Molecular Mechanisms Involved in Chromosome Segregation
Eukaryotic organisms use large and intricate macromolecular machines to accurately pass chromosomes to two daughter cells during cell division. Errors during chromosome segregation are frequently deleterious to the cell and often result in aneuploidy leading to the formation of cancer cells. Kinetochores are large macromolecular machines that assemble specifically at the centromeric chromatin and act as a structural scaffold to support chromosome segregation. The inner kinetochore (CCAN) recognises centromere-specific CENPA nucleosome (CENP-ANuc) and couples it to the microtubules (MTs) of the mitotic spindle. In human cells, the details of CCAN-CENP-ANuc assembly are unknown. This thesis investigates the structure and assembly of the human CCAN-CENP- ANuc complex, showing that CCAN tightly grips the linker DNA of the CENPANuc. CENP-C emerges as the main recognition protein of the inner kinetochore. CCAN topologically entraps the linker DNA, explaining how it can withstand both pushing and pulling forces applied by the mitotic spindle. Kinetochores are also platforms for spindle assembly checkpoint (SAC) signalling, a pathway that ensures all chromosomes are correctly attached to the mitotic spindle prior to their segregation. The anaphase promoting complex/cyclosome (APC/C) is a key regulator of the anaphase transition, and SAC directly inhibits APC/C via a soluble mitotic checkpoint complex (MCC). While many molecular details of APC/C function are now known, recent data suggest that the APC/C is also SUMOylated but the function of this modification is unknown. To address the role of this modification, the APC/C was SUMOylated in vitro and the function of SUMOylated APC/C was investigated. We show that SUMOylation results in repositioning of a small domain of APC2, which sterically competes and displaces the MCC from APC/C. This allows a more robust APC/C reactivation during anaphase onset. Microtubule organising centres (MTOCs) nucleate and organise the mitotic spindle. The γ-tubulin Ring Complex (γ-TuRC) is a highly conserved part of MTOCs in most eukaryotes and it is essential for MT nucleation. The molecular mechanism of MT nucleation from γ-TuRC remains unclear. We have purified MTOC from yeast (known as Spindle Pole Body) and performed cryo-electron tomography analysis of native γ-TuRC complexes nucleating microtubules. We observed that γ-TuRC is a perfect symmetry match for MT nucleation and that additional factors allow efficient MT nucleation at native γ-TuRCs.
Structural biology, kinetochores, γ-TuRC, gamma-Tubulin Ring Complex, anaphase-promoting complex (APC/C)
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