16 research outputs found

    Chromatids segregate without centrosomes during Caenorhabditis elegans mitosis in a Ran- and CLASP-dependent manner

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    This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License.During mitosis, chromosomes are connected to a microtubule-based spindle. Current models propose that displacement of the spindle poles and/or the activity of kinetochore microtubules generate mechanical forces that segregate sister chromatids. Using laser destruction of the centrosomes during Caenorhabditis elegans mitosis, we show that neither of these mechanisms is necessary to achieve proper chromatid segregation. Our results strongly suggest that an outward force generated by the spindle midzone, independently of centrosomes, is sufficient to segregate chromosomes in mitotic cells. Using mutant and RNAi analysis, we show that the microtubule-bundling protein SPD-1/MAP-65 and BMK-1/kinesin-5 act as a brake opposing the force generated by the spindle midzone. Conversely, we identify a novel role for two microtubule-growth and nucleation agents, Ran and CLASP, in the establishment of the centrosome-independent force during anaphase. Their involvement raises the interesting possibility that microtubule polymerization of midzone microtubules is continuously required to sustain chromosome segregation during mitosis.This work was supported by an ATIP grant from Centre National de la Recherche Scientifique and Human Frontiers Science Program Grant RGP0034/2010 to M.D. W.N. is the recipient of a PhD fellowship from the French Government.Peer Reviewe

    Spindle elongation in C. elegans embryos : characterization of a new pushing force

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    A la fin de la vie d’une cellule, différentes forces mécaniques permettent la séparation des chromosomes. Nos données préliminaires suggèrent l’existence d’un autre mécanisme provenant du centre du fuseau mitotique, non décrit dans l’embryon une cellule de C. elegans qui permettrait la séparation des chromosomes. Dans cette cellule, les microtubules kinétochoriens n’appliquent aucune force mécaniques sur les chromosomes durant l’anaphase. Il a été décrit que les chromosomes sont séparés grâce au déplacement des centrosomes via les forces de traction corticales. A l’aide de la microchirurgie laser dans les embryons une cellule de C. elegans, j’ai montré qu’en détruisant physiquement un ou deux centrosomes, les chromosomes continuent de se séparer, révélant l’existence d’une force de propulsion interne au fuseau mitotique (Nahaboo et al., 2015). En combinant la destruction de centrosomes et l’inactivation génétique, nous avons caractérisé les rôles de gènes favorisant ou freinant cette force de propulsion. J’ai observé que la kinésine-5, BMK-1, et le crosslinker MAP-65/SPD-1 freinent cette force de propulsion. Alors que dans d’autres espèces ces protéines favorisent la séparation des chromosomes. Nous avons remarqué que les protéines RanGTP et CLASP, favorisant de la nucléation et la polymérisation des microtubules, aident cette force de propulsion. Ces propriétés suggèrent que la polymérisation des microtubules au centre du fuseau est requise pour permettre la séparation des chromosomes durant la mitose.Par manque d’outils adéquats afin d’altérer la dynamique des microtubules, nous avons collaboré avec l’équipe de biochimistes du Dr. D. Trauner à Munich en Allemagne. Ils ont synthétisé la molécule photoactivable, Photostatin (PST), permettant la dépolymérisation des microtubules en quelques secondes (Borowiak et al., 2015). Entre 390 - 430 nm, PST est activé, dépolymérisant les microtubules, alors qu’entre 500 – 530 nm, PST est inactivé, permettant la polymérisation normale des microtubules. J’ai mesuré que la croissance des microtubules avec PST actif est absente dans des cellules Hela. J’ai montré que le cycle cellulaire dans l’embryon de C. elegans est arrêté localement en présence de PST actif. Nous avons alors montré que PST contrôle optiquement la dynamique des microtubules, in vitro, in cellulo et in vivo, de manière non invasive, rapide, locale et réversible. En résume, j’ai identifié une nouvelle force permettant la séparation des chromosomes à l’aide des approches moléculaires et biophysiques, et j’ai aidé à la caractérisation PST, un antimicrotubule photoactivable de manière locale et réversible.In mitosis, different mechanical forces are involved in chromosome segregation. In C. elegans one-cell embryos, preliminary data suggest that an unknown mechanism, coming from inside the mitotic spindle, could influence chromosome separation. In those cells, it has been showed that kinetochore microtubule activity is absent. Thanks to external pulling forces, centrosome separation drives chromosome segregation. By using microsurgery inside the one-cell C. elegans embryos, we have shown that destroying one or two centrosomes did not prevent chromosome separation, revealing the existence of an outward pushing force (Nahaboo et al., 2015). By combining gene inactivation and centrosome destruction, we showed that the kinesin-5 and the crosslinker SPD-1 act as a brake on this pushing force, whereas they enhance chromosome segregation in other species. Moreover, we identified a novel role for the two microtubule-growth and nucleation agents, RanGTP and CLASP, in the establishment of the centrosome-independent force during anaphase. Their involvement raises the interesting possibility that microtubule polymerization of midzone microtubules is required to sustain chromosome segregation during mitosis. Then, we aim to reversibility affect microtubule dynamics in the central spindle. Because of the lack of adequate tools, we have collaborated with biochemists from Dr. D. Trauner lab, in Munich, Germany, who are specialized in photoactivable drugs. They have synthetized a photoswitable drug, Photostatin (PST), which can depolymerize microtubules in few seconds in an on/off manner (Borowiak et al., 2015). Under blue light (390 - 430 nm), PST is activated leading to microtubule depolymerization, whereas under green light (500 - 530 nm), PST is activated which does not affect microtubule dynamics. I measured that microtubule growing is absent in presence of activated PST in Hela cells. I also showed that cell cycle can be stopped thank to activated PST in multiple cell C. elegans embryos. We have shown that PST can control microtubule dynamics thanks to visible light in vitro, in cellulo and in vivo, as an on/off switch, in a non-invasive, local and reversible manner

    Elongation du fuseau mitotique dans l'embryon de C. elegans: caractérisation d'une nouvelle force de propulsion

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    info:eu-repo/semantics/nonPublishe

    RNASeq_mouse embryo mesoderm

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    This file contains the following excel files: Sample list, Expression levels, Ranked list of differential expressio

    Distinct mesoderm migration phenotypes in extra-embryonic and embryonic regions of the early mouse embryo

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    In mouse embryo gastrulation, epiblast cells delaminate at the primitive streak to form mesoderm and definitive endoderm, through an epithelial-mesenchymal transition. Mosaic expression of a membrane reporter in nascent mesoderm enabled recording cell shape and trajectory through live imaging. Upon leaving the streak, cells changed shape and extended protrusions of distinct size and abundance depending on the neighboring germ layer, as well as the region of the embryo. Embryonic trajectories were meandrous but directional, while extra-embryonic mesoderm cells showed little net displacement. Embryonic and extra-embryonic mesoderm transcriptomes highlighted distinct guidance, cytoskeleton, adhesion, and extracellular matrix signatures. Specifically, intermediate filaments were highly expressed in extra-embryonic mesoderm, while live imaging for F-actin showed abundance of actin filaments in embryonic mesoderm only. Accordingly, Rhoa or Rac1 conditional deletion in mesoderm inhibited embryonic, but not extra-embryonic mesoderm migration. Overall, this indicates separate cytoskeleton regulation coordinating the morphology and migration of mesoderm subpopulations.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

    Regionally specific levels and patterns of keratin 8 expression in the mouse embryo visceral endoderm emerge upon anterior-posterior axis determination

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    The mechanical properties of the different germ layers of the early mammalian embryo are likely to be critical for morphogenesis. Cytoskeleton components (actin and myosin, microtubules, intermediate filaments) are major determinants of epithelial plasticity and resilience to stress. Here, we take advantage of a mouse reporter for Keratin 8 to record the pattern of the keratin intermediate filaments network in the first epithelia of the developing mouse embryo. At the blastocyst stage, Keratin 8 is strongly expressed in the trophectoderm, and undetectable in the inner cell mass and its derivatives, the epiblast and primitive endoderm. Visceral endoderm cells that differentiate from the primitive endoderm at the egg cylinder stage display apical Keratin 8 filaments. Upon migration of the Anterior Visceral Endoderm and determination of the anterior-posterior axis, Keratin 8 becomes regionally distributed, with a stronger expression in embryonic, compared to extra-embryonic, visceral endoderm. This pattern emerges concomitantly to a modification of the distribution of Filamentous (F)-actin, from a cortical ring to a dense apical shroud, in extra-embryonic visceral endoderm only. Those regional characteristics are maintained across gastrulation. Interestingly, for each stage and region of the embryo, adjacent germ layers display contrasted levels of keratin filaments, which may play a role in their adaptation to growth and morphological changes.info:eu-repo/semantics/publishe

    Asymmetry in the frequency and position of mitosis in the mouse embryo epiblast at gastrulation.

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    At gastrulation, a subpopulation of epiblast cells constitutes a transient posteriorly located structure called the primitive streak, where cells that undergo epithelial-mesenchymal transition make up the mesoderm and endoderm lineages. Mouse embryo epiblast cells were labelled ubiquitously or in a mosaic fashion. Cell shape, packing, organization and division were recorded through live imaging during primitive streak formation. Posterior epiblast displays a higher frequency of rosettes, some of which associate with a central cell undergoing mitosis. Cells at the primitive streak, in particular delaminating cells, undergo mitosis more frequently than other epiblast cells. In pseudostratified epithelia, mitosis takes place at the apical side of the epithelium. However, mitosis is not restricted to the apical side of the epiblast, particularly on its posterior side. Non-apical mitosis occurs specifically in the streak even when ectopically located. Posterior non-apical mitosis results in one or two daughter cells leaving the epiblast layer. Cell rearrangement associated with mitotic cell rounding in posterior epiblast, in particular when non-apical, might thus facilitate cell ingression and transition to a mesenchymal phenotype.info:eu-repo/semantics/publishe
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