The dual use of cohesin and its protector Sgo1 contributes to the choreography of the chromosome and the centrosome cycle

Supernumerous centrosomes cause chromosome mis-segregation and genomic instability, thereby likely contributing to the development of cancer. Centrosome duplication in S phase requires the preceding licensing step in late mitosis/early G1 phase, centriole disengagement. Reminiscent of the control of chromosome number, this dependence usually ensures that centrosomes are duplicated only once per cell cycle. The multi-subunit protein complex cohesin forms a tripartite Scc1-Smc1-Smc3-ring around sister chromatids. In early mitosis cohesin is removed from chromosome arms by the phosphorylation-dependent prophase pathway. During this time, centromeric cohesin is protected by shugoshin 1 and protein phosphatase 2A (Sgo1-PP2A). It is opened only in anaphase by separase-dependent cleavage of Scc1, which triggers chromosome segregation. Shortly thereafter, centrioles loosen their tight orthogonal arrangement, which licenses later centrosome duplication in S-phase. While a role of separase in centriole disengagement has been reported, the molecular details of this process remain enigmatic. Extending recent studies on cultured cells, this work reveals in a reconstituted system that the proteolytic activity of separase is required for centriole disengagement, while its other known function as Cdk1-inhibitor is dispensable. Consistent with previous reports, cohesin is found to be associated with centrosomes and its centrosomal localization is further fine-mapped by electron microscopy. Importantly, a hitherto unknown function of cohesin in centriole engagement is unraveled. Both premature sister chromatid separation and centriole disengagement are induced in vivo by premature activation of separase or depletion of Sgo1. These unscheduled events are suppressed by expression of non-cleavable Scc1 or inhibition of the prophase pathway. Moreover, centriole disengagement can be artificially triggered by a site-specific protease unrelated to separase when endogenous Scc1 has previously been replaced by a correspondingly engineered variant. Separation of centrioles can even be induced by ectopic cleavage of cohesin, i.e. within an engineered Smc3. Thus, the chromosome and centrosome cycles exhibit extensive parallels and are coordinated with each other by dual use of the cohesin ring complex. The second part of this thesis comprises the analysis and functional characterization of differently spliced Sgo1 isoforms. The data presented in this thesis identified a short alternatively spliced exon that not only directs human Sgo1 to centrosomes but at the same time abrogates also its association with centromeres. The change of just three consecutive amino acids within the corresponding peptide inactivates both the pro-centrosomal as well as the anti-centromeric targeting effect. Importantly, localization closely correlates with function as revealed by knockdown-rescue experiments: Depletion of all Sgo1 isoforms by RNAi resulted in unscheduled loss of sister chromatid cohesion as well as centriole engagement. Selective expression of individual Sgo1 isoforms from siRNA resistant transgenes demonstrated that centromere-associated Sgo1 variants shield only sister chromatid cohesion. Contrary, centrosomally bound isoforms of Sgo1 exclusively preserve centriole engagement. Expression of the relevant exon in fusion with eGFP or shugoshin 2 (Sgo2) directs both proteins to centrosomes but enables only the Sgo2-based chimera to now protect centriole engagement. This demonstrates that 1) the centrosome localization signal of Sgo1 is transferable, and 2) targeting per se is necessary but not sufficient for protection of centrosomal cohesin. Consistent with shugoshin´s mode of action at centromeres, centrosome-associated variants with an altered PP2A binding site are compromised in their ability to sustain centriole engagement. Based on these findings, it is tempting to speculate that an expression imbalance between the differently specialized Sgo1 isoforms could interfere with the crucial synchrony between the chromosome- and the centrosome cycles.

Level thesis:degree_level

thesis.doctoral

Grantor dc:publisher

Universität Bayreuth

Year

2012

Author dc:creator

• Schöckel, Laura

Contributors dc:contributor

• Stemmann, Olaf

Repository record source_url

https://epub.uni-bayreuth.de/id/eprint/1672/

OAI identifier oai:identifier

oai:epub.uni-bayreuth.de:1672