As a major microtubule-organizing center in mammalian cells, the centrosome consists of two centrioles that are surrounded by the pericentriolar material. Starting from G1/S in the cell cycle, the centrosome duplicates and the two duplicated centrosomes are kept close to each other by a fibrous centrosome linker until late G2 phase. The two duplicated centrosomes start to separate in late G2 and eventually form the poles of the bipolar spindle during mitosis. The separation of centrosomes is initiated by centrosome disjunction, which is the dissolution of the centrosome linker mediated by NIMA-related kinase 2A (Nek2A)-induced phosphorylation. Timely separation of centrosomes is crucial for proper formation of a bipolar spindle and subsequently for error-free chromosome segregat...[ Read more ]

As a major microtubule-organizing center in mammalian cells, the centrosome consists of two centrioles that are surrounded by the pericentriolar material. Starting from G1/S in the cell cycle, the centrosome duplicates and the two duplicated centrosomes are kept close to each other by a fibrous centrosome linker until late G2 phase. The two duplicated centrosomes start to separate in late G2 and eventually form the poles of the bipolar spindle during mitosis. The separation of centrosomes is initiated by centrosome disjunction, which is the dissolution of the centrosome linker mediated by NIMA-related kinase 2A (Nek2A)-induced phosphorylation. Timely separation of centrosomes is crucial for proper formation of a bipolar spindle and subsequently for error-free chromosome segregation. Until now, the precise mechanism of centrosome separation has not yet been fully understood. On the other hand, GAS2L1 (growth arrest-specific protein 2 like 1) is an actin- and microtubule- binding protein with unclear cellular functions, and this thesis research uncovers the important function of GAS2L1 in centrosome disjunction and the regulatory mechanism of GAS2L1 function by Nek2A-mediated phosphorylation. Furthermore, this research also provides a comprehensive understanding on the molecular mechanisms underlying centrosome disjunction mediated by Nek2A. First, the results revealed that GAS2L1 localizes to the proximal end of centrioles and participates in centrosome disjunction in late G2 by tethering cytoskeletons to centrioles. This GAS2L1 function requires its association with F-actin, microtubules, and the microtubule end-binding proteins (EBs). Second, the mechanism to regulate GAS2L1’s function has been investigated. GAS2L1 contains an F-actin binding calponin-homology (CH) domain and a microtubule-binding GAS2-related (GAR) domain, and the CH and GAR domains bind to each other to inhibit the functions of both domains. GAS2L1 undergoes Nek2A-mediated phosphorylation at Ser352 in G2/M, which is required for centrosome disjunction. Ser352 phosphorylation of GAS2L1 disrupts the interaction between the two domains and relieves the autoinhibition, promoting GAS2L1 binding to F-actin and microtubule. Therefore, in late G2 phase of the cell cycle, Ser352 phosphorylation of GAS2L1 promotes centrosome disjunction presumably by inducing centrosomal tethering of cytoskeletons. Third, the roles of the two Nek2A-mediated events, GAS2L1 phosphorylation and centrosome-linker disassembly, in triggering centrosome disjunction have been dissected. These two events together are required to trigger centrosome disjunction in late G2, and neither action alone is sufficient to initiate splitting of centrosomes. Furthermore, centrosome disjunction induced by GAS2L1 and its phosphorylation at Ser352 mediated by Nek2A are required for establishing symmetrical bipolar spindles and for the subsequent chromosome segregation with high fidelity. Overall, the study presented in this thesis not only provides a comprehensive mechanistic model of centrosome disjunction mediated by Nek2A, but also highlights the importance of centrosome separation in chromosome segregation and the maintenance of genome stability.