Meiosis is a specialized cell division that produces haploid gametes from a diploid progenitor cell. It consists of one round of DNA replication followed by two consecutive rounds of chromosome segregation. Homologous chromosomes segregate in meiosis I and sister chromatids segregate in meiosis II. Failure to correctly regulate meiosis can result in aneuploidy, where daughter cells inherit an incorrect number of chromosomes. Aneuploidy is usually poorly tolerated in eukaryotes, and is associated with infertility, miscarriages and birth defects. At the meiosis I to meiosis II transition, DNA replication does not occur between chromosome segregation steps despite the need for Spindle Pole Bodies (SPBs) to be re-licensed in order to build meiosis II spindles. The mechanisms that make this distinction are not yet known. In budding yeast, the protein phosphatase Cdc14 is essential for the progression of cells into meiosis II. Cdc14 is sequestered for the majority of the cell cycle in the nucleolus by the inhibitor Cfi1/Net, and is only released in anaphase. We have observed Cdc14 localizing to and interacting with SPB components when nucleolar sequestration is inhibited. Through fluorescence microscopy and EM analysis, we have determined that Cdc14 is required for the re-duplication of SPBs after meiosis I. Our data implies a role for Cdc14 in the phospho-regulation of SPB half-bridge component Sfi1. Cdc14 is therefore essential for the relicensing of SPB duplication, a crucial step necessary to ensure accurate chromosome segregation in meiosis.

<p dir="ltr">The Cdc14 phosphatase family is highly conserved in fungi. In Saccharomyces cerevisiae, Cdc14 is essential for down-regulation of cyclin-dependent kinase activity at mitotic exit. However, this essential function is not broadly conserved and requires only a small fraction of normal Cdc14 activity. In general, few conserved functions of Cdc14 phosphatase have been defined. Here, I present mechanistic biochemical and phenotypic characterization of Cdc14 phosphatases in fungi. I have demonstrated that fungal Cdc14 phosphatases possess an invariant motif in the disordered C-terminal tail that is required for full enzymatic activity. This motif, termed substrate-like catalytic enhancer (SLiCE), functions during the rate-limiting step of Cdc14-directed catalysis, by binding to the active site and supporting phospho-enzyme hydrolysis. Adjacent to the SLiCE motif exists a conserved minimal Cdk consensus motif that likely serves a regulatory function as phosphorylation of this site inhibits Cdc14 activity in vitro. Vertebrate Cdc14 enzymes also possess a distinct, but mechanistically similar SLiCE motif, which may be the first described biochemical difference between Cdc14 enzymes. Moreover, the vertebrate SLiCE motif lacks an adjacent Cdk consensus motif, which may point to differences in how Cdc14 activity is regulated in higher eukaryotes.</p><p dir="ltr">Mutation of this motif in vivo served as a tool to discover biological processes that require high Cdc14 activity. In S. cerevisiae strains expressing this hypomorphic mutant allele (cdc14hm), I discovered a novel sensitivity to cell wall stresses, including chitin-binding compounds and echinocandin antifungal drugs. This sensitivity was also observed in the distantly related fungi Schizosaccharomyces pombe deletion strain and the human fungal pathogen Candida albicans hypomorphic and deletion strains, suggesting that this phenotype reflects a conserved function of Cdc14 orthologs in mediating fungal cell wall integrity. I also revealed that high Cdc14 activity is required for C. albicans ability to develop hyphae, which is an important virulence trait. This led to our determination that high Cdc14 activity is critical for virulence in two animal models of invasive candidiasis. Together, these results argue that Cdc14 would be an excellent antifungal drug target for the treatment of invasive Candida infections and sensitization to existing antifungal drugs.</p><p dir="ltr">Lastly, I implemented the auxin-inducible degradation system in C. albicans. Using this system, we were able to deplete Cdc14 and other target protein levels to >95% within minutes. Depletion of Cdc14 was robust enough to phenocopy gene deletions, confirming previous results and demonstrating the utility of rapid target protein inactivation. This system will serve as a powerful tool for future functional characterization of Cdc14 in C. albicans and other pathogenic fungal species.</p>

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Meiosis is the process by which haploid gametes are produced from a diploid cell. It is a specialised form of cell division which involves one round of DNA replication followed by two rounds of chromosome segregation. Errors in the segregation process can give rise to aneuploidy, which can result in miscarriages and birth defects. In the first meiotic division homologous chromosomes are segregated, and sister chromatids are segregated in the second division. This is coordinated with two rounds of spindle microtubule assembly and disassembly. How these two processes are coordinated is unknown. In my PhD, I study the role of the protein phosphatase 2A (PP2A) regulatory subunit, Cdc55, in budding yeast meiosis. PP2A is a conserved heterotrimeric enzyme that has important roles in mitosis and meiosis. These roles are dictated by binding to either of its two regulatory subunits, Rts1 and Cdc55, in budding yeast . I show that Cdc55 is required for the proper assembly of a meiotic spindle in meiosis I, through the maintenance of the Cdc14 phosphatase in the nucleolus early in meiosis. In addition, Cdc55 is also required to limit the formation of PP2A complexes with the Rts1 regulatory subunit, and this is essential for the timely dissolution of sister chromatid cohesion. Thus, Cdc55 couples spindle assembly with chromosome segregation through its interactions with Cdc14 and PP2ARts1. Finally, I show some preliminary studies looking at the possible downstream effectors of Cdc14 that are important in this mechanism.