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

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.