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.

Alterations in the actin cytoskeleton following the formation of cell-matrix and cell-cell junctions are orchestrated by vinculin. Vinculin associates with a large number of cytoskeletal and signaling proteins, and this flexibility is thought to contribute to rapid dissociation and reassociations of adhesion complexes. Intramolecular interactions between vinculin's head (Vh) and tail (Vt) domains limit access of its binding sites for other adhesion proteins. While the crystal structures of the Vh and Vt domains are known, these domains represent less than half of the entire protein and are separated by a large central region of unknown structure and function. Here we report the crystal structure of human full-length vinculin to 2.85 A resolution. In its resting state, vinculin is a loosely packed collection of alpha-helical bundles held together by Vh-Vt interactions. The three new well ordered alpha-helical bundle domains are similar in their structure to either Vh (Vh2 and Vh3) or to Vt (Vt2) and their loose packing provides the necessary flexibility that allows vinculin to interact with its various protein partners at sites of cell adhesion.

The number of recombination events per meiosis varies extensively among individuals. This recombination phenotype differs between female and male, and also among individuals of each gender. In this study, we used high-density SNP genotypes of over 2,300 individuals and their offspring in two datasets to characterize recombination landscape and to map the genetic variants that contribute to variation in recombination phenotypes. We found six genetic loci that are associated with recombination phenotypes. Two of these (RNF212 and an inversion on chromosome 17q21.31) were previously reported in the Icelandic population, and this is the first replication in any other population. Of the four newly identified loci (KIAA1462, PDZK1, UGCG, NUB1), results from expression studies provide support for their roles in meiosis. Each of the variants that we identified explains only a small fraction of the individual variation in recombination. Notably, we found different sequence variants associated with female and male recombination phenotypes, suggesting that they are regulated by different genes. Characterization of genetic variants that influence natural variation in meiotic recombination will lead to a better understanding of normal meiotic events as well as of non-disjunction, the primary cause of pregnancy loss.

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 ␣-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 ␣-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 ␣-actinin and disassembling focal adhesions. In their native (inactive) states the vinculin binding sites of talin and ␣-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 ␣-actinin, by provoking structural changes that allow their vinculin binding sites to swing out, which are then sufficient to bind to and activate vinculin.Adhesion complexes form on the cell membrane when cells come in contact with each other or with components of the extracellular matrix and trigger elaborate signaling networks that direct dynamic and rapid rearrangements of the actin cytoskeleton (1, 2). Vinculin is a highly conserved and critical regulator of both cell-cell and cell-matrix junctions, as it anchors these junctions to the actin cytoskeleton by binding to talin and ␣-actinin, which directly interact with integrin and/or cadherin transmembrane receptors in focal adhesions and adherens junctions, respectively (3, 4). Overall, vinculin appears to stabilize these junctions, as vinculin overexpression augments the formation of adhesion complexes and prevents cell migration (5), whereas vinculin loss leads to marked defects in adhesion with the extracellular matrix, cell spreading, and concomitant increases in the rates of (chaotic) cell migration (6, 7). Furthermore, vinculin appears to play an important role in cancer, where it functions as a tumor suppressor that inhibits cell invasion and metastasis (8), and in other pathophysiological scenarios, including wound healing, ischemia, and apoptosis (9 -11).Vinculin is a 117-kDa modular protein that is comprised of five helical bundle domains (Vh1, Vh2, Vh3, Vt2, and Vt, Ref. 12). Intramolecular interactions of its N-terminal seven-helical bundle (Vh1) domain with its C-terminal five-helical bundle tail (Vt) domain clamp vinculin in its inactive closed conformation (12-15). Biochemical studies have shown high affinity interactions of isolated Vh1 and Vt domains (14, 16), and additional interdomain interactions of Vt with the Vt2 domain have been proposed to further ...

Activation of the transcription factor FKHR (Forkhead in human rhabdomyosarcoma, FOXO1a) in various established cell lines induces cell cycle arrest followed by apoptosis. These effects are inhibited through activation of the phosphatidylinositol 3-kinase/Akt pathway, resulting in FKHR phosphorylation and its export from the nucleus, thus blocking its pro-apoptotic activity. Here we report that FKHR regulates fusion of differentiating primary myoblasts. We demonstrate that FKHR is localized in the cytoplasm of proliferating myoblasts, yet translocates to the nucleus by a phosphorylation-independent pathway following serum starvation, a condition that induces myoblast differentiation. FKHR phosphorylation during terminal differentiation appears to downregulate its fusion activity, as a dominant-active non-phosphorylatable FKHR mutant dramatically augments the rate and extent of myotube fusion. However, this FKHR mutant exerts its effects only after other events initiated the differentiation process. Conversely, enforced expression of a dominantnegative FKHR mutant blocks myotube formation whereas wild-type FKHR has no effect. We conclude that in addition to the role of FoxO proteins in regulating cell cycle progress and apoptosis, FKHR controls the rate of myotube fusion during myogenic differentiation.