Mechanisms that Regulate Kinetochore Assembly
The generation and survival of all organisms depends on the faithful execution of cell division. A complete understanding of the regulation and controls over cell division is critical to elucidate the mechanisms that govern self-renewal, proliferation, and development. A key event in the cell cycle is the precise partitioning of every pair of duplicated chromosomes to daughter cells. Defects in chromosome segregation are associated with cancer and other diseases. Chromosome segregation is directed by the kinetochore, the macromolecular protein structure that assembles onto centromeric chromatin. Kinetochores contain hundreds of proteins that assemble on centromeres to create a microtubule-binding site, but the mechanism and regulation of how cells assemble such a complex structure is not yet understood. To elucidate the mechanisms that regulate kinetochore assembly, we have developed a cell-free method for kinetochore assembly in which centromeric DNA from budding yeast is used as a template to assemble kinetochores from whole cell extracts. This method generates kinetochore particles that contain components of all kinetochore subcomplexes and exhibit microtubule-binding activity. A summer project is available that will apply this assay to address an outstanding question about the assembly process, such as how conserved cell cycle kinases regulate kinetochore assembly or how individual proteins facilitate the assembly process. We will tailor the project to the interests of the student, and it can utilize biochemistry, genetics, cell biology, and single molecule biophysics techniques.