Philippe R.J. Bois

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 α-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 α-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, talins effects on Vh structure establish helical bundle conversion as a signalling mechanism by which proteins direct cellular responses.

FKHR (FOXO1a) is required for myotube fusion of primary mouse myoblasts

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 pro cess. Conversely, enforced expression of a dominant‐negative 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.

Genetic Analysis of Variation in Human Meiotic Recombination

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.

The Vinculin Binding Sites of Talin and α-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 α-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 α-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 α-actinin and disassembling focal adhesions. In their native (inactive) states the vinculin binding sites of talin and α-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 α-actinin, by provoking structural changes that allow their vinculin binding sites to swing out, which are then sufficient to bind to and activate vinculin.

Structural Dynamics of α-Actinin-Vinculin Interactions

ABSTRACT α-Actinin and vinculin orchestrate reorganization of the actin cytoskeleton following the formation of adhesion junctions. α-Actinin interacts with vinculin through the binding of an α-helix (αVBS) present within the R4 spectrin repeat of its central rod domain to vinculins N-terminal seven-helical bundle domain (Vh1). The Vh1:αVBS structure suggests that αVBS first unravels from its buried location in the triple-helical R4 repeat to allow it to bind to vinculin. αVBS binding then induces novel conformational changes in the N-terminal helical bundle of Vh1, which disrupt its intramolecular association with vinculins tail domain and which differ from the alterations in Vh1 provoked by the binding of talin. Surprisingly, αVBS binds to Vh1 in an inverted orientation compared to the binding of talins VBSs to vinculin. Importantly, the binding of αVBS and talins VBSs to vinculins Vh1 domain appear to also trigger distinct conformational changes in full-length vinculin, opening up distant regions that are buried in the inactive molecule. The data suggest a model where vinculins Vh1 domain acts as a molecular switch that undergoes distinct structural changes provoked by talin and α-actinin binding in focal adhesions versus adherens junctions, respectively.

High‐resolution sperm typing of meiotic recombination in the mouse MHC Eβ gene

Meiotic crossovers detected by pedigree analysis in the mouse MHC cluster into hotspots. To explore the properties of hotspots, we subjected the class II Eβ gene to high‐resolution sperm crossover analysis. We confirm the presence of a highly localized hotspot 1.0–1.6 kb wide in the second intron of Eβ and show that it is flanked by DNA which is almost completely recombinationally inert. Mice heterozygous for haplotype s and another MHC haplotype show major haplotype‐dependant variation in crossover rate but always the same hotspot, even in crosses including the highly diverged p haplotype. Crossovers in reciprocal orientations occur at similar rates but show different distributions across the hotspot, with the position of centre points in the two orientations shifted on average by 400 bp. This asymmetry results in crossover products showing biased gene conversion in favour of hotspot markers from the non‐initiating haplotype, and supports the double‐strand break repair model of recombination, with haplotype s as the most efficient crossover initiator. The detailed behaviour of the Eβ hotspot, including evidence for highly localized recombination initiation, is strikingly similar to human hotspots.

Shigella applies molecular mimicry to subvert vinculin and invade host cells.

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 vinculins functions, where S. flexneri utilizes a remarkable level of molecular mimicry of the talin–vinculin interaction to activate vinculin. Mimicry of vinculins interactions may therefore be a general mechanism applied by pathogens to infect the host cell.

Nucleosome occupancy landscape and dynamics at mouse recombination hotspots

During meiosis, paternal and maternal homologous chromosomes recombine at specific recombination sites named hotspots. What renders 2% of the mammalian genomes permissive to meiotic recombination by allowing Spo11 endonuclease to initiate double‐strand breaks is largely unknown. Work in yeast has shown that chromatin accessibility seems to be important for this activity. Here, we define nucleosome profiles and dynamics at four mouse recombination hotspots by purifying highly enriched fractions of meiotic cells. We found that nucleosome occupancy is generally stable during meiosis progression. Interestingly, the cores of recombination hotspots have largely open chromatin structure, and the localization of the few nucleosomes present in these cores correlates precisely with the crossover‐free zones in recombinogenic domains. Collectively, these high‐resolution studies suggest that nucleosome occupancy seems to direct, at least in part, how meiotic recombination events are processed.

Hypermutable minisatellites, a human affair?

Minisatellites are a class of highly polymorphic GC-rich tandem repeats. They include some of the most variable loci in the human genome, with mutation rates ranging from 0.5% to >20% per generation. Structurally, they consist of 10- to 100-bp intermingled variant repeats, making them ideal tools for dissecting mechanisms of instability at tandem repeats. Distinct mutation processes generate rare intra-allelic somatic events and frequent complex conversion-like germline mutations in these repeats. Furthermore, turnover of repeats at human minisatellites is controlled by intense recombinational activity in DNA flanking the repeat array. Surprisingly, whereas other mammalian genomes possess minisatellite-like sequences, hypermutable loci have not been identified that suggest human-specific turnover processes at minisatellite arrays. Attempts to transfer minisatellite germline instability to the mouse have failed. However, yeast models are now revealing valuable information regarding the mechanisms regulating instability at these tandem repeats. Finally, minisatellites and tandem repeats provide exquisitely sensitive molecular tools to detect genomic insults such as ionizing radiation exposure. Surprisingly, by a mechanism that remains elusive, there are transgenerational increases in minisatellite instability.

FoxO1a-cyclic GMP-dependent kinase I interactions orchestrate myoblast fusion.

ABSTRACT The regulatory circuits that orchestrate mammalian myoblast cell fusion during myogenesis are poorly understood. The transcriptional activity of FoxO1a directly regulates this process, yet the molecular mechanisms governing FoxO1a activity during muscle cell differentiation remain unknown. Here we show an autoregulatory loop in which FoxO1a directly activates transcription of the cyclic GMP-dependent protein kinase I (cGKI) gene and where the ensuing cGKI activity phosphorylates FoxO1a and abolishes its DNA binding activity. These findings establish the FoxO1a-to-cGKI pathway as a novel feedback loop that allows the precise tuning of myoblast fusion. Interestingly, this pathway appears to operate independently of muscle cell differentiation programs directed by myogenic transcription factors.