Kinetochore component function in C. elegans oocytes revealed by 4D tracking of holocentric chromosomes

During cell division, chromosome congression to the spindle center, their orientation along the spindle long axis and alignment at the metaphase plate depend on interactions between spindle microtubules and kinetochores, and are pre-requisite for chromosome bi-orientation and accurate segregation. How these successive phases are controlled during oocyte meiosis remains elusive. Here we provide 4D live imaging during the first meiotic division in C. elegans oocytes with wild-type or disrupted kinetochore protein function. We show that, unlike in monocentric organisms, holocentric chromosome bi-orientation is not strictly required for accurate chromosome segregation. Instead, we propose a model in which initial kinetochore-localized BHC module (comprised of BUB-1Bub1, HCP-1/2CENP-F and CLS-2CLASP)-dependent pushing acts redundantly with Ndc80 complex-mediated pulling for accurate chromosome segregation in meiosis. In absence of both mechanisms, homologous chromosomes tend to co-segregate in anaphase, especially when initially mis-oriented. Our results highlight how different kinetochore components cooperate to promote accurate holocentric chromosome segregation in oocytes of C. elegans.This work was supported by CNRS and University ParisCité, by NIHR01GM117407 and R01GM130764 (J.C.C.), and by grants from the European Research Council ERC-CoG ChromoSOMe 819179 and from the Agence Nationale de la Recherche ANR-19-CE13-0015 (J.D.).Peer ReviewedPostprint (published version

Mécanismes de ségrégation des chromosomes dans l'ovocyte de C. elegans

Les gamètes femelles appelés ovocytes sont produits par un type spécifique de division cellulaire appelée méiose. Afin de produire des gamètes haploïdes, et contrairement aux divisions mitotiques des cellules somatiques, la méiose implique une seule étape de réplication du génome suivie de deux étapes de ségrégation des chromosomes. La fidélité de la ségrégation des chromosomes pendant la méiose est cruciale pour éviter l’aneuploïdie embryonnaire qui entraînerait des défauts de développement ou un avortement spontané. Dans la plupart des types cellulaires, la ségrégation des chromosomes repose sur un fuseau composé de microtubules. En parallèle à l'assemblage du fuseau, des complexes multi-protéiques appelés kinétochores s’assemblent sur le côté des chromosomes et leur permettent d’interagir avec les microtubules dynamiques du fuseau. Étonnamment, la ségrégation des chromosomes dans l'ovocyte de C. elegans se déroule d'une manière atypique indépendante des kinétochores. Le mécanisme alternatif utilisé dans ces oocytes pour la ségrégation des chromosomes est cependant inconnu. Au cours de mon doctorat, j'ai utilisé une combinaison d'imagerie photonique à haute résolution temporelle, corrélée à de la microscopie électronique à haute résolution spatiale. J’ai également utilisé de la photoablation par laser des microtubules et réalisé l'inhibition ciblée de protéines clés pour disséquer le mécanisme atypique de ségrégation des chromosomes dans l'ovocyte de C. elegans. Mes résultats montrent que la ségrégation des chromosomes est produite par une force dépendante des microtubules qui pousse les chromosomes. Par une analyse détaillée de l’organisation des microtubules dans des fuseaux en anaphase partiellement reconstruits par microscopie électronique en tomographie, je propose un modèle impliquant la génération de force par l'allongement d’un réseau de courts microtubules formant le fuseau central. De plus, je démontre que l'activité de l'orthologue de CLASP chez C. elegans (CLS-2) est essentielle pour l'assemblage du fuseau en anaphase. Ce travail est actuellement sous presse dans le journal Nature Communications. Parallèlement, j'ai disséqué le rôle de CLS-2 dans l'assemblage du fuseau d'ovocytes et la ségrégation chromosomique. J'ai perturbé de manière systématique les domaines individuels et les résidus conservés de manière évolutive dans CLS-2 pour déterminer leur contribution à la fonction et à la localisation de cette protéine pendant la première méiose femelle. Dans l'ensemble, mes résultats montrent que la ségrégation chromosomique dans l'ovocyte de C. elegans consiste en un mécanisme de poussée chromosomique atypique et dépendant de CLS-2.Female gametes called oocytes are produced through a specific type of celldivision termed meiosis. In order to produce haploid gametes, and unlike mitoticdivisions of somatic cells, meiosis involves a single round of genome replication followed by two rounds of chromosome segregation. Accuracy of chromosome segregation during meiosis is crucial to avoiding embryonic aneuploidy that wouldlead to developmental defects or spontaneous abortion. In most cell types,chromosome segregation relies on a microtubule-based spindle. Concomitant tospindle assembly, multi-protein complexes termed kinetochores assemble on the side of chromosomes and couple microtubule dynamics to chromosomal movements. Strikingly, in the C. elegans oocyte chromosome segregation occurs in an atypical kinetochore-independent manner. The alternative mechanism used in these oocytes for chromosome segregation is however unknown. During my PhD, I used a combination of high spatial and temporal resolution live imaging, correlated light and electron tomography, laser-mediated photoablation of microtubules, and targeted inhibition of key proteins to dissect this a typical mechanism of chromosome segregation in the C. elegans oocyte. Myresults show that chromosome segregation is driven by a microtubule-dependent force that pushes the segregating chromosomes apart during anaphase. Aftercareful analysis of partially reconstructed anaphase spindles by electrontomography for microtubule quantity, length, orientation, and overlaps, I proposea model involving the elongation and/or sliding of tiled microtubules in the central spindle as the candidate structure responsible for this force generation. Additionally, I demonstrate that the activity of the C. elegans CLASP ortholog CLS-2 is essential for proper anaphase spindle assembly. This work is currently in press at Nature Communications.In parallel, I have more closely examined the role of the C. elegans CLS-2 in oocyte spindle assembly and chromosome segregation. I have thoroughly and systematically perturbed the individual domains and evolutionarily conserved residues in CLS-2 to determine their contribution to the function and localization ofthis protein during the first female meiosis. Overall my results show that chromosome segregation in the C. elegan

Kinetochore components are required for central spindle assembly.

International audienceA critical structure poised to coordinate chromosome segregation with division plane specification is the central spindle that forms between separating chromosomes after anaphase onset. The central spindle acts as a signalling centre that concentrates proteins essential for division plane specification and contractile ring constriction. However, the molecular mechanisms that control the initial stages of central spindle assembly remain elusive. Using Caenorhabditis elegans zygotes, we found that the microtubule-bundling protein SPD-1(PRC1) and the motor ZEN-4(MKLP-1) are required for proper central spindle structure during its elongation. In contrast, we found that the kinetochore controls the initiation of central spindle assembly. Specifically, central spindle microtubule assembly is dependent on kinetochore recruitment of the scaffold protein KNL-1, as well as downstream partners BUB-1, HCP-1/2(CENP-F) and CLS-2(CLASP); and is negatively regulated by kinetochore-associated protein phosphatase 1 activity. This in turn promotes central spindle localization of CLS-2(CLASP) and initial central spindle microtubule assembly through its microtubule polymerase activity. Together, our results reveal an unexpected role for a conserved kinetochore protein network in coupling two critical events of cell division: chromosome segregation and cytokinesis

High-Resolution Temporal Analysis Reveals a Functional Timeline for the Molecular Regulation of Cytokinesis

International audienceTo take full advantage of fast-acting temperature-sensitive mutations, thermal control must be extremely rapid. We developed the Therminator, a device capable of shifting sample temperature in ∼17 s while simultaneously imaging cell division in vivo. Applying this technology to six key regulators of cytokinesis, we found that each has a distinct temporal requirement in the Caenorhabditis elegans zygote. Specifically, myosin-II is required throughout cytokinesis until contractile ring closure. In contrast, formin-mediated actin nucleation is only required during assembly and early contractile ring constriction. Centralspindlin is required to maintain division after ring closure, although its GAP activity is only required until just prior to closure. Finally, the chromosomal passenger complex is required for cytokinesis only early in mitosis, but not during metaphase or cytokinesis. Together, our results provide a precise functional timeline for molecular regulators of cytokinesis using the Therminator, a powerful tool for ultra-rapid protein inactivation