limited regions of actin filaments.

Actinin↗
PubMed1990

The PMA-induced specific association of LFA-1 and talin in intact cloned T helper cells.

Previous experiments with individual cell couples formed between cloned T helper (Th) cells and antigen-presenting cells have led us to suggest that the cytoskeletal protein talin may be associated with the cell surface protein LFA-1 in the Th cell. In order to examine this suggestion, we induced the surface capping of LFA-1 with suitable specific antibody reagents on the intact Th cells, and then determined by double immunofluorescence microscopic experiments, whether talin was co-clustered with the LFA-1 caps. With untreated Th cells, capping of LFA-1 did not result in any redistribution of intracellular talin. However, if the intact Th cells were treated with the phorbol ester PMA, the capping of LFA-1 resulted in a co-clustering of talin with the LFA-1 caps, but not a alpha-actinin. The capping of TCR or CD4 on the Th cells with or without pretreatment with PMA did not lead to any such co-clustering of talin with these caps. PMA treatment of the Th cells therefore induces a direct or indirect association of talin with LFA-1 underneath the Th cell surface. PMA treatment of the Th cells also increased their polarized spreading and adherence to substrata, as had been observed before. We found, furthermore, that this increased adherence upon PMA-treatment was inhibited by the presence of antibodies to LFA-1. The association of talin, and very likely also F-actin microfilaments, with LFA-1 appears to mediate a generalized increased adhesivity of the Th cells. The relevance of these findings with isolated Th cells to the interaction of Th cells with specific antigen-presenting cells is discussed.

Animals↗
PubMed1999

The behavior of calpain-generated N- and C-terminal fragments of talin in integrin-mediated signaling pathways.

Our previous results showed that the binding of an adhesive ligand to integrin alphaIIbbeta(3) on the surface of platelets triggers the activation of calpain and the limited proteolysis of talin by calpain. To explore the physiological significance of the calpain-mediated cleavage of talin, we analyzed the behavior of the calpain-generated fragments of talin (N-terminal 47 kDa and C-terminal 190 kDa) during platelet activation by biochemical and immunoelectron microscopic studies. Intact talin and micro-calpain translocate from the Triton X-100-soluble fraction to the insoluble fraction upon platelet stimulation by thrombin, and the limited proteolysis of talin occurs in the Triton X-100-insoluble fraction, the cytoskeletal fraction. The fully autolyzed 76-kDa micro-calpain (active form) is found predominantly in the Triton X-100-insoluble fraction in stimulated platelets. While the N-terminal 47-kDa fragment remains in the Triton X-100-insoluble fraction, the C-terminal 190-kDa fragment is released into the Triton X-100-soluble fraction in a time-dependent manner. Immunoelectron microscopic observations revealed that the 47-kDa fragment locates on the submembrane zone just beneath the plasma membrane, including the open canalicular systems, while most of the 190-kDa fragment exists diffusely in the cytoplasm in thrombin-stimulated platelets. These findings suggest that calpain may contribute to the reorganization of the cytoskeleton in an integrin-mediated signaling pathway through the redistribution of the functional domain of talin.

Amino Acid Sequence↗
PubMed1996

Talin can crosslink actin filaments into both networks and bundles.

The talin-actin interaction was examined by using negative staining and cosedimentation assays. At pH 6.4 and low ionic strength, talin extensively crosslinked actin filaments into both networks and bundles. The bundles consist of parallel actin filaments with a center-to-center distance of 13 nm, and talin crossbridges spaced at 36-nm intervals along the bundles. As pH was increased stepwise from 6.4 to 7.3, talin's bundling activity was decreased first, then its networking activity. Qualitatively similar results were obtained at pH 6.4 by increasing ionic strength. Chemical crosslinking indicated talin was present as a dimer from pH 6.4 to 7.3, with or without added KC1. The results show that talin can interact directly with actin filaments by formation of actin filament networks and bundles, with the bundles more sensitive to dissolution by increase in pH or ionic strength.

Actins↗
PubMed1991

Human platelet P-235, a talin-like actin binding protein, binds selectively to mixed lipid bilayers.

The interaction of platelet talin (P-235) with mixtures of dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG) and dimyristoylphosphatidylserine (DMPS) as well as with pure lipids was studied in reconstituted lipid bilayers. Incorporation of platelet talin into vesicles was achieved by self-assembly during cycles of freeze-thawing of co-dispersions containing vesicles and the purified protein. The yield of protein incorporation as a function of lipid composition was determined by measuring the protein/lipid ratio using protein assay, phosphate determination and gel electrophoresis in parallel. Protein-lipid interactions are monitored by high sensitive differential scanning calorimetry (DSC) measuring (i) the shifts of transition states delta Ts* and delta Tl*, where Ts represents the solidus line, the onset of lipid chain melting, and Tl the liquidus line, the endpoint of chain melting, and (ii) the heats of transition. Cytoplasmic talin differs from a membrane bound form by its ability and mode of lipid interaction. The latter partially penetrates into the hydrophobic region of the bilayer, which renders a low incorporation rate even into neutral lipids. This interaction is greatly enhanced in the presence of charged lipids: a marked shift of Tl occurs due to a selective electrostatic interaction of the protein with the membrane surface. Evidence for a selective binding is also provided by Fourier transform infrared spectroscopy (FTIR). Right-side-out oriented platelet talin can be cleaved by proteinases, which truncate the extrinsic electrostatic binding domain but not the hydrophobic. In addition, reconstituted platelet talin, like in vivo, can be cleaved by thrombin. The interaction of cytoplasmic platelet talin with lipid bilayers is purely electrostatic. Our data suggest that protein reconstitution by freeze-thawing is an equilibrium process and that the protein distribution between the membrane and water is determined by the Nernst distribution law. Consequently, the work of protein transfer from water into the bilayer can be measured as a function of charged lipids.

Blood Proteins↗
PubMed1990

Spatio-temporal distribution of the adherens junction-associated molecules vinculin and talin in the early avian embryo.

To gain an insight into the possible involvement of the cytoskeletal components and cellular junctions in morphogenetic processes during development, we have studied the spatio-temporal distribution of two major adherens-junction-associated molecules, vinculin and talin, during avian embryogenesis, using immunofluorescence microscopy and immunoblotting. Both molecules were detected at very early stages during morphogenesis and were found in a wide variety of tissues deriving from the three primary germ layers. A number of tissues, including smooth and striated muscles, endothelia, and some hemopoietic precursors, expressed vinculin and talin at especially high levels either transiently or permanently. Conversely, only a few cell types, e.g., circulating erythrocytes and neurones in the central nervous system lacked or expressed them at very low levels. In addition, expression of vinculin and talin was in some cases modulated in connection with morphological rearrangements of tissues. In particular, they were transiently enhanced in restricted areas of the ectoderm and endoderm undergoing extensive foldings. However, other morphogenetic events such as local disruptions of epithelia were not accompanied by extensive modifications in their expression. Finally, it appeared that, in most cases, vinculin and talin overlapped in their distribution, and the level of their expression was regulated coincidently with the notable exceptions of the primordium of the central nervous system, the nephron, and the liver where each molecule followed independent regulatory patterns. It appears from this study that the spatio-temporal distribution of vinculin and talin correlates frequently with that of the adhesion molecules A-CAM (or N-cadherin), L-CAM, and of integrin receptors. Thus, vinculin and talin, in association with the membrane components of adherens junctions, may actively participate both in the control of cellular interactions during early embryonic development and in cell differentiation.

Animals↗
PubMed2007

Structural basis of integrin activation by talin.

Regulation of integrin affinity (activation) is essential for metazoan development and for many pathological processes. Binding of the talin phosphotyrosine-binding (PTB) domain to integrin beta subunit cytoplasmic domains (tails) causes activation, whereas numerous other PTB-domain-containing proteins bind integrins without activating them. Here we define the structure of a complex between talin and the membrane-proximal integrin beta3 cytoplasmic domain and identify specific contacts between talin and the integrin tail required for activation. We used structure-based mutagenesis to engineer talin and beta3 variants that interact with comparable affinity to the wild-type proteins but inhibit integrin activation by competing with endogenous talin. These results reveal the structural basis of talin's unique ability to activate integrins, identify an interaction that could aid in the design of therapeutics to block integrin activation, and enable engineering of cells with defects in the activation of multiple classes of integrins.

Amino Acid Sequence↗
PubMed2005

A vinculin binding domain from the talin rod unfolds to form a complex with the vinculin head.

The cytoskeletal protein talin plays a key role in activating integrins and in coupling them to the actin cytoskeleton. Its N-terminal globular head, which binds beta integrins, is linked to an extended rod having a C-terminal actin binding site and several vinculin binding sites (VBSs). The NMR structure of residues 755-889 of the rod (containing a VBS) is shown to be an amphipathic four-helix bundle with a left-handed topology. A talin peptide corresponding to the VBS binds the vinculin head; the X-ray crystallographic structure of this complex shows that the residues which interact with vinculin are buried in the hydrophobic core of the talin fragment. NMR shows that the interaction involves a major structural change in the talin fragment, including unfolding of one of its helices, making the VBS accessible to vinculin. Interestingly, the talin 755-889 fragment binds more than one vinculin head molecule, suggesting that the talin rod may contain additional as yet unrecognized VBSs.

Actins↗
PubMed2003

Vinculin activation by talin through helical bundle conversion.

Vinculin is a conserved component and an essential regulator of both cell-cell (cadherin-mediated) and cell-matrix (integrin-talin-mediated focal adhesions) junctions, and it anchors these adhesion complexes to the actin cytoskeleton by binding to talin in integrin complexes or to alpha-actinin in cadherin junctions. In its resting state, vinculin is held in a closed conformation through interactions between its head (Vh) and tail (Vt) domains. The binding of vinculin to focal adhesions requires its association with talin. Here we report the crystal structures of human vinculin in its inactive and talin-activated states. Talin binding induces marked conformational changes in Vh, creating a novel helical bundle structure, and this alteration actively displaces Vt from Vh. These results, as well as the ability of alpha-actinin to also bind to Vh and displace Vt from pre-existing Vh-Vt complexes, support a model whereby Vh functions as a domain that undergoes marked structural changes that allow vinculin to direct cytoskeletal assembly in focal adhesions and adherens junctions. Notably, talin's effects on Vh structure establish helical bundle conversion as a signalling mechanism by which proteins direct cellular responses.

Adherens Junctions↗
PubMed2004

Calpain-mediated proteolysis of talin regulates adhesion dynamics.

Dynamic regulation of adhesion complexes is required for cell migration and has therefore emerged as a key issue in the study of cell motility. Recent progress has been made in defining some of the molecular mechanisms by which adhesion disassembly is regulated, including the contributions of adhesion adaptor proteins and tyrosine kinases. However, little is known about the potential contribution of proteolytic mechanisms to the regulation of adhesion complex dynamics. Here, we show that proteolysis of talin by the intracellular calcium-dependent protease calpain is critical for focal adhesion disassembly. We have generated a single point mutation in talin that renders it resistant to proteolysis by calpain. Quantification of adhesion assembly and disassembly rates demonstrates that calpain-mediated talin proteolysis is a rate-limiting step during adhesion turnover. Furthermore, we demonstrate that disassembly of other adhesion components, including paxillin, vinculin and zyxin, is also dependent on the ability of calpain to cleave talin, suggesting a general role for talin proteolysis in regulating adhesion turnover. Together, these findings identify calpain-mediated proteolysis of talin as a mechanism by which adhesion dynamics are regulated.

Animals↗
PubMed1986

Talin is phosphorylated on tyrosine in chicken embryo fibroblasts transformed by Rous sarcoma virus.

We have examined the extent of tyrosine phosphorylation of talin, a component of the cytoskeleton localized in the focal adhesions and, therefore, a potential substrate of p60v-src, the transforming protein of Rous sarcoma virus. p60v-src is a tyrosine kinase that induces high levels of phosphotyrosine and the disorganization of the cytoskeleton in transformed cells. With a polyclonal antibody utilized in a previous study [Maher, P. A., Pasquale, E. B., Wang, J. Y. J. & Singer, S. J. (1985) Proc. Natl. Acad. Sci. USA 82, 6576-6580] for the detection of tyrosine-phosphorylated proteins, we have detected phosphotyrosine residues in talin molecules immunoprecipitated from Rous sarcoma virus-transformed, but not normal, chicken embryo fibroblasts. Phospho amino acid analysis of talin from the infected cells confirmed the presence of phosphotyrosine, in addition to phosphoserine and phosphothreonine. The extent of tyrosine modification in talin was compared to that in vinculin, the other focal adhesion component previously found to contain enhanced levels of phosphotyrosine in various retrovirus-transformed cells. A considerably (3 times) larger fraction of the talin than of the vinculin molecules was found to be phosphorylated on tyrosine. The phosphorylation of talin on tyrosine may be crucial for the expression of the abnormal morphology characteristic of cells transformed by Rous sarcoma virus.

Animals↗
PubMed2004

A fluorescence cell biology approach to map the second integrin-binding site of talin to a 130-amino acid sequence within the rod domain.

The cytoskeletal protein talin, which provides a direct link between integrins and actin filaments, has been shown to contain two distinct binding sites for integrin beta subunits. Here, we report the precise delimitation and a first functional analysis of the talin rod domain integrin-binding site. Partially overlapping cDNAs covering the entire human talin gene were transiently expressed as DsRed fusion proteins in Chinese hamster ovary cells expressing alpha(IIb)beta(3), linked to green fluorescent protein (GFP). Two-color fluorescence analysis of the transfected cells, spread on fibrinogen, revealed distinct subcellular staining patterns including focal adhesion, actin filament, and granular labeling for different talin fragments. The rod domain fragment G (residues 1984-2344), devoid of any known actin- or vinculin-binding sites, colocalized with beta(3)-GFP in focal adhesions. Direct in vitro interaction of fragment G with native platelet integrin alpha(IIb)beta(3) or with the recombinant wild type, but not the Y747A mutant beta(3) cytoplasmic tail, linked to glutathione S-transferase, was demonstrated by surface plasmon resonance analysis and pull-down assays, respectively. Here, we demonstrate for the first time the in vivo relevance of this interaction by fluorescence resonance energy transfer between beta(3)-GFP and DsRed-talin fragment G. Further in vitro pull-down studies allowed us to map out the integrin-binding site within fragment G to a stretch of 130 residues (fragment J, residues 1984-2113) that also localized to focal adhesions. Finally, we show by a cell biology approach that this integrin-binding site within the talin rod domain is important for beta(3)-cytoskeletal interactions but does not participate in alpha(IIb)beta(3) activation.

Actins↗
PubMed2004

Competition for talin results in trans-dominant inhibition of integrin activation.

The ability of integrin adhesion receptors to undergo rapid changes in affinity for their extracellular ligands (integrin activation) is essential for the development and function of multicellular animals and is dependent on interactions between the integrin beta subunit-cytoplasmic tail and the cytoskeletal protein talin. Cross-talk among different integrins and between integrins and other receptors impacts many cellular processes including adhesion, spreading, migration, clot retraction, proliferation, and differentiation. One form of integrin cross-talk, transdominant inhibition of integrin activation, occurs when ligand binding to one integrin inhibits the activation of a second integrin. This may be relevant clinically in a number of settings such as during platelet adhesion, leukocyte trans-migration, and angiogenesis. Here we report that competition for talin underlies the trans-dominant inhibition of integrin activation. This conclusion is based on our observations that (i). beta tails selectively defective in talin binding are unable to mediate trans-dominant inhibition, (ii). trans-dominant inhibition can be reversed by overexpression of integrin binding and activating fragments of talin, and (iii). expression of another non-integrin talin-binding protein, phosphatidylinositol phosphate kinase type Igamma-90, also inhibits integrin activation. Thus, the sequestration of talin by the suppressive species is both necessary and sufficient for trans-dominant inhibition of integrin activation.

Animals↗
PubMed2004

Structural basis for amplifying vinculin activation by talin.

Talin interactions with vinculin are essential for focal adhesions. Curiously, talin contains three noncontiguous vinculin binding sites (VBS) that can bind individually to the vinculin head (Vh) domain. Here we report the crystal structure of the human Vh.VBS1 complex, a validated model of the Vh.VBS2 structure, and biochemical studies that demonstrate that all of talin VBSs activate vinculin by provoking helical bundle conversion of the Vh domain, which displaces the vinculin tail (Vt) domain. Thus, helical bundle conversion is a structurally conserved response in talin-vinculin interactions. Furthermore, talin VBSs bind to Vh in a mutually exclusive manner but do differ in their affinity for Vh and in their ability to displace Vt, suggesting that the strengths of these interactions could lead to differences in signaling outcome. These findings support a model in which talin binds to and activates multiple vinculin molecules to provoke rapid reorganization of the actin cytoskeleton.

Amino Acid Sequence↗
PubMed1986

Talin at myotendinous junctions.

Junctions formed by skeletal muscles where they adhere to tendons, called myotendinous junctions, are sites of tight adhesion and where forces generated by the cell are placed on the substratum. In this regard, myotendinous junctions and focal contacts of fibroblasts in vitro are analogues. Talin is a protein located at focal contacts that may be involved in force transmission from actin filaments to the plasma membrane. This study investigates whether talin is also found at myotendinous junctions. Protein separations on SDS polyacrylamide gels and immunolabeling procedures show that talin is present in skeletal muscle. Immunofluorescence microscopy using anti-talin indicates that talin is found concentrated at myotendinous junctions and in lesser amounts in periodic bands over nonjunctional regions. Electron microscopic immunolabeling shows talin is a component of the digitlike processes of muscle cells that extend into tendons at myotendinous junctions. These findings indicate that there may be similarities in the molecular composition of focal contacts and myotendinous junctions in addition to functional analogies.

Animals↗
PubMed1997

Talin-null cells of Dictyostelium are strongly defective in adhesion to particle and substrate surfaces and slightly impaired in cytokinesis.

Dictyostelium discoideum contains a full-length homologue of talin, a protein implicated in linkage of the actin system to sites of cell-to-substrate adhesion in fibroblasts and neuronal growth cones. Gene replacement eliminated the talin homologue in Dictyostelium and led to defects in phagocytosis and cell-to-substrate interaction of moving cells, two processes dependent on a continuous cross talk between the cell surface and underlying cytoskeleton. The uptake rate of yeast particles was reduced, and only bacteria devoid of the carbohydrate moiety of cell surface lipopolysaccharides were adhesive enough to be recruited by talin-null cells in suspension and phagocytosed. Cell-to-cell adhesion of undeveloped cells was strongly impaired in the absence of talin, in contrast with the cohesion of aggregating cells mediated by the phospholipid-anchored contact site A glycoprotein, which proved to be less talin dependent. The mutant cells were still capable of moving and responding to a chemoattractant, although they attached only loosely to a substrate via small areas of their surface. With their high proportion of binucleated cells, the talin-null mutants revealed interactions of the mitotic apparatus with the cell cortex that were not obvious in mononucleated cells.

Animals↗
PubMed1990

Spatial and temporal distribution of vinculin and talin in migrating avian neural crest cells and their derivatives.

Neural crest cells express different adhesion modes at each phase of their development starting with their separation from the neural tube, followed by migration along definite pathways throughout the embryo, and finally to settlement and differentiation in elected embryonic regions. In order to determine possible changes in the cytoskeleton organization and function during these processes, we have studied the in situ distribution of two major cytoskeleton-associated elements involved in the membrane anchorage of actin microfilaments, i.e. vinculin and talin, during the ontogeny of the neural crest and its derivatives in the avian embryo. Prior to emigration, neural crest cells exhibited both vinculin and talin at levels similar to the neighbouring neural epithelial cells, and this expression apparently did not change as cells became endowed with migratory properties. However, vinculin became selectively enhanced in neural crest cells as they further migrated towards their final destination. This increase in vinculin amount was particularly striking in vagal and truncal neural crest cells entering cellular environments, such as the sclerotome and the gut mesenchyme. Talin was also expressed by neural crest cells but, in contrast to vinculin, staining was not conspicuous compared to neighbouring mesenchymal cells. High levels of vinculin persisted throughout embryogenesis in almost all neural derivatives of the neural crest, including the autonomous and sensory ganglia and Schwann cells along the peripheral nerves. In contrast, the non-neural derivatives of the neural crest rapidly lost their prominent vinculin staining after migration. The pattern of talin in the progeny of the neural crest was complex and varied with the cell types: for example, some cranial sensory ganglia expressed high amounts of the molecule whereas autonomic ganglia were nearly devoid of it. Our results suggest that (i) vinculin and talin may follow independent regulatory patterns within the same cell population, (ii) the level of expression of vinculin and talin in neural crest cells may be consistent with the rapid, constant modulations of their adhesive properties, and (iii) the expression patterns of the two molecules may also be correlated with the genesis of the peripheral nervous system.

Animals↗
PubMed1992

Further characterisation of the talin-binding site in the cytoskeletal protein vinculin.

The cytoskeletal protein vinculin is a component of adherens-type junctions where it is one of a number of interacting proteins thought to link the cytoplasmic domain of adhesion receptors to F-actin. Vinculin has been shown to bind to at least three other cytoskeletal proteins, talin, paxillin and alpha-actinin. In this study, we further characterise the talin-binding domain in vinculin using a series of chick vinculin polypeptides expressed as glutathione-S-transferase fusion proteins in Escherichia coli. Thus 125I-talin bound to a fusion protein spanning residues 1-398, but not to those spanning residues 399-881 or 881-1066 in an SDS-PAGE gel-blot assay. We have previously characterised two chick vinculin cDNAs (2.89 kb cDNA and cVin5) which are identical in the region of overlap except that cVin5 lacks coding sequence for residues 167-207. Interestingly, a fusion protein spanning residues 1-398, but lacking residues 167-207, was unable to bind talin. However, further analysis showed that residues 167-207 are insufficient to support binding, and deletion of as few as 31 N-terminal residues abolished binding activity. The results of the gel-blot assay were essentially confirmed using purified fusion proteins adsorbed to glutathione-agarose beads. The smallest vinculin fusion protein able to bind talin contained residues 1-258. This fusion protein was as effective as whole vinculin in inhibiting the binding of 125I-vinculin to talin-coated microtitre wells. Interestingly, mutations which altered the charge characteristics of the highly conserved residues 178 and 181 abolished binding, whereas conservative substitutions were without effect. However, such mutations did not abolish the ability of mutant polypeptides spanning residues 1-398 to target to cell-matrix junctions in Cos cells. We have investigated the possible origin of the cDNA clone cVin5 by defining the structure of a 5' portion of the chicken vinculin gene, and by analysing vinculin transcripts in a variety of adult tissues and embryonic fibroblasts using reverse transcriptase and polymerase chain reaction. Although residues 167-207 are encoded on a separate exon, we have been unable to identify a tissue where this exon is alternatively spliced.

Amino Acid Sequence↗