Investigation of FEAR and MEN pathway homologs in multinucleate cells

Abstract

The cell cycle is a sequence of events enabling a cell to replicate and proliferate. Common landmark events in most eukaryotic cell cycles are duplication of the DNA, mitosis, and cell separation. The cell cycle lays the basis for development in multicellular organisms, and is of course important for cell or tissue renewal. Disregulation of the cell cycle can lead to uncontrolled growth and tumor formation. Mitosis, or nuclear division, is tightly regulated to ensure proper segregation of nuclei to daughter cells. In budding yeast (S. cerevisiae), the timing of mitosis is coupled to cytokinesis by the action of the FEAR and MEN regulatory networks, which mediate exit from mitosis by activation of the phosphatase ScCdc14. In this thesis, we investigate the function of homologs of these pathways in the filamentous fungus, Ashbya gossypii. This organism provides a unique cellular setting to study these matters, as it is a close relative of S.cerevisiae and shares most of the molecular machinery. However within its multinucleate hyphae, mitosis is uncoupled from cytokinesis, and cytokinesis is not followed by cell separation, thus leading to compartments containing 8-10 nuclei, separated by septa. We look into how exit from mitosis is affected in such a cellular environment where nuclei are free to divide without the spatial and temporal constraints as described in budding yeast, where each mitosis is followed by cytokinesis and cell separation. First we study the core component of exit from mitosis signalling. We examinethe phosphatase AgCdc14 and discover that its regulation is very similar to S. cerevisiae, with the phosphatase being sequestered within the nucleolus throughout interphase, and released during anaphase. Furthermore, we find it to be an essential component in nuclear cycle progression in A. gossypii, with nuclei in null mutants failing to undergo mitosis. We then look at MEN homologs in A. gossypii and present evidence that the kinase cascade function of the pathway is likely conserved. We however show MEN homologs to be non-essential and play no role in AgCdc14 regulation. On the other hand, we detect sporulation deficiencies in our mutants, which we could attribute to septation defects. More interestingly, we observe a partial mitotic arrest in MEN deficient cells. MEN homologs thus seem to have diverged from the primary role of their counter-parts in budding yeast. The final chapter deals with homologs of the FEAR pathway. It had been previously suggested that, in primitive cells, the task of Cdc14 phosphatase regulation may be solely bestowed upon this network of genes. We find this to hold true for A. gossypii, where we report severe mitotic defects upon deletion of FEAR homologs, and in particular, disruption of control over AgCdc14 release. We interpret the results as evidence for a simpler system regulating exit from mitosis in A. gossypii and lay out potential implications for the more complex system in S. cerevisiae.