| Statement of the problem. During cell division, accurate chromosome segregation in eukaryotic cells is ensured by the assembly of the kinetochore, a microtubule-binding site that attaches to the mitotic spindle, on each chromosome. The kinetochore is specifically assembled on the centromere, a specialized region of each chromosome that is consitutively bound by >15 centromere-speccific proteins. These proteins, which include centromere proteins A and C (CENP-A and CENP-C), are required for kinetochore assembly and proper chromosome segregation. Centromere assembly, and how the centromere promotes kinetochore formation in mitosis, are poorly understood.;Results and conclusions. We show that, unlike the histone variant CENP-A, CENP-C is not maintained at centromeres through spermatogenesis but is assembled at sperm centromeres from the egg cytoplasm. Kinetochore formation on sperm chromatin is prevented by immunodepletion of CENP-C from metaphase egg extract, and in vitro translated CENP-C can complement depleted extracts. Using this assay, we identified CENP-C mutants that localize to centromeres but do not support kinetochore assembly. We find that the amino terminus of CENP-C promotes kinetochore assembly by ensuring proper targeting of the Mis12/MIND complex and CENP-K. The second chapter of this thesis describes observations of the assembly of CENP-C when a conserved arginine residue in the signature motif has been mutated. This mutant CENP-C assembles at centromeres in interphase egg extract and does not require the presence of endogenous CENP-C. Our work demonstrates the power of the Xenopus egg extract system for studying centromere biology and begins to ascertain CENP-C's role in assembling the mitotic kinetochore during cell division.;Procedure and methods. This thesis describes work utilizing the Xenopus egg extract as an in vitro system to study CENP-C and its role in centromere and kinetochore assembly. |
