Meiosis is a specialized form of cell division where homologous chromosomes are segregated in meiosis I before sister chromatids are segregated in meiosis II. To establish this pattern, a number of changes to the mitotic chromosome segregation machinery are put in place. Firstly, sister kinetochores orient towards the same pole in meiosis I (mono-orientation). Secondly, homologue recombination creates chiasmata, which link homologues together. And thirdly, cohesin, the molecule that holds sister chromatids together, is cleaved in a step-wise manner. This is achieved because the Shugoshin (Sgo1) protein recruits protein phosphatase 2A (PP2A) to centromeres to counteract cohesin phosphorylation, which is required for its cleavage. The work presented here has investigated two critical aspects of cohesin protection: firstly, how cohesin protection is deactivated in meiosis II and, secondly, how a meiosis-specific protein called Spo13 helps to set up cohesin protection in meiosis I. Previously, our lab had shown that Sgo1 is removed from chromosomes when sister chromatids come under tension during mitosis. I therefore sought to investigate whether sister kinetochore mono-orientation allows Sgo1 to stay on centromeres during meiosis I and carry out its protective function. To this end, I modified meiosis I chromosomes to lack both chiasmata and mono-oriented kinetochores. Under these conditions, where sister chromatids are forced to be under tension in metaphase I, Sgo1 is undetectable on chromosomes. As a consequence, centromeric cohesin is largely lost in anaphase I leading to the premature separation of sister chromatids in a fraction of cells. Since mono-orientation of sister kinetochores is exclusive to meiosis I, these findings suggest that Sgo1 localisation is influenced by sister kinetochore tension in both mitosis and meiosis. Therefore, our findings suggest a mechanism that could contribute to the deprotection of cohesin in meiosis II. However, loss of cohesin protection upon bi-orientation is not complete, suggesting that other factors are involved in the efficient protection and deprotection of cohesin. One such factor is the meiosis-specific protein Spo13, which had previously been shown to be required for cohesin protection as well as kinetochore monoorientation. Although it had been suggested that Spo13 regulates Sgo1 recruitment to centromeres, I could not find any evidence to support a loss of Sgo1, or PP2A, in spo13Δ cells. Additionally, even when Sgo1 is stabilised and clearly visible in anaphase I of spo13Δ mutants, pericentromeric cohesion is still defective. Therefore, I investigated the effect that polo kinase Cdc5, an interactor of Spo13, has on Sgo1. While cellular Sgo1 levels are increased in response to Cdc5 loss, this effect seems to be independent of Spo13. However, Spo13 is required for proper levels of Cdc5 at centromeres and the centromeric recruitment of Cdc5 by Spo13 is likely to be functionally important because tethering of Cdc5 to kinetochores rescued the mono-orientation phenotype of spo13Δ cells. In contrast, I found no evidence that the Spo13-Cdc5 interaction is required for cohesin protection. Meiotic overexpression of SPO13 enhances cohesin protection in meiosis I, apparently independent of its robust interaction with Cdc5, and causes increased Sgo1 enrichment at centromeres. This suggested that Spo13 might recruit Sgo1 to cohesin itself to facilitate its protection. Although I could not detect a loss of Sgo1-cohesin interaction in spo13Δ cells, tethering of Sgo1 to cohesin restores pericentromeric Rec8 to spo13Δ mutants in anaphase I. Surprisingly, sister chromatids still segregate in this case, suggesting that pericentromeric cohesion is defective, despite maintenance of Rec8. Furthermore, inhibition of either one of the cohesin kinases, DDK and Hrr25, restores sister chromatid cohesion to spo13Δ cells. Therefore, the findings in this study suggest that Spo13 is at the centre of a complex regulatory network that coordinates cohesin protection and sister chromatid cohesion in meiosis I.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:726596 |
| Date | January 2017 |
| Creators | Galander, Stefan |
| Contributors | Marston, Adele ; Hardwick, Kevin |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/25395 |
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