Mutual Antagonism Between the Anaphase Promoting Complex and the Spindle Assembly Checkpoint Contributes to Mitotic Timing in Caenorhabditis elegans

The anaphase promoting complex/cyclosome (APC/C) triggers the separation of sister chromatids and exit from mitosis across eukaryotic evolution. The APC/C is inhibited by the spindle assembly checkpoint (SAC) until all chromosomes have achieved bipolar attachment, but whether the APC/C reciprocally regulates the SAC is less understood. Here, we report the characterization of a novel allele of the APC5 component SUCH-1 in Caenorhabditis elegans. We find that some such-1(t1668) embryos lack paternally contributed DNA and centrioles and assemble a monopolar spindle in the one-cell stage. Importantly, we show that mitosis is drastically prolonged in these embryos, as well as in embryos that are otherwise compromised for APC/C function and assemble a monopolar spindle. This increased duration of mitosis is dependent on the SAC, since inactivation of the SAC components MDF-1/MAD1 or MDF-2/MAD2 rescues proper timing in these embryos. Moreover, partial depletion of the E1 enzyme uba-1 significantly increases mitosis duration upon monopolar spindle assembly. Taken together, our findings raise the possibility that the APC/C negatively regulates the SAC and, therefore, that the SAC and the APC/C have a mutual antagonistic relationship in C. elegans embryos.

Mutual Inhibition Between the Anaphase-promoting Complex and the Spindle Assembly Checkpoint in C. Elegans Embryos

Alexandra Bezler

The cell cycle orchestrates timely duplication and distribution of cellular components to daughter cells. Checkpoints operate to survey the faithful completion of specific steps during the cell cycle, thus ensuring the equal segregation of genetic material. During mitosis, the anaphase promoting complex/cyclosome (APC/C) triggers the separation of sister chromatids and the exit from mitosis. The APC/C is inhibited by the spindle assembly checkpoint (SAC) until all chromosomes have achieved bipolar attachment, but whether the APC/C reciprocally regulates the SAC is less understood. The principal line of work of this thesis is the characterization of a novel allele of the APC5 component SUCH-1 in C. elegans. We find that some such-1(t1668) embryos lack paternally-contributed DNA and centrioles and assemble a monopolar spindle in the one-cell stage. Importantly, we show that mitosis is drastically prolonged in these embryos, as well as in embryos that are otherwise compromised for APC/C function and assemble a monopolar spindle. This increased duration of mitosis is dependent on the SAC, since inactivation of the SAC components MDF-1/Mad1 or MDF-2/Mad2 rescues proper timing in these embryos. Taken together, our findings support a model whereby the APC/C negatively regulates the SAC and, therefore, that the SAC and the APC/C have a mutual antagonistic relationship in C. elegans embryos. In a second line of work, we set out to ask whether the difference in the pace of cell cycle progression at the two-cell stage in early C. elegans embryos is important for proper development. The pace of cell cycle progression changes in specific lineages during development of metazoan organisms, and in C. elegans this is already apparent at the two-cell stage, since the anterior blastomere AB divides 2 min before the posterior blastomere P1. Since cell cycle duration is temperature-dependent in nematodes, we set out to induce a synchronous division between the two cells by heating preferentially P1, and thus accelerating specifically its cell cycle. To this end, in collaboration with the Renaud laboratory, we developed a microfluidic chip that can accommodate C. elegans embryos and generate a transient temperature gradient between AB and P1. So far, we have managed to achieve a temperature difference of ∼2 °C over the 50 µm that correspond to the length of a C. elegans embryo. Analysis of embryonic development at different temperatures indicates that this should be increased to ∼3-5 °C by further optimization to achieve the desired synchronous division.

EPFL2011

Characterizing the localization and role of lin-5 and era-1 mRNAs in early C. elegans embryos

Zoltán Péter Spiró

Asymmetric cell division is essential for the generation of diversity during development and the function of stem cell lineages. The Caenorhabditis elegans zygote is an attractive model to investigate the mechanisms of spindle positioning during asymmetric cell division. In this polarized cell, the asymmetric distribution of cortical force generators along the antero-posterior axis and pulling on astral microtubules leads to the unequal cleavage of the one-cell embryo. The mechanisms underlying such cortical force generation are thought to act strictly at the protein level. In this thesis work we report that the mRNA encoding the cortical force generator component LIN-5 is enriched around centrosomes in early embryos, in a manner that depends on microtubules and dynein. We established that the lin-5 coding sequence is necessary and sufficient for mRNA enrichment around centrosomes in C. elegans. In addition, we found that lin-5 mRNA is mislocalized in lin-5(ev571) mutant embryos, which harbor a 9 nucleotide insertion in the coding sequence. Moreover, an intragenic revertant of lin-5(ev571), lin-5(ev571he63), also exhibits mislocalized lin-5 mRNA distribution. We demonstrated that this is accompanied by diminished pulling forces on the posterior spindle pole, suggesting that centrosomal localization of lin-5 mRNA is important for robust pulling forces. We found also that lin-5 mRNA centrosomal enrichment is slightly asymmetric during anaphase, with more transcripts present on the anterior side. We developed a novel FRAP-based assay, which revealed that lin-5 is translated/folded preferentially in the cytoplasm compared to centrosomes. Furthermore, we found that morpholino-mediated inhibition of lin-5 translation diminishes pulling forces on the posterior side during anaphase. Together, these findings lead us to propose that preferential translation/folding of lin-5 in the posterior cytoplasm following release of the mRNA from the posterior centrosome contributes to asymmetric cortical distribution of force generators, and thus to proper spindle positioning. Moreover, we found that the mRNA of an uncharacterized gene, era-1 is enriched on the anterior side of the zygote and is inherited by the anterior blastomeres. Similar to era-1 mRNA, a YFP fusion of ERA-1 protein is also asymmetrically distributed. Moreover, asymmetric distribution of both era-1 mRNA and YFP-ERA-1 protein requires the era-1 3'UTR. Furthermore, the RNA-binding protein MEX-5 is needed for both asymmetric era-1 mRNA localization and for its translational activation. Furthermore, we report that the clathrin heavy chain CHC-1 negatively regulates pulling forces acting on centrosomes during interphase and on spindle poles during mitosis in one-cell C. elegans embryos. We establish a similar role for the cytokinesis/apoptosis/RNA-binding protein CAR-1 and uncover that CAR-1 is needed to maintain normal levels of CHC-1. We demonstrate that CHC-1 is necessary for proper organization of the cortical acto-myosin network and for full cortical tension. Furthermore, we establish that the centrosome positioning phenotype of embryos depleted of CHC-1 is alleviated by stabilizing the acto-myosin network. Conversely, we demonstrate that slight perturbations of the acto-myosin network results in excess centrosome movements. Overall, our findings lead us to propose that clathrin plays a critical role in centrosome positioning by promoting acto-myosin cortical tension.

EPFL2014

Mutual Inhibition Between the Anaphase-promoting Complex and the Spindle Assembly Checkpoint in C. Elegans Embryos

Alexandra Bezler

EPFL2011

Characterizing the localization and role of lin-5 and era-1 mRNAs in early C. elegans embryos

Zoltán Péter Spiró

EPFL2014