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PubMed1996

Talin contains three actin-binding sites each of which is adjacent to a vinculin-binding site.

We have determined the sequence of chicken talin (2,541 amino acids, M(r) 271,881) which is very similar (89% identity) to that of the mouse protein. Alignments with the Caenorhabditis elegans and Dictyostelium discoideum talin sequences show that the N- and C-terminal regions of the protein are conserved whereas the central part of the molecule is more divergent. By expressing overlapping talin polypeptides as fusion proteins, we have identified at least three regions of the protein which can bind F-actin: residues 102-497, 951-1,327 and 2,269-2,541. The N-terminal binding site contains a region with homology to the ERM family of actin-binding proteins, and the C-terminal site is homologous to the yeast actin-binding protein Sla2p. Each of the actin-binding sites is close to, but distinct from a binding site for vinculin, a protein which also binds actin. The Pro1176 to Thr substitution found in talin from Wistar-Furth rats does not destroy the capacity of this region of the protein to bind actin or vinculin. Microinjection studies showed that a fusion protein containing the N-terminal actin-binding site localised weakly to stress fibres, whereas one containing the C-terminal site initially localised predominantly to focal adhesions. The former was readily solubilised, and the latter was resistant to Triton extraction. The N-terminal talin polypeptide eventually disrupted actin stress fibres whereas the C-terminal polypeptide was without effect. However, a larger C-terminal fusion protein also containing a vinculin-binding site did disrupt stress fibres and focal adhesions. The results suggest that, although both the N- and C-terminal regions of talin bind actin, the properties of these two regions of the protein are distinct.

Actins↗

PubMed1987

Properties of talin from chicken gizzard smooth muscle.

This paper describes the structural and biochemical characterization of talin, a protein localized to various cellular sites where bundles of actin filaments attach to the plasma membrane. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein has a molecular mass of 225,000 +/- 5,000 daltons. Hydrodynamic measurements at protein concentrations less than 0.72 mg/ml indicate a monomeric protein with a native molecular mass of 213,000 +/- 15,000 daltons. Sedimentation equilibrium experiments indicate self-association at protein concentrations of 0.72 mg/ml and higher. The data suggest that this self-association is a simple monomer:dimer equilibrium over the range of concentrations observed. At low protein concentrations where talin is a monomer, the Stokes radius and sedimentation coefficient vary with ionic strength. Under low ionic strength conditions (5-20 mM NaCl), talin has a Stokes radius of 6.5 nm and a sedimentation value of 9.4, suggesting an asymmetric globular molecule; whereas under high ionic strength conditions (200 mM NaCl), the Stokes radius increases to 7.7 nm and the sedimentation coefficient decreases to 8.8, suggesting a more elongated protein. This conformation change is confirmed by electron microscopy which reveals a more globular protein at low ionic strength which unfolds to become an elongated flexible molecule as the ionic strength is increased to physiological and higher levels. The amino acid composition of talin indicates a low level of aromatic residues, consistent with its relatively low extinction coefficient, talin has an isoelectric point between pH 6.7 and 6.8 based on isoelectric focusing. The detailed purification of talin is described.

Amino Acids↗

PubMed1983

Talin: a cytoskeletal component concentrated in adhesion plaques and other sites of actin-membrane interaction.

Talin is a recently identified cytoskeletal protein with a polypeptide molecular weight of 215,000 daltons. In cultured fibroblasts talin has been localized by immunofluorescence in adhesion plaques (focal contacts), in the ruffling membranes and leading lamellae of the cell periphery, and in fibrillar patterns that align with microfilament bundles and/or with cell surface fibronectin. These cellular locations suggest that the protein could function either in the attachment of microfilaments to the plasma membrane or in the organization of microfilaments close to membrane attachment sites. Cell transformation by viruses such as Rous sarcoma virus disrupts the normal organization of talin, and in most transformed cells talin appears distributed diffusely through the cytoplasm. In a few cells talin is detected in doughnut-shaped aggregates, as a ring surrounding a central core of actin. The significance of these structures is uncertain, but in some cells the individual structures will condense to form much larger aggregates with a striking appearance when viewed by immunofluorescence microscopy.

Actins↗

PubMed1993

The marked pH dependence of the talin-actin interaction.

The effect of pH on the interaction between talin and actin was examined by using cosedimentation assays, low shear viscometry, and electron microscopy. When the pH at which the interaction was tested was lowered from 7.5 to 7.0 and below, the ability of talin to cosediment with F-actin was greatly enhanced, with maximal binding at pH 6.6. Talin's ability to increase the low shear viscosity of F-actin solutions was also significantly elevated as pH was decreased from 7.4 to 6.6. Electron microscope observations of negatively stained actin controls and talin-actin mixtures supported these results by demonstrating an increase in the degree of crosslinking of actin filaments in the presence of talin at pH values of 7 and lower.

Actins↗

PubMed1999

Characterization of the human talin (TLN) gene: genomic structure, chromosomal localization, and expression pattern.

Talin is a high-molecular-weight cytoskeletal protein, localized at cell-extracellular matrix associations known as focal contacts. In these regions, talin is thought to link integrin receptors to the actin cytoskeleton. Talin plays a key role in the assembly of actin filaments and in spreading and migration of various cell types. Talin proteins are found in a wide variety of organisms, from slime molds to humans. The human Talin (HGMW-approved symbol TLN) gene was previously mapped to chromosome 9p, but little was known of its sequence and genomic structure. To characterize human TLN further, we have isolated a single bacterial artificial chromosome clone, harboring the entire gene. The gene extends over more than 23 kb and consists of 57 exons. We have localized TLN to human chromosome band 9p13 by both fluorescence in situ hybridization and radiation hybrid mapping. Northern blot analysis detected TLN expression in various human tissues, including leukocytes, lung, placenta, liver, kidney, spleen, thymus, colon, skeletal muscle, and heart. Based on its chromosomal location, expression pattern, and protein function, we considered TLN as a candidate gene for cartilage-hair hypoplasia (CHH), an autosomal recessive metaphyseal chondrodysplasia, previously mapped to 9p13. We sequenced the entire TLN coding sequence in several CHH patients, but no functional mutations were detected.

Cartilage↗

PubMed1990

Talin and vinculin in the oocytes, eggs, and early embryos of Xenopus laevis: a developmentally regulated change in distribution.

We have investigated the expression and distribution of talin and vinculin in the oocytes, eggs, and embryos of Xenopus laevis. Antibodies to the previously characterized avian proteins stain several different Xenopus cell types identically by immunofluorescence: adhesion plaques of cultured kidney (A6) cells, the cell peripheries of oviduct cells, and the postsynaptic neuromuscular junctions of tadpole tail muscle fibers. These antibodies also identify cognate proteins of the appropriate sizes on immunoblots of A6 cell and oviduct lysates. Using these antibodies on ovarian tissue, we find talin to be highly localized at the cortices of oocytes and vinculin to be in the oocyte cytoplasm and absent from the oocyte cortex. In the cells of the ovarian layers that surround the oocytes, talin and vinculin can be detected as soluble and cytoskeletal components. Vinculin is first detectable as a cytoskeletal component in eggs, appearing some time during or between oocyte maturation and oviposition. During early embryo development, talin and vinculin are colocalized in the cortex of cleavage furrows and blastomeres. Thus, Xenopus oocytes and eggs display different distributions of talin and vinculin. The change from unlinked localization to colocalization appears to be developmentally regulated, occurring during the transition from oocyte to egg.

Animals↗

PubMed2004

Talin: an emerging focal point of adhesion dynamics.

The adhesion protein talin and the phosphoinositide PIP2 are emerging as key modulators of adhesion dynamics. Recent genetic studies on talin demonstrate its physiological role in organizing adhesions, stabilizing integrin-actin linkages and mediating integrin signaling in vivo. Biophysical force measurements provide further evidence that it is required for the reinforcement of the extracellular matrix-integrin-actin connection. Knockdown data along with structural analyses establish a major role for talin in 'inside-out' integrin activation through its direct interaction with integrin cytoplasmic domains. A recently uncovered role for talin is the recruitment of a PIPKI gamma isoform to adhesions. This introduces a novel connection between talin and PIP2 generation. Finally, PIP2 also stimulates the transient, direct binding interaction of the Arp2/3 complex with vinculin and thus may couple adhesion to actin assembly.

Animals↗

PubMed2006

Structural basis for the phosphorylation-regulated focal adhesion targeting of type Igamma phosphatidylinositol phosphate kinase (PIPKIgamma) by talin.

Phosphatidylinositol-4,5-bisphosphate (PIP2) is a key lipid messenger that regulates myriad diverse cellular signaling pathways. To ensure specificity in disparate cellular events, PIP2 must be localized to specific sub-cellular sites. At PIP2-regulated focal adhesion (FA) sites, such localization is in part mediated via the recruitment and activation of PIP2-producing enzyme, type Igamma phosphatidylinositol phosphate kinase (PIPKIgamma), by a phosphotyrosine binding (PTB) domain of talin. Transient phosphorylation of PIPKIgamma at Y644 regulates the interaction and efficient FA targeting of PIPKIgamma; however, the underlying structural basis remains elusive. We have determined the NMR structure of talin-1 PTB in complex with the Y644-phosphorylated PIPKIgamma fragment (WVpYSPLH). As compared to canonical PTB domains that typically recognize the NPXpY turn motif from a variety of signaling proteins, our structure displays an unusual non-NPXpY-based recognition mode for talin-1 PTB where K(357)RW in beta5 strand forms an antiparallel beta-sheet with the VpYS of PIPKIgamma. A specific electrostatic triad between K357/R358 of talin-1 PTB and the pY644 of PIPKIgamma was observed, which is consistent with the mutagenesis and isothermal calorimetry data. Combined with previous in vivo data, our results provide a framework for understanding how phosphorylation of Y644 in PIPKIgamma promotes its specific interaction with talin-1, leading to efficient local synthesis of PIP2 and dynamic regulation of integrin-mediated FA assembly.

Animals↗

PubMed1998

Peptide-specific antibodies localize the major lipid binding sites of talin dimers to oppositely arranged N-terminal 47 kDa subdomains.

Using ultrastructural analysis and labeling with polyclonal antibodies that recognize peptide sequences specific for phospholipid binding, we mapped the functional domain structure of intact platelet talin and its proteolytic fragments. The talin dimer, which is crucial for actin and lipid binding, is built of a backbone containing the 200 kDa rod portions, at both ends of which a 47 kDa globular domain is attached. Peptide-specific polyclonal antibodies were raised against three potential lipid binding sequences residing within the N-terminal 47 kDa domain (i.e. S19, amino acids 21-39; H18, amino acids 287-304; and H17, amino acids 385-406). Antibodies H17 and H18 localize these lipid binding sequences within the N-terminal 47 kDa globular talin subdomains opposed at the outer 200 kDa rod domains within talin dimers. Hence, we conclude that in its dimeric form, which is used in actin and lipid binding, talin is a dumbbell-shaped molecule built of two antiparallel subunits.

Amino Acid Sequence↗

PubMed1997

Perineurium talin immunoreactivity decreases in diabetic neuropathy.

We studied the immunolocalization of Dp116 (a 116 kDa protein product of the dystrophin gene), vinculin, talin, vimentin, desmin, spectrin and titin in the sural nerve biopsies of 25 patients with peripheral neuropathies of different origin. 4 patients presented with HMSN type 1, 4 with HMSN type 2, 2 with HNPP, 4 with CIDP, 5 with chronic axonal neuropathy of unknown origin, 3 with vasculitic neuropathy, 3 with diabetic neuropathy. Expression and localization of Dp116, vinculin, vimentin, desmin, spectrin and titin did not differ from normal control cases. Spectrin and titin immunoreactivities were absent and desmin was occasionally found in few epineurial vessels. A thin rim of Dp116 binding surrounded the outermost layer of myelin sheaths. Perineurium and epineurial vessels stained deeply for vinculin. Vimentin immunoreactivity was seen in all endoneurial, perineurial and epineurial cells. Immunoreactivity for talin was normally found at endoneurial and epineurial vessel walls, perineurial cells and epineurial fibroblasts in all the sural nerves except diabetic nerves. In the latter, whereas talin binding was normal in the vessel walls and epineurial fibroblasts, it was markedly reduced in the perineurium. On immunoblot, two bands at 235 and 190 kDa were found in the sural nerves with the antibody anti-talin, and both were reduced only in the patients with diabetic neuropathy. We postulate that decreased perineurium talin in diabetic polyneuropathy may be related to the known alterations of the tight junctions of the perineurial cells, which have been proposed to be a contributory factor to impaired permeability barrier properties.

Case-Control Studies↗

PubMed1999

A unique talin homologue with a villin headpiece-like domain is required for multicellular morphogenesis in Dictyostelium.

Molecules involved in the interaction between the extracellular matrix, cell membrane and cytoskeleton are of central importance in morphogenesis. Talin is a large cytoskeletal protein with a modular structure consisting of an amino-terminal membrane-interacting domain, with sequence similarities to members of the band 4.1 family, and a carboxy-terminal region containing F-actin-binding and vinculin-binding domains [1] [2]. It also interacts with the cytoplasmic tail of beta integrins which, on the external face of the membrane, bind to extracellular matrix proteins [3]. The possible roles of talin in multicellular morphogenesis in development remain largely unexplored. In Dictyostelium, a eukaryotic microorganism capable of multicellular morphogenesis, a talin homologue (TALA) has previously been identified and shown to play an important role in cell-to-substrate adhesion and maintenance of normal elastic properties of the cell [4] [5] [6]. Here, we describe a second talin homologue (TALB) that is required for multicellular morphogenesis in the development of Dictyostelium. Unlike any other talin characterised to date, it contains an additional carboxy-terminal domain homologous to the villin headpiece.

Amino Acid Sequence↗

PubMed2002

Talin is essential for integrin function in Drosophila.

We show that the Drosophila gene rhea, isolated because its wing blister phenotype is typical of mutants affecting integrin function, encodes talin. Embryos deficient in talin have very similar phenotypes to integrin (betaPS) null embryos, including failure in germ band retraction and muscle detachment. We demonstrate that talin is not required for the presence of integrins on the cell surface or their localization at muscle termini. However, talin is required for formation of focal adhesion-like clusters of integrins on the basal surface of imaginal disc epithelia and junctional plaques between muscle and tendon cells. These results indicate that talin is essential for integrin function and acts by stably linking clusters of ECM-linked integrins to the cytoskeleton.

Animals↗

PubMedDate not supplied

Identification of talin as a major cytoplasmic protein implicated in platelet activation.

During platelet activation there is a major reorganization in the platelet cytoskeleton that accompanies a rapid change in platelet shape. Many of the events associated with activation are attributed to a rise in calcium concentration within the platelet cytoplasm. One direct consequence of the elevated calcium is the activation of a calcium-dependent protease that cleaves a major platelet protein of relative molecular mass (Mr) approximately 235,000 (235K) to 200K. This protein, P235, has been purified and reported to interact with actin, but the significance of the proteolytic cleavage is unknown. Talin, a cytoskeletal protein in smooth muscle and fibroblasts, binds vinculin and, together with vinculin, is localized in fibroblasts at sites of actin-membrane attachment. Talin and P235 have similar purification procedures, sedimentation coefficients and Stokes' radii (ref. 6 and Molony et al., unpublished observations). Of particular significance, talin is readily cleaved by proteases from approximately 215K to a fragment of approximately 190K. Given these similarities we have investigated the possible relationship between these proteins. Here we demonstrate that platelet P235 is recognized by anti-talin antibody and that it binds vinculin. Both proteins are cleaved in vitro by the calcium-activated protease to yield similar fragments. We conclude that P235 corresponds to the platelet form of talin.

Actins↗

PubMed2004

Talin B is required for force transmission in morphogenesis of Dictyostelium.

Talin plays a key role in the assembly and stabilisation of focal adhesions, but whether it is directly involved in force transmission during morphogenesis remains to be elucidated. We show that the traction force of Dictyostelium cells mutant for one of its two talin genes talB is considerably smaller than that of wild-type cells, both in isolation and within tissues undergoing morphogenetic movement. The motility of mutant cells in tightly packed tissues in vivo or under strong resistance conditions in vitro was lower than that of wild-type cells, but their motility under low external force conditions was not impaired, indicating inefficient transmission of force in mutant cells. Antibody staining revealed that the talB gene product (talin B) exists as small units subjacent to the cell membrane at adhesion sites without forming large focal adhesion-like assemblies. The total amount of talin B on the cell membrane was larger in prestalk cells, which exert larger force than prespore cells during morphogenesis. We conclude that talin B is involved in force transmission between the cytoskeleton and cell exterior.

Actins↗

PubMed1998

Opening-up of liposomal membranes by talin.

Morphological changes of liposomes caused by interactions between liposomal membranes and talin, a cytoskeletal submembranous protein, were studied by direct, real-time observation by using high-intensity dark-field microscopy. Surprisingly, when talin was added to a liposome solution, liposomes opened stable holes and were transformed into cup-shaped liposomes. The holes became larger with increasing talin concentration, and finally the cup-shaped liposomes were transformed into lipid bilayer sheets. These morphological changes were reversed by protein dilution, i.e., the sheets could be transformed back into closed spherical liposomes. We demonstrated that talin was localized mainly along the membrane verges, presumably avoiding exposure of its hydrophobic portion at the edge of the lipid bilayer. This is the first demonstration that a lipid bilayer can stably maintain a free verge in aqueous solution. This finding refutes the established dogma that all lipid bilayer membranes inevitably form closed vesicles and suggests that talin is a useful tool for manipulating liposomes.

Animals↗

PubMed2004

Structural basis for phosphatidylinositol phosphate kinase type Igamma binding to talin at focal adhesions.

The cytoskeletal protein talin binds to a short C-terminal sequence in phosphatidylinositol phosphate kinase type Igamma (PIPKIgamma), activating the enzyme and promoting the local production of phosphatidylinositol 4,5 bisphosphate, which regulates focal adhesion dynamics as well as clathrin-mediated endocytosis in neuronal cells. Here we show by crystallographic, NMR, and calorimetric analysis that the phosphotyrosine binding (PTB)-like domain of talin engages the PIPKIgamma C terminus in a mode very similar to that of integrin binding. However, PIPKIgamma binds in the canonical PTB-peptide mode with an SPLH motif replacing the classic NPXY motif. The tighter packing of the SPLH motif against the hydrophobic core of talin may explain the stronger binding of PIPKIgamma. Two tyrosine residues flanking the SPLH motif (Tyr-644 and Tyr-649) have been implicated in the regulation of talin binding. We show that phosphorylation at Tyr-644, a Src phosphorylation site in vivo, has little effect on the binding mode or strength, which is consistent with modeling studies in which the phosphotyrosine makes surface-exposed salt bridges, and we suggest that its strong activating effect arises from the release of autoinhibitory restraints in the full-length PIPKIgamma. Modeling studies suggest that phosphorylation of Tyr-649 will likewise have little effect on talin binding, whereas phosphorylation of the SPLH serine is predicted to be strongly disruptive. Our data are consistent with the proposal that Src activity promotes a switch from integrin binding to PIPKIgamma binding that regulates focal adhesion turnover.

Amino Acid Motifs↗

PubMed2005

Mapping and consensus sequence identification for multiple vinculin binding sites within the talin rod.

The interaction between the cytoskeletal proteins talin and vinculin plays a key role in integrin-mediated cell adhesion and migration. Three vinculin binding sites (VBS1-3) have previously been identified in the talin rod using a yeast two-hybrid assay. To extend these studies, we spot-synthesized a series of peptides spanning all the alpha-helical regions predicted for the talin rod and identified eight additional VBSs, two of which overlap key functional regions of the rod, including the integrin binding site and C-terminal actin binding site. The talin VBS alpha-helices bind to a hydrophobic cleft in the N-terminal vinculin Vd1 domain. We have defined the specificity of this interaction by spot-synthesizing a series of 25-mer talin VBS1 peptides containing substitutions with all the commonly occurring amino acids. The consensus for recognition is LXXAAXXVAXX- VXXLIXXA with distinct classes of hydrophobic side chains at positions 1, 4, 5, 8, 9, 12, 15, and 16 required for vinculin binding. Positions 1, 8, 12, 15, and 16 require an aliphatic residue and will not tolerate alanine, whereas positions 4, 5, and 9 are less restrictive. These preferences are common to all 11 VBS sequences with a minor variation occurring in one case. A crystal structure of this variant VBS peptide in complex with the vinculin Vd1 domain reveals a subtly different mode of vinculin binding.

Amino Acid Sequence↗

PubMed2006

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.

Actinin↗