Role of Spo13 and Cdc5 in the establishment of sister kinetochore mono-orientation
Item statusRestricted Access
Embargo end date17/06/2023
Meiosis is a type of cell division that leads to the creation of gametes. Unlike mitosis, which produces exact copies of the mother cell, the aim of meiosis is to halve the cell’s chromosomal content. To achieve this, the mechanisms governing the division processes have been heavily modified. This applies particularly to the first of two divisions, when genome reduction happens. At the beginning of meiosis I, the homologous chromosomes pair and create linked bivalents, which generates tension between the homologues and leads them to be segregated towards the opposite spindle poles. This type of segregation, which is referred to as “reductional”, is further biased by the fusion of the sister kinetochores. In budding yeast, the fused kinetochore allows the attachment of only one microtubule, leading to kinetochore mono-orientation. Kinetochore mono-orientation, in cooperation with the pericentromeric cohesin, precludes precocious segregation of sister chromatids. It has been proposed that kinetochore fusion depends on the monopolin complex, which provides a bridge between the sister kinetochores. In turn, the recruitment of monopolin complex depends on Spo13 and the polo-like kinase Cdc5. While the current model of the Spo13 role indicates that it functions as a platform for kinetochore localisation of Cdc5, the exact functions of both proteins in sister-kinetochore mono-orientation remain obscure. During my project, I tried to learn the precise localisation of Spo13. Using mass spectrometry, I have shown that Spo13 is recruited to the inner kinetochore and that this localisation depends on Spo13 interaction with Cdc5. However, the abundance of dispersed monopolin complex precluded studying it by the same method. Cdc5 is a versatile kinase with plenty of possible substrates. To discover the phosphorylation sites that may play role in the mono-orientation establishment, I immunoprecipitated the kinetochore and submitted it to be analysed by mass spectrometry. I utilised a Cdc5 kinetochore-tethered (Cdc5-kt) system which allows Cdc5 to localise on kinetochores independently of Spo13. Because CDC5-kt cells co-segregate the sister chromatids in the absence of Spo13 and the monopolin complex, I hoped to understand why Cdc5-kt helps rescue the phenotype. I found that several components of the spindle assessment and error correction pathways are enriched in spo13Δ cells, while Cdc5-kt lowers their kinetochore occupancy to wild type levels. Therefore, Spo13 likely participates in two parallel pathways to establish sister kinetochore mono-orientation: firstly, it recruits monopolin to provide the kinetochore fusion and secondly, it modulates the mechanisms involved in microtubule-kinetochore attachment tension sensing.