Regulation of chromosome congression : focus on the function of hSpindly and the kinetochore recruitment of the Ska complex

Mitosis is a process in which cells divide their genetic materials equally into two daughter cells. During mitosis, specialized structures called kinetochores (KTs), located at the centromeric regions of chromosomes, are captured by microtubules (MTs) radiated from spindle poles. Subsequently, the chromosomes align at the metaphase plate, a process called congression. This process needs to be tightly controlled in order to maintain genomic stability, as subsequent chromosome segregation depends critically on KT-MT interactions. However, how KT-MT attachments are regulated remains largely unknown. Therefore, identification of novel KT/spindle proteins and thorough examination of their functions and regulation will increase our understanding on chromosome congression mechanisms and thus mitotic progression.
This thesis focuses on the study of two KT and spindle components previously identified in a survey of the human mitotic spindle and it is therefore divided into two parts. The first part describes the functional characterization of a novel KT and spindle localizing protein, human Spindly (hSpindly, previously called CCDC99), which is the human homologue of Drosophila Spindly. We show that hSpindly specifically recruits dynein/dynactin to KTs. Localization of hSpindly is in turn controlled by the Rod/ZW10/Zwilch (RZZ) complex and Aurora B kinase. hSpindly depletion results in reduced inter-KT tension, unstable KT-MT fibers (K-fibers), and extensive prometaphase delay and severe chromosome misalignment. Moreover, depletion of hSpindly induces a striking spindle rotation, which can be rescued by co-depletion of dynein. However, in contrast to Drosophila, hSpindly depletion does not abolish the removal of MAD2 and ZW10 from KTs. Collectively, our data reveal hSpindly-mediated dynein functions and highlight a critical role of KT dynein in spindly orientation.
In the second part of this thesis, the regulation of the Ska complex (composed of Ska1, Ska2 and Ska3) has been studied. We show that Aurora B activity negatively regulates the localization of the Ska complex to KTs. Furthermore, recruitment of the Ska complex to KTs depends on the KNL-1/Mis12/Ndc80 (KMN) network. In agreement with this, we have identified interactions between members of the KMN and Ska complexes and demonstrate that the interaction between the two complexes is regulated by Aurora B activity. Aurora B can directly phosphorylate Ska1 and Ska3 in vitro, and expression of phosphomimetic mutants of Ska1 and Ska3 impairs Ska KT recruitment and formation of stable K-fibers, disrupting mitotic progression. We propose that Aurora B phosphorylation antagonizes the interaction between the Ska complex and the KMN network, thereby controlling Ska KT recruitment and stabilization of KT-MT attachments.
Together, we conclude that hSpindly and the Ska complex, two important components of KTs in metazoans, are involved in the regulation of chromosome congression by recruiting KT dynein and stabilizing KT-MT attachments, respectively. Both their function and localization are tightly regulated by mitotic kinases and upstream structural components.