Le facteur de restriction viral BST2/Tetherin ancre les Midbody post-cytokinétiques à la surface cellulaire

The Midbody Remnant (MBR) is a structure that arises once cytokinetic abscission, the last step in cell division, is completed. Then, the MBR interacts with the cell surface and can play various roles in development, polarisation or cell proliferation. I first characterized a new MBR purification method. This study revealed that BST2, a protein known to anchor enveloped viruses to the cell surface, is enriched at the MBR. I thus focused on BST2 and its role at the MBR, especially in the interaction with the recipient cell plasma membrane. I fist confirmed by microscopy the enrichment of BST2 t the MBR. Similarly to viruses, the absence of BST2 increases the detachment of MBRs from the cell surface. They are thus released in the extracellular medium, increasing their transfer to neighbouring cells. Mechanistically, in parallel with virion restriction, BST2 dimerization and GPI anchor are both required for proper localization and functions of BST2 at the MBR. Using purified MBRs, we showed that BST2 at the midbody membrane -but not at the plasma membrane of the cell- is important for MBR retention to the cell surface. Altogether, these results show that BST2 localizes at the midbody remnant to promote its retention at the cell surface of non-infected cells. I propose that, in a way analogous to enveloped virions, BST2 tethers midbody remnants and participates in promoting their proper interaction with recipient cells.Le Midbody (MB) post-cytokinétique est une structure créée suite à la séparation physique des cellules filles après la dernière étape de la division cellulaire, la cytokinèse. Le MB interagit ensuite avec la surface cellulaire afin de jouer des rôles variés dans le développement, la polarisation ou la prolifération cellulaire. J’ai d’abord caractérisé une nouvelle méthode de purification de MB post-cytokinétiques. Cette étude a révélé que BST2, une protéine connue pour ancrer les virus enveloppés à la surface cellulaire, est enrichie au MB. Je me suis donc concentré sur BST2 et son rôle au MB, notamment dans l’interaction avec la membrane plasmique de la cellule réceptrice. J’ai d’abord confirmé par microscopie l’enrichissement de BST2 au MB. De manière similaire aux virus, l’absence de BST2 augmente le détachement des MB de la surface cellulaire. Ils sont ainsi relâchés dans le milieu extracellulaire, augmentant leur transfert aux cellules avoisinantes. Mécaniquement, le domaine de dimérisation et l’ancre GPI de BST2 sont requis pour la localisation et la fonction au MB. En utilisant des MB purifiés, j’ai montré que BST2 à la membrane du MB -mais pas à la membrane plasmique- est important pour la rétention de ceux-ci à la surface cellulaire. En conclusion, ces résultats montrent une nouvelle fonction cellulaire de BST2. En effet, 1/ BST2 localise au MB et 2/BST2 permet de mieux retenir le MB à la surface cellulaire. Je propose donc que BST2 ancre les MB et participe à promouvoir leur étroite interaction avec la surface cellulaire, de manière analogue à son activité de restriction virale dans les cellules infectées

Le facteur de restriction viral BST2/Tetherin ancre les Midbody post-cytokinétiques à la surface cellulaire

The Midbody Remnant (MBR) is a structure that arises once cytokinetic abscission, the last step in cell division, is completed. Then, the MBR interacts with the cell surface and can play various roles in development, polarisation or cell proliferation. I first characterized a new MBR purification method. This study revealed that BST2, a protein known to anchor enveloped viruses to the cell surface, is enriched at the MBR. I thus focused on BST2 and its role at the MBR, especially in the interaction with the recipient cell plasma membrane. I fist confirmed by microscopy the enrichment of BST2 t the MBR. Similarly to viruses, the absence of BST2 increases the detachment of MBRs from the cell surface. They are thus released in the extracellular medium, increasing their transfer to neighbouring cells. Mechanistically, in parallel with virion restriction, BST2 dimerization and GPI anchor are both required for proper localization and functions of BST2 at the MBR. Using purified MBRs, we showed that BST2 at the midbody membrane -but not at the plasma membrane of the cell- is important for MBR retention to the cell surface. Altogether, these results show that BST2 localizes at the midbody remnant to promote its retention at the cell surface of non-infected cells. I propose that, in a way analogous to enveloped virions, BST2 tethers midbody remnants and participates in promoting their proper interaction with recipient cells.Le Midbody (MB) post-cytokinétique est une structure créée suite à la séparation physique des cellules filles après la dernière étape de la division cellulaire, la cytokinèse. Le MB interagit ensuite avec la surface cellulaire afin de jouer des rôles variés dans le développement, la polarisation ou la prolifération cellulaire. J’ai d’abord caractérisé une nouvelle méthode de purification de MB post-cytokinétiques. Cette étude a révélé que BST2, une protéine connue pour ancrer les virus enveloppés à la surface cellulaire, est enrichie au MB. Je me suis donc concentré sur BST2 et son rôle au MB, notamment dans l’interaction avec la membrane plasmique de la cellule réceptrice. J’ai d’abord confirmé par microscopie l’enrichissement de BST2 au MB. De manière similaire aux virus, l’absence de BST2 augmente le détachement des MB de la surface cellulaire. Ils sont ainsi relâchés dans le milieu extracellulaire, augmentant leur transfert aux cellules avoisinantes. Mécaniquement, le domaine de dimérisation et l’ancre GPI de BST2 sont requis pour la localisation et la fonction au MB. En utilisant des MB purifiés, j’ai montré que BST2 à la membrane du MB -mais pas à la membrane plasmique- est important pour la rétention de ceux-ci à la surface cellulaire. En conclusion, ces résultats montrent une nouvelle fonction cellulaire de BST2. En effet, 1/ BST2 localise au MB et 2/BST2 permet de mieux retenir le MB à la surface cellulaire. Je propose donc que BST2 ancre les MB et participe à promouvoir leur étroite interaction avec la surface cellulaire, de manière analogue à son activité de restriction virale dans les cellules infectées

The viral restriction factor BST2/Tetherin tethers Cytokinetic Midbody Remnants to the cell surface

Le Midbody (MB) post-cytokinétique est une structure créée suite à la séparation physique des cellules filles après la dernière étape de la division cellulaire, la cytokinèse. Le MB interagit ensuite avec la surface cellulaire afin de jouer des rôles variés dans le développement, la polarisation ou la prolifération cellulaire. J’ai d’abord caractérisé une nouvelle méthode de purification de MB post-cytokinétiques. Cette étude a révélé que BST2, une protéine connue pour ancrer les virus enveloppés à la surface cellulaire, est enrichie au MB. Je me suis donc concentré sur BST2 et son rôle au MB, notamment dans l’interaction avec la membrane plasmique de la cellule réceptrice. J’ai d’abord confirmé par microscopie l’enrichissement de BST2 au MB. De manière similaire aux virus, l’absence de BST2 augmente le détachement des MB de la surface cellulaire. Ils sont ainsi relâchés dans le milieu extracellulaire, augmentant leur transfert aux cellules avoisinantes. Mécaniquement, le domaine de dimérisation et l’ancre GPI de BST2 sont requis pour la localisation et la fonction au MB. En utilisant des MB purifiés, j’ai montré que BST2 à la membrane du MB -mais pas à la membrane plasmique- est important pour la rétention de ceux-ci à la surface cellulaire. En conclusion, ces résultats montrent une nouvelle fonction cellulaire de BST2. En effet, 1/ BST2 localise au MB et 2/BST2 permet de mieux retenir le MB à la surface cellulaire. Je propose donc que BST2 ancre les MB et participe à promouvoir leur étroite interaction avec la surface cellulaire, de manière analogue à son activité de restriction virale dans les cellules infectées.The Midbody Remnant (MBR) is a structure that arises once cytokinetic abscission, the last step in cell division, is completed. Then, the MBR interacts with the cell surface and can play various roles in development, polarisation or cell proliferation. I first characterized a new MBR purification method. This study revealed that BST2, a protein known to anchor enveloped viruses to the cell surface, is enriched at the MBR. I thus focused on BST2 and its role at the MBR, especially in the interaction with the recipient cell plasma membrane. I fist confirmed by microscopy the enrichment of BST2 t the MBR. Similarly to viruses, the absence of BST2 increases the detachment of MBRs from the cell surface. They are thus released in the extracellular medium, increasing their transfer to neighbouring cells. Mechanistically, in parallel with virion restriction, BST2 dimerization and GPI anchor are both required for proper localization and functions of BST2 at the MBR. Using purified MBRs, we showed that BST2 at the midbody membrane -but not at the plasma membrane of the cell- is important for MBR retention to the cell surface. Altogether, these results show that BST2 localizes at the midbody remnant to promote its retention at the cell surface of non-infected cells. I propose that, in a way analogous to enveloped virions, BST2 tethers midbody remnants and participates in promoting their proper interaction with recipient cells

The viral restriction factor tetherin/BST2 tethers cytokinetic midbody remnants to the cell surface

International audienceThe midbody at the center of the intercellular bridge connecting dividing cells recruits the machinery essential for the final steps of cytokinesis.1-5 Successive abscission on both sides of the midbody generates a free midbody remnant (MBR) that can be inherited and accumulated in many cancer, immortalized, and stem cells, both in culture and in vivo.6-12 Strikingly, this organelle was recently shown to contain information that induces cancer cell proliferation, influences cell polarity, and promotes dorso-ventral axis specification upon interaction with recipient cells.13-16 Yet the mechanisms by which the MBR is captured by either a daughter cell or a distant cell are poorly described.10,14 Here, we report that BST2/tetherin, a well-established restriction factor that blocks the release of numerous enveloped viruses from the surface of infected cells,17-20 plays an analogous role in retaining midbody remnants. We found that BST2 is enriched at the midbody during cytokinesis and localizes at the surface of MBRs in a variety of cells. Knocking out BST2 induces the detachment of MBRs from the cell surface, their accumulation in the extracellular medium, and their transfer to distant cells. Mechanistically, the localization of BST2 at the MBR membrane is both necessary and sufficient for the interaction between MBRs and the cell surface. We thus propose that BST2 tethers post-cytokinetic midbody remnants to the cell surface. This finding reveals new parallels between cytokinesis and viral biology21-26 that unexpectedly extend beyond the ESCRT-dependent abscission step

Introns Protect Eukaryotic Genomes from Transcription-Associated Genetic Instability

International audienceTranscription is a source of genetic instability that can notably result from the formation of genotoxic DNA:RNA hybrids, or R-loops, between the nascent mRNA and its template. Here we report an unexpected function for introns in counteracting R-loop accumulation in eukaryotic genomes. Deletion of endogenous introns increases R-loop formation, while insertion of an intron into an intronless gene suppresses R-loop accumulation and its deleterious impact on transcription and recombination in yeast. Recruitment of the spliceosome onto the mRNA, but not splicing per se, is shown to be critical to attenuate R-loop formation and transcription-associated genetic instability. Genome-wide analyses in a number of distant species differing in their intron content, including human, further revealed that intron-containing genes and the intron-richest genomes are best protected against R-loop accumulation and subsequent genetic instability. Our results thereby provide a possible rationale for the conservation of introns throughout the eukaryotic lineage

The mammalian midbody and midbody remnant are assembly sites for RNA and localized translation

International audienceLong ignored as a vestigial remnant of cytokinesis, the mammalian midbody (MB) is released post-abscission inside large extracellular vesicles called MB remnants (MBRs). Recent evidence suggests that MBRs can modulate cell proliferation and cell fate decisions. Here, we demonstrate that the MB matrix is the site of ribonucleoprotein assembly and is enriched in mRNAs that encode proteins involved in cell fate, oncogenesis, and pluripotency, which we are calling the MB granule. Both MBs and post-abscission MBRs are sites of spatiotemporally regulated translation, which is initiated when nascent daughter cells re-enter G1 and continues after extracellular release. MKLP1 and ARC are necessary for the localization and translation of RNA in the MB dark zone, whereas ESCRT-III is necessary to maintain translation levels in the MB. Our work reveals a unique translation event that occurs during abscission and within a large extracellular vesicle

The Flemmingsome reveals an ESCRT-to-membrane coupling via ALIX/syntenin/syndecan-4 required for completion of cytokinesis

International audienceCytokinesis requires the constriction of ESCRT-III filaments on the side of the midbody, where abscission occurs. After ESCRT recruitment at the midbody, it is not known how the ESCRT-III machinery localizes to the abscission site. To reveal actors involved in abscission, we obtained the proteome of intact, post-abscission midbodies (Flemmingsome) and identified 489 proteins enriched in this organelle. Among these proteins, we further characterized a plasma membrane-to-ESCRT module composed of the transmembrane proteoglycan syndecan-4, ALIX and syntenin, a protein that bridges ESCRT-III/ALIX to syndecans. The three proteins are highly recruited first at the midbody then at the abscission site, and their depletion delays abscission. Mechanistically, direct interactions between ALIX, syntenin and syndecan-4 are essential for proper enrichment of the ESCRT-III machinery at the abscission site, but not at the midbody. We propose that the ESCRT-III machinery must be physically coupled to a membrane protein at the cytokinetic abscission site for efficient scission, uncovering common requirements in cytokinesis, exosome formation and HIV budding