Characterisation of conditional alleles of the Schizosaccharomyces pombe cytokinesis regulator byr4

Regulation of cytokinesis is essential for the cell during its division cycle. Failure to do so can lead to aneuploidy, which can be fatal and lead to senescence or cancer. A useful model organism for studying cytokinesis in eukaryotes is Schizosaccharomyces pombe (fission yeast) which undergoes medial division similar to animal cells. For S. pombe to undergo cytokinesis, a septum has to be made. The signalling pathway which controls this septum formation is the septation initiation network (SIN). The SIN consists of 13 components. The three scaffold proteins, Ppc89p, Sid4p, and Cdc11p, localise SIN components to the spindle pole body (SPB), where SIN signalling takes place. The furthest upstream regulator of the SIN is Plo1p. The main regulator of the SIN is the GTPase Spg1p which is controlled by a GAP complex consisting of Byr4p and Cdc16p. The component Etd1p is also part of the SIN. Though its role remains elusive, it is generally agreed that it is involved in SIN activation. When the SIN is active, it is commonly believed that the signal is transmitted linearly from GTP-bound Spg1p and its effector Cdc7p to the Sid1p and Cdc14p complex, which passes on the signal to the Sid2p and Mob1p complex, which leads to septum formation. Conditional alleles allow for the manipulation of essential processes. Since most SIN components are essential, the availability of conditional SIN alleles has been useful for studying the signalling pathway. At the time of writing, no conditional alleles of byr4 have been published. Before this Ph.D. study, several conditional byr4 alleles were obtained in the lab of Prof. Viesturs Simanis. In this Ph.D. thesis, I characterised the conditional byr4 alleles and used them to gain novel insights into the SIN pathway. The conditional byr4 alleles gave rise to multiseptated cells at 36°C, which in most cases had Cdc7p and Mob1p present at the SPBs, while Sid1p presence was infrequent. One of the mutants, byr4-CST, encodes a C-terminally truncated Byr4p fragment, which lacks a domain considered essential for GAP function. byr4-CST was found to be viable independently of Cdc16p. In addition to the multiseptated phenotype at 36°C, byr4-CST became multinucleated at 19°C with Spg1p, Cdc7p, Sid1p, and Mob1p present at the SPBs. The byr4-CST mutant also gave new insight into the role of Etd1p, suggesting that Etd1p has a function related to the GAP complex. Although a definitive explanation for the viability of byr4-CST remains elusive, the experiments in this thesis suggest that byr4-CST viability and phenotypes could be related to changes in Spg1p levels and the possibility for Byr4p-CST to regulate Spg1p by titrating it off the SPB and into the cytosol. Alternatively, byr4-CST could be regulated through an attenuating mechanism downstream of the core SIN kinases, which remains to be uncovered.