|About the Book|
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 isMoreStatement 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.-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.-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-Cs role in assembling the mitotic kinetochore during cell division.