Talin is a large flexible rod-shaped protein that activates the integrin family of cell adhesion molecules and couples them to cytoskeletal actin. Its rod region consists of a series of helical bundles. Here we show that residues 1815–1973 form a 5-helix bundle, with a topology unique to talin which is optimally suited for formation of a long rod such as talin. This is much more stable than the 4-helix (1843–1973) domain described earlier and as a result its vinculin binding sequence is inaccessible to vinculin at room temperature, with implications for the overall mechanism of the talin-vinculin interaction.
MINT-7722300, MINT-7760951: Talin-1 (uniprotkb:P26039) and Vinculin (uniprotkb:P12003) bind (MI:0407) by molecular sieving (MI:0071)
HSQC, heteronuclear single quantum coherence; SCOP, structural classification of proteins; VBS, vinculin-binding site; Talin; Vinculin; NMR; Domain structure; Helical bundle
Focal adhesions (FAs) regulate cell migration. Vinculin, with its many potential binding partners, can interconnect signals in FAs. Despite the well-characterized structure of vinculin, the molecular mechanisms underlying its action have remained unclear. Here, using vinculin mutants, we separate the vinculin head and tail regions into distinct functional domains. We show that the vinculin head regulates integrin dynamics and clustering and the tail regulates the link to the mechanotransduction force machinery. The expression of vinculin constructs with unmasked binding sites in the head and tail regions induces dramatic FA growth, which is mediated by their direct interaction with talin. This interaction leads to clustering of activated integrin and an increase in integrin residency time in FAs. Surprisingly, paxillin recruitment, induced by active vinculin constructs, occurs independently of its potential binding site in the vinculin tail. The vinculin tail, however, is responsible for the functional link of FAs to the actin cytoskeleton. We propose a new model that explains how vinculin orchestrates FAs.
Upon cell adhesion, talin physically couples the cytoskeleton via integrins to the extracellular matrix, and subsequent vinculin recruitment is enhanced by locally applied tensile force. Since the vinculin binding (VB) sites are buried in the talin rod under equilibrium conditions, the structural mechanism of how vinculin binding to talin is force-activated remains unknown. Taken together with experimental data, a biphasic vinculin binding model, as derived from steered molecular dynamics, provides high resolution structural insights how tensile mechanical force applied to the talin rod fragment (residues 486–889 constituting helices H1–H12) might activate the VB sites. Fragmentation of the rod into three helix subbundles is prerequisite to the sequential exposure of VB helices to water. Finally, unfolding of a VB helix into a completely stretched polypeptide might inhibit further binding of vinculin. The first events in fracturing the H1–H12 rods of talin1 and talin2 in subbundles are similar. The proposed force-activated α-helix swapping mechanism by which vinculin binding sites in talin rods are exposed works distinctly different from that of other force-activated bonds, including catch bonds.
For cell survival, most eukaryotic cells need to be mechanically anchored to their environment. This is done by transmembrane proteins, including integrins, which externally bind to the extracellular matrix and on the cell interior to the contractile cytoskeleton via scaffolding proteins. One essential scaffolding protein is talin, which binds to integrins via its head and to the cytoskeletal filament f-actin via its rodlike tail. As cells apply tensile forces to newly formed adhesion sites, proteins that are part of such force-bearing networks get stretched and might change their structure and thus function. One of many proteins that are recruited to newly formed adhesions is vinculin, and vinculin recruitment is upregulated by tensile mechanical force—but how? Since talin's vinculin binding sites are buried in its native structure, we used steered molecular dynamics here to derive a high resolution structural model of how tensile mechanical forces might activate talin's vinculin binding sites. Once tensile forces break up the talin rod into helix subbundles, an event that we find here to constitute the main energy barrier, we propose how the strain-induced gradual exposure of the vinculin-binding helices finally allows for their activation and enables helix swapping with the vinculin head.
The translational machinery of the cell re-localizes to focal adhesions following the activation of integrin receptors. This response allows for rapid, local production of components needed for adhesion complex assembly and signaling. Vinculin links focal adhesions to the actin cytoskeleton following its activation by integrin signaling, which severs intramolecular interactions of the vinculin head and tail (Vt) domains. Our vinculin:raver1 crystal structures and binding studies show that activated Vt selectively interacts with one of the three RNA recognition motifs (RRM) of raver1, that the vinculin:raver1 complex binds to F-actin, and that raver1 binds selectively to RNA, including a sequence found in vinculin mRNA. Further, mutation of residues that mediate interaction of raver1 with vinculin abolish their co-localization in cells. These findings suggest a feed-forward model where vinculin activation at focal adhesions provides a scaffold for recruitment of raver1 and its mRNA cargo to facilitate the production of components of adhesion complexes.
focal adhesion; actin cytoskeleton; crystallography; RNP motif; RNA binding
We used quick-freeze, deep-etch, rotary replication and immunogold cytochemistry to identify a new structure at focal contacts. In Xenopus fibroblasts, elongated aggregates of particles project from the membrane to contact bundles of actin microfilaments. Before terminating, a single bundle of microfilaments interacts with several aggregates that appear intermittently over a distance of several microns. Aggregates are enriched in proteins believed to mediate actin- membrane interactions at focal contacts, including beta 1-integrin, vinculin, and talin, but they appear to contain less alpha-actinin and filamin. We also identified a second, smaller class of aggregates of membrane particles that contained beta 1-integrin but not vinculin or talin and that were not associated with actin microfilaments. Our results indicate that vinculin, talin, and beta 1-integrin are assembled into distinctive structures that mediate multiple lateral interactions between microfilaments and the membrane at focal contacts.
Relative to vinculin, a unique 68-residue insert in the C-terminal tail of metavinculin results in a loss of actin filament-bundling activity but gain of actin filament-severing activity.
Vinculin and its splice variant, metavinculin (MV), are key elements of multiple protein assemblies linking the extracellular matrix to the actin cytoskeleton. Vinculin is expressed ubiquitously, whereas MV is mainly expressed in smooth and cardiac muscle tissue. The only difference in amino acid sequence between the isoforms is a 68-residue insert in the C-terminal tail domain of MV (MVt). Although the functional role of this insert remains elusive, its importance is exemplified by point mutations that are associated with dilated and hypertrophic cardiomyopathy. In vinculin, the actin binding site resides in the tail domain. In this paper, we show that MVt binds actin filaments similarly to the vinculin tail domain. Unlike its splice variant, MVt did not bundle actin filaments. Instead, MVt promoted severing of actin filaments, most efficiently at substoichiometric concentrations. This surprising and seemingly contradictory alteration of vinculin function by the 68-residue insert may be essential for modulating compliance of vinculin-induced actin bundles when exposed to rapidly increasing external forces.
Vinculin is an important protein for the linkage between adhesion molecules and the actin cytoskeleton. The activation mechanism of vinculin is still controversial. In order to provide useful information for a better understanding of its activation, we analyze the motion mode of vinculin with elastic network model in this work. The results show that, to some extent, the five domains will present structural rigidity in the motion process. The differences between the structure fluctuations of these domains are significant. When vinculin interacted with other partners, the central long alpha-helix of the first domain becomes bent. This bending deformation can weaken the interaction between the first domain and the tail domain. This motion mode of the first domain is in good agreement with the information extracted from some realistic complex structures. With the aid of the anisotropy elastic network mode, we analyze the motion directions of these domains. The fourth domain has a rotational motion. This rotation is favorable for the releasing of the tail domain from the pincer-like clamp, which is formed by the first and the third domain. All these motion modes are an inherent feature of the structure, and these modes mainly depend on the topology character of the structure.
vinculin; motion mode; elastic network model; activation mechanism
Vinculin binds to multiple focal adhesion and cytoskeletal proteins and has been implicated in transmitting mechanical forces between the actin cytoskeleton and integrins or cadherins. It remains unclear to what extent the mechano-coupling function of vinculin also involves signaling mechanisms. We report the effect of vinculin and its head and tail domains on force transfer across cell adhesions and the generation of contractile forces. The creep modulus and the adhesion forces of F9 mouse embryonic carcinoma cells (wild-type), vinculin knock-out cells (vinculin −/−), and vinculin −/− cells expressing either the vinculin head domain, tail domain, or full-length vinculin (rescue) were measured using magnetic tweezers on fibronectin-coated super-paramagnetic beads. Forces of up to 10 nN were applied to the beads. Vinculin −/− cells and tail cells showed a slightly higher incidence of bead detachment at large forces. Compared to wild-type, cell stiffness was reduced in vinculin −/− and head cells and was restored in tail and rescue cells. In all cell lines, the cell stiffness increased by a factor of 1.3 for each doubling in force. The power-law exponent of the creep modulus was force-independent and did not differ between cell lines. Importantly, cell tractions due to contractile forces were suppressed markedly in vinculin −/− and head cells, whereas tail cells generated tractions similar to the wild-type and rescue cells. These data demonstrate that vinculin contributes to the mechanical stability under large external forces by regulating contractile stress generation. Furthermore, the regulatory function resides in the tail domain of vinculin containing the paxillin-binding site.
Binding of the cytoskeletal protein vinculin to talin is one of a number of interactions involved in linking F-actin to cell-matrix junctions. To identify the talin binding domain in vinculin, we expressed the NH2-terminal region of the molecule encoded by two closely similar, but distinct vinculin cDNAs, using an in vitro transcription translation system. The 5' Eco RI-Bam HI fragment of a partial 2.89-kb vinculin cDNA encodes a 45-kD polypeptide containing the first 398 amino acids of the molecule. The equivalent restriction enzyme fragment of a second vinculin cDNA (cVin5) lacks nucleotides 746- 867, and encodes a 41-kD polypeptide missing amino acids 167-207. The radiolabeled 45-kD vinculin polypeptide bound to microtiter wells coated with talin, but not BSA, and binding was inhibited by unlabeled vinculin. In contrast, the 41-kD vinculin polypeptide was devoid of talin binding activity. The role of residues 167-207 in talin binding was further analyzed by making a series of deletions spanning this region, each deletion of seven amino acids contiguous with the next. Loss of residues 167-173, 174-180, 181-187, 188-194, or 195-201 resulted in a marked reduction in talin binding activity, although loss of residues 202-208 had much less effect. When the 45-kD vinculin polypeptide was expressed in Cos cells, it localized to cell matrix junctions, whereas the 41-kD polypeptide, lacking residues 167-207, was unable to do so. Interestingly, some deletion mutants with reduced ability to bind talin in vitro, were still able to localize to cell matrix junctions.
α-Actinin is an actin crosslinking molecule that can serve as a scaffold and maintain dynamic actin filament networks. As a crosslinker in the stressed cytoskeleton, α-actinin can retain conformation, function, and strength. α-Actinin has an actin binding domain and a calmodulin homology domain separated by a long rod domain. Using molecular dynamics and normal mode analysis, we suggest that the α-actinin rod domain has flexible terminal regions which can twist and extend under mechanical stress, yet has a highly rigid interior region stabilized by aromatic packing within each spectrin repeat, by electrostatic interactions between the spectrin repeats, and by strong salt bridges between its two anti-parallel monomers. By exploring the natural vibrations of the α-actinin rod domain and by conducting bending molecular dynamics simulations we also predict that bending of the rod domain is possible with minimal force. We introduce computational methods for analyzing the torsional strain of molecules using rotating constraints. Molecular dynamics extension of the α-actinin rod is also performed, demonstrating transduction of the unfolding forces across salt bridges to the associated monomer of the α-actinin rod domain.
The cell interacts with its environment in both biochemical and mechanical ways. In this study we explore one of the ways in which the cell interacts mechanically with its environment. α-Actinin is a cytoskeletal crosslinker: it functions to scaffold the cytoskeletal actin filaments that provide mechanical reinforcement to the cell. In its functional environment α-actinin is exposed to a multitude of mechanical stresses as it attaches itself to a dynamic network of actin filaments. The actin filaments extend, rotate, and bend the α-actinin crosslinkers. In this study we employ molecular dynamics techniques to understand the structural characteristics of α-actinin that underlie its ability to provide a scaffold in such a stressed environment. We analyzed the natural frequencies of α-actinin and simulated force-induced bending, extension, and twisting. Our results suggest that α-actinin has structural flexibility facilitating crosslinking in a dynamic environment and also structural rigidity stabilizing the linkage in the stressed environment. We have discovered novel natural bending movements of the rod domain that enhance its function as a crosslinker. We have also demonstrated the specific structural characteristics of α-actinin that give it the previously suggested property of having partial flexibility. Our results enhance the understanding of structural mechanics in the cytoskeletal molecules.
Fluorescently labeled vinculin binds to focal contact areas in permeabilized cells independent of actin (Avnur, Z., J. V. Small, and B. Geiger, 1983, J. Cell Biol., 96:1622-1630), but the nature of the binding site is unknown. In this study we have examined the interaction of vinculin with these sites in permeabilized L6 myoblasts to define conditions that perturb the binding and subsequently to reconstitute it. Mild treatment with low concentrations of protease prevents vinculin incorporation without gross changes in the cytoskeleton or extensive protein breakdown. Exposure to buffers of moderate ionic strength also reduces subsequent vinculin binding without large morphological effects. These extraction conditions were used to obtain a fraction from gizzard which was able to restore the vinculin localization. Talin, actin, and vinculin itself were able to alter the binding of labeled vinculin to permeabilized cells and each also interacted with vinculin in gel overlays; however, they were unable to reconstitute the binding site in treated permeabilized cells. The results show a requirement for an as yet unidentified protein to capacitate vinculin binding to focal contact sites and suggest that this protein is peripheral and interacts directly with the binding site.
The leukocyte adhesion molecule L-selectin mediates binding to lymph node high endothelial venules (HEV) and contributes to leukocyte rolling on endothelium at sites of inflammation. Previously, it was shown that truncation of the L-selectin cytoplasmic tail by 11 amino acids abolished binding to lymph node HEV and leukocyte rolling in vivo, but the molecular basis for that observation was not determined. This study examined potential interactions between L-selectin and cytoskeletal proteins. We found that the cytoplasmic domain of L- selectin interacts directly with the cytoplasmic actin-binding protein alpha-actinin and forms a complex with vinculin and possibly talin. Solid phase binding assays using the full-length L-selectin cytoplasmic domain bound to microtiter wells demonstrated direct, specific, and saturable binding of purified alpha-actinin to L-selectin (Kd = 550 nM), but no direct binding of purified talin or vinculin. Interestingly, talin potentiated binding of alpha-actinin to the L- selectin cytoplasmic domain peptide despite the fact that direct binding of talin to L-selectin could not be measured. Vinculin binding to the L-selectin cytoplasmic domain peptide was detectable only in the presence of alpha-actinin. L-selectin coprecipitated with a complex of cytoskeletal proteins including alpha-actinin and vinculin from cells transfected with L-selectin, consistent with the possibility that alpha- actinin binds directly to L-selectin and that vinculin associates by binding to alpha-actinin in vivo to link actin filaments to the L- selectin cytoplasmic domain. In contrast, a deletion mutant of L- selectin lacking the COOH-terminal 11 amino acids of the cytoplasmic domain failed to coprecipitate with alpha-actinin or vinculin. Surprisingly, this mutant L-selectin localized normally to the microvillar projections on the cell surface. These data suggest that the COOH-terminal 11 amino acids of the L-selectin cytoplasmic domain are required for mediating interactions with the actin cytoskeleton via a complex of alpha-actinin and vinculin, but that this portion of the cytoplasmic domain is not necessary for proper localization of L- selectin on the cell surface. Correct L-selectin receptor positioning is therefore insufficient for leukocyte adhesion mediated by L- selectin, suggesting that this adhesion may also require direct interactions with the cytoskeleton.
Vinculin is a conserved actin binding protein localized in focal adhesions and cell-cell junctions. Here, we report that vinculin is tyrosine phosphorylated in platelets spread on fibrinogen and that the phosphorylation is Src kinases dependent. The phosphorylation of vinculin on tyrosine was reconstituted in vanadate treated COS-7 cells coexpressing c-Src. The tyrosine phosphorylation sites in vinculin were mapped to residues 100 and 1065. A phosphorylation-specific antibody directed against tyrosine residue 1065 reacted with phosphorylated platelet vinculin but failed to react with vinculin from unstimulated platelet lysates. Tyrosine residue 1065 located in the vinculin tail domain was phosphorylated by c-Src in vitro. When phosphorylated, the vinculin tail exhibited significantly less binding to the vinculin head domain than the unphosphorylated tail. In contrast, the phosphorylation did not affect the binding of vinculin to actin in vitro. A double vinculin mutant protein Y100F/Y1065F localized to focal adhesion plaques. Wild-type vinculin and single tyrosine phosphorylation mutant proteins Y100F and Y1065F were significantly more effective at rescuing the spreading defect of vinculin null cells than the double mutant Y100F/Y1065F. The phosphorylation of vinculin by Src kinases may be one mechanism by which these kinases regulate actin filament assembly and cell spreading.
Vinculin, a major structural component of vertebrate cell-cell and cell- matrix adherens junctions, has been found to interact with several other junctional components. In this report, we have identified and characterized a binding site for filamentous actin. These results included studies with gizzard vinculin, its proteolytic head and tail fragments, and recombinant proteins containing various gizzard vinculin sequences fused to the maltose binding protein (MBP) of Escherichia coli. In cosedimentation assays, only the vinculin tail sequence mediated a direct interaction with actin filaments. The binding was saturable, with a dissociation constant value in the micromolar range. Experiments with deletion clones localized the actin-binding domain to a region confined by residues 893-1016 in the 170-residue-long carboxyterminal segment, while the proline-rich hinge connecting the globular head to the rodlike tail was not required for this interaction. In fixed and permeabilized cells (cell models), as well as after microinjection, proteins containing the actin-binding domain specifically decorated stress fibers and the cortical network of fibroblasts and epithelial cells, as well as of brush border type microvilli. These results corroborated the sedimentation experiments. Our data support and extend previous work showing that vinculin binds directly to actin filaments. They are consistent with a model suggesting that in adhesive cells, the NH2-terminal head piece of vinculin directs this molecule to the focal contact sites, while its tail segment causes bundling of the actin filament ends into the characteristic spear tip-shaped structures.
Dystrophin and utrophin link the F-actin cytoskeleton to the cell membrane via an associated glycoprotein complex. This functionality results from their domain organization having an N-terminal actin-binding domain followed by multiple spectrin-repeat domains and then C-terminal protein-binding motifs. Therapeutic strategies to replace defective dystrophin with utrophin in patients with Duchenne muscular dystrophy require full-characterization of both these proteins to assess their degree of structural and functional equivalence. Here the high resolution structures of the first spectrin repeats (N-terminal repeat 1) from both dystrophin and utrophin have been determined by x-ray crystallography. The repeat structures both display a three-helix bundle fold very similar to one another and to homologous domains from spectrin, α-actinin and plectin. The utrophin and dystrophin repeat structures reveal the relationship between the structural domain and the canonical spectrin repeat domain sequence motif, showing the compact structural domain of spectrin repeat one to be extended at the C-terminus relative to its previously defined sequence repeat. These structures explain previous in vitro biochemical studies in which extending dystrophin spectrin repeat domain length leads to increased protein stability. Furthermore we show that the first dystrophin and utrophin spectrin repeats have no affinity for F-actin in the absence of other domains.
A number of cytoskeletal-associated proteins that are concentrated in focal contacts, namely alpha-actinin, vinculin, talin, and integrin, have been shown to interact in vitro such that they suggest a potential link between actin filaments and the membrane. Because some of these interactions are of low affinity, we suspect the additional linkages also exist. Therefore, we have used a synthetic peptide corresponding to the cytoplasmic domain of beta 1 integrin and affinity chromatography to identify additional integrin-binding proteins. Here we report our finding of an interaction between the cytoplasmic domain of beta 1 integrin and the actin-binding protein alpha-actinin. Beta 1- integrin cytoplasmic domain peptide columns bound several proteins from Triton extracts of chicken embryo fibroblasts. One protein at approximately 100 kD was identified by immunoblot analysis as alpha- actinin. Solid phase binding assays indicated that alpha-actinin bound specifically and directly to the beta 1 peptide with relatively high affinity. Using purified heterodimeric chicken smooth muscle integrin (a beta 1 integrin) or the platelet integrin glycoprotein IIb/IIIa complex (a beta 3 integrin), binding of alpha-actinin was also observed in similar solid phase assays, albeit with a lower affinity than was seen using the beta 1 peptide. alpha-Actinin also bound specifically to phospholipid vesicles into which glycoprotein IIb/IIIa had been incorporated. These results lead us to suggest that this integrin-alpha- actinin linkage may contribute to the attachment of actin filaments to the membrane in certain locations.
Cells require distinct adhesion complexes to form contacts with their neighbors or the extracellular matrix, and vinculin links these complexes to the actin cytoskeleton. Metavinculin, an isoform of vinculin that harbors a unique 68-residue insert in its tail domain, has distinct actin bundling and oligomerization properties and plays essential roles in muscle development and homeostasis. Moreover, patients with sporadic or familial mutations in the metavinculin-specific insert invariably develop fatal cardiomyopathies. Here we report the high resolution crystal structure of the metavinculin tail domain, as well as the crystal structures of full-length human native metavinculin (1,134 residues) and of the full-length cardiomyopathy-associated ΔLeu954 metavinculin deletion mutant. These structures reveal that an α-helix (H1′) and extended coil of the metavinculin insert replace α-helix H1 and its preceding extended coil found in the N-terminal region of the vinculin tail domain to form a new five-helix bundle tail domain. Further, biochemical analyses demonstrate that this helix replacement directs the distinct actin bundling and oligomerization properties of metavinculin. Finally, the cardiomyopathy associated ΔLeu954 and Arg975Trp metavinculin mutants reside on the replaced extended coil and the H1′ α-helix, respectively. Thus, a helix replacement mechanism directs metavinculin's unique functions.
Over the past decade our laboratory has focused on understanding how soluble cytoskeleton-associated proteins interact with membranes and other lipid aggregates. Many protein domains mediating specific cell membrane interactions appear by fluorescence microscopy and other precision techniques to be partially inserted into the lipid bilayer. It is unclear whether these protein-lipid-interactions are dependent on shared protein motifs or unique regional physiochemistry, or are due to more global characteristics of the protein.
We have developed a novel computational program that predicts a protein's lipid-binding site(s) from primary sequence data. Hydrophobic labeling, Fourier transform infrared spectroscopy (FTIR), film balance, T-jump, CD spectroscopy and calorimetry experiments confirm that the interfaces predicted for several key cytoskeletal proteins (alpha-actinin, Arp2, CapZ, talin and vinculin) partially insert into lipid aggregates. The validity of these predictions is supported by an analysis of the available three-dimensional structural data. The lipid interfaces predicted by our algorithm generally contain energetically favorable secondary structures (e.g., an amphipathic alpha-helix flanked by a flexible hinge or loop region), are solvent-exposed in the intact protein, and possess favorable local or global electrostatic properties.
At present, there are few reliable methods to determine the region of a protein that mediates biologically important interactions with lipids or lipid aggregates. Our matrix-based algorithm predicts lipid interaction sites that are consistent with the available biochemical and structural data. To determine whether these sites are indeed correctly identified, and whether use of the algorithm can be safely extended to other classes of proteins, will require further mapping of these sites, including genetic manipulation and/or targeted crystallography.
Integrins promote formation of focal adhesions and trigger intracellular signaling pathways through cytoplasmic proteins such as talin, alpha-actinin, and focal adhesion kinase (FAK). The beta 1 integrin subunit has been shown to bind talin and alpha-actinin in in vitro assays, and these proteins may link integrin to the actin cytoskeleton either directly or through linkages to other proteins such as vinculin. However, it is unknown which of these associations are necessary in vivo for formation of focal contacts, or which regions of beta 1 integrin bind to specific cytoskeletal proteins in vivo. We have developed an in vivo assay to address these questions. Microbeads were coated with anti-chicken beta 1 antibodies to selectively cluster chicken beta 1 integrins expressed in cultured mouse fibroblasts. The ability of cytoplasmic domain mutant beta 1 integrins to induce co-localization of proteins was assessed by immunofluorescence and compared with that of wild-type integrin. As expected, mutant beta 1 lacking the entire cytoplasmic domain had a reduced ability to induce co-localization of talin, alpha-actinin, F-actin, vinculin, and FAK. The ability of beta 1 integrin to co-localize talin and FAK was found to require a sequence near the C-terminus of beta 1. The region of beta 1 required to co-localize alpha-actinin was found to reside in a different sequence, several amino acids further from the C-terminus of beta 1. Deletion of 13 residues from the C-terminus blocked co-localization of talin, FAK, and actin, but not alpha-actinin. Association of alpha-actinin with clustered integrin is therefore not sufficient to induce the co-localization of F-actin.
In cultured cells, the 230-kDa protein talin is found at discrete plasma membrane foci known as focal adhesions, sites that anchor the intracellular actin cytoskeleton to the extracellular matrix. The regulated assembly of focal adhesions influences the direction of cell migrations or the reorientation of cell shapes. Biochemical studies of talin have shown that it binds to the proteins integrin, vinculin, and actin in vitro. To understand the function of talin in vivo and to correlate its in vitro and in vivo biochemical properties, various genetic approaches have been adopted. With the intention of using genetics in the study of talin, we identified a homologue to mouse talin in a genetic model system, the nematode Caenorhabditis elegans. C. elegans talin is 39% identical and 59% similar to mouse talin. In wild-type adult C. elegans, talin colocalizes with integrin, vinculin, and alpha-actinin in the focal adhesion-like structures found in the body-wall muscle. By examining the organization of talin in two different C. elegans mutant strains that do not make either beta-integrin or vinculin, we were able to determine that talin does not require vinculin for its initial organization at the membrane, but that it depends critically on the presence of integrin for its initial assembly at membrane foci.
The possible role of a 140K membrane-associated protein complex (140K) in fibronectin-cytoskeleton associations has been examined. The 140K was identified by the monoclonal antibody JG22E. Monoclonal and polyclonal antibodies to the 140K showed identical patterns of binding to the cell membranes of fixed and permeabilized chicken embryonic fibroblasts; localization was diffuse, but with marked concentration in cell-to-extracellular matrix contact sites. Correlative localization with interference reflection microscopy and double-label or triple- label immunofluorescence showed that 140K co-distributed with extracellular fibronectin fibrils and intracellular alpha-actinin in microfilament bundles at extracellular matrix contact sites but tended not to co-localize with tropomyosin present in bundles at sites farther from adhesion sites. In addition, binding of antibodies to 140K, alpha- actinin, and fibronectin was excluded from vinculin-rich focal adhesion sites at the cellular periphery. A progressive development of cell surface alpha-actinin-140K-fibronectin associations was observed in early spreading cells. The anti-140K monoclonal antibody JG22E inhibited the attachment and spreading of both normal and Rous sarcoma virus-transformed chicken embryonic fibroblasts to a fibronectin substratum. However, the anti-140K monoclonal antibody became a positive mediator of cell attachment and spreading if it was adsorbed or cross-linked to the substratum. Our results provide the first description of a membrane-associated protein complex that co-localizes with fibronectin and microfilament bundles, and they suggest that the 140K complex may be part of a cell surface linkage between fibronectin and the cytoskeleton.
Talin is a large cytoskeletal protein (2541 amino acid residues) which plays a key role in integrin-mediated events that are crucial for cell adhesion, migration, proliferation and survival. This review summarises recent work on the structure of talin and on some of the structurally better defined interactions with other proteins. The N-terminal talin head (approx. 50 kDa) consists of an atypical FERM domain linked to a long flexible rod (approx. 220 kDa) made up of a series of amphipathic helical bundle domains. The F3 FERM subdomain in the head binds the cytoplasmic tail of integrins, but this interaction can be inhibited by an interaction of F3 with a helical bundle in the talin rod, the so-called “autoinhibited form” of the molecule. The talin rod contains a second integrin-binding site, at least two actin-binding sites and a large number of binding sites for vinculin, which is important in reinforcing the initial integrin–actin link mediated by talin. The vinculin binding sites are defined by hydrophobic residues buried within helical bundles, and these must unfold to allow vinculin binding. Recent experiments suggest that this unfolding may be mediated by mechanical force exerted on the talin molecule by actomyosin contraction.
Electronic supplementary material
The online version of this article (doi:10.1007/s12551-009-0009-4) contains supplementary material, which is available to authorized users.
Cell adhesion; Extracellular matrix interactions; Integrin–actin link; Integrins; Talin
The retinal pigmented epithelium (RPE) is a simple cuboidal epithelium with apical processes which, unlike many epithelia, do not extend freely into a lumen but rather interdigitate closely with the outer segments of the neural retina. To determine whether this close association was reflected in the cytoskeletal organization of the RPE, we studied the components of the cytoskeleton of the RPE and their localization in the body of the cell and in the apical processes. By relative mobility on SDS gels and by immunoblotting, we identified actin, vimentin, myosin, spectrin (240/235), and alpha-actinin as major components, and vinculin as a minor component. In addition, the RPE cytoskeleton contains polypeptides of Mr 280,000 and 250,000; the latter co-electrophoreses with actin-binding protein. By immunofluorescence, the terminal web region appeared similar to the comparable region of the intestinal epithelium that consists of broad belts of microfilaments containing myosin, actin, spectrin, and alpha- actinin. However, the components of the apical processes were very different from those of intestinal microvilli. We observed staining along the process for myosin, actin, spectrin, alpha-actinin, and vinculin. The presence in the apical processes of contractile proteins and also of proteins typically found at sites of cell attachments suggests that the RPE may actively adhere to, and exert tension on, the neural retina.
Shigella flexneri, the causative agent of bacillary dysentery, injects invasin proteins through a type III secretion apparatus upon contacting the host cell, which triggers pathogen internalization. The invasin IpaA is essential for S. flexneri pathogenesis and binds to the cytoskeletal protein vinculin to facilitate host cell entry. We report that IpaA harbors two vinculin-binding sites (VBSs) within its C-terminal domain that bind to and activate vinculin in a mutually exclusive fashion. Only the highest affinity C-terminal IpaA VBS is necessary for efficient entry and cell–cell spread of S. flexneri, whereas the lower affinity VBS appears to contribute to vinculin recruitment at entry foci of the pathogen. Finally, the crystal structures of vinculin in complex with the VBSs of IpaA reveal the mechanism by which IpaA subverts vinculin's functions, where S. flexneri utilizes a remarkable level of molecular mimicry of the talin–vinculin interaction to activate vinculin. Mimicry of vinculin's interactions may therefore be a general mechanism applied by pathogens to infect the host cell.
Meta-vinculin, a vinculin-related protein, has been isolated from human uterus smooth muscle. Specific antibodies to meta-vinculin, which distinguish between meta-vinculin and vinculin, were prepared by absorption of anti-meta-vinculin serum on vinculin coupled to nitrocellulose. Meta-vinculin specific antibody demonstrates only smooth and cardiac muscle specificity and is able to cross-react with a small 21-kD fragment of the meta-vinculin polypeptide chain. This antibody does not interact with protease resistant 95-kD core shared by vinculin and meta-vinculin. Meta-vinculin specific antibody was used for the localization of meta-vinculin in smooth and cardiac muscles by the indirect immunofluorescence method. At the light microscopy resolution level it was found that meta-vinculin and vinculin are localized in the same cellular adhesive structures. Meta-vinculin is present in membrane-associated microfilament-bound plaques of smooth muscle, in intercalated discs and costameres of cardiac muscle. In primary culture of smooth muscle cells from human aorta, meta-vinculin and vinculin were found to be present in focal contacts of the cells. During the cultivation of smooth muscle cells, the quantity of meta- vinculin decreased progressively and finally meta-vinculin completely disappeared from the focal contacts. The data show that in smooth and cardiac muscles meta-vinculin could be a structural component of microfilament-membrane attachment sites, defined earlier by the localization of vinculin.