Investigating the regulation of fission yeast cytokinesis by the anillin mid1, the ESCRT regulator vps4 and the Aurora kinase ark1

Mammalian and fission yeast cells determine the site of division by the medial placement of an actomyosin contractile ring. Contraction of this ring then leads to cytokinesis and the final separation of the equal-size daughter cells. In the fission yeast Schizosaccharomyces pombe (S. pombe) the anillin Mid1p, aurora Ark1p and the ESCRT family of proteins are involved in regulating specification of division site, mitotic entry and establishment of the final separation, respectively. In this Thesis, for the first time, we provide evidence of genetic interactions between both the vps4+ and ark1+ genes with the mid1+ gene. Combinatorial mutants revealed a synthetic growth phenotype indicating their requirement for S. pombe survival. Therefore, this research investigated Mid1p potential regulation by the ESCRT regulator Vps4p (Chapters 3 and 4). Initially, a physical interaction between Vps4p and Mid1p recombinant proteins was detected and mapped to the "C-term" domain of Mid1p. Next, the effects of absence of this interaction on the cellular localization of Mid1p were determined; this analysis revealed mis-localization of Mid1p and suggested a potential mechanism whereby Vps4p interacts with Mid1p through the PH domain to regulate Mid1p localization to cortical nodes. This hypothetical mechanism was investigated by determining the effects of Mid1p PH domain absence on its localization in S. pombe. Such analysis revealed mis-localization of Mid1p, but it was inconclusive in confirming the interaction between Mid1p-PH domain and Vps4p. Interestingly, the absence of the PH domain caused a Mid1p nuclear exclusion phenotype, suggesting that Mid1p PH domain is involved not only in its known role in Mid1p cortical anchorage but also in Mid1p nuclear localization. Therefore, it is possible that Mid1p nuclear localization regulates its interaction with Vps4p. To test this hypothesis, the well-characterized NLS regions in Mid1p were utilized, and their requirement for the interaction with Vps4p was tested. Such analysis revealed that the interaction between Vps4p and Mid1p does not depend on Mid1p nuclear localization. Mid1p potential regulation by the aurora kinase Ark1p was also tested (Chapters 5 and 6). Initially, in vitro phosphorylation of recombinant Mid1p by mammalian recombinant aurora A and plk1 kinases was tested; such analysis confirmed this interaction and mapped it to the "N-term" and "Middle" domains of Mid1p. Next, Mid1p "N-term" and "Middle" domains were mapped to yield six potential phospho-sites (S167, S328, S331, S332, S523 and S531), which were utilized to confirm the in vitro phosphorylation by the aurora kinase Ark1p. The results were inconclusive, so an in vivo integration S. pombe approach was utilized to analyze the phospho-sites. This approach involved the production of several versions of the mid1 gene, each bearing a single or multiple phospho-mimetic or phospho-resistant mutation/s. These mid1 gene versions were integrated into the chromosomal DNA of wild-type (W-T) or an ark1-T11 S. pombe strain at the endogenous mid1+ locus. Cell morphology phenotypes were then characterized for two selected Mid1p potential phospho-sites, S523 and S531. Defects in cell morphology were observed in the case of all phospho-resistant mutants but were not observed in the case of all phospho-mimetic mutants. Therefore, we concluded that the two Mid1p sites, S523 and S531, are important for its function. Overall, the work reported in this thesis provides insights into S. pombe cell cycle regulation. Using in vivo S. pombe genetic and microscopic approaches in addition to in vitro pull-down and phosphorylation experiments, we discovered novel Mid1p-dependent pathways for S. pombe cell cycle regulation.