Title: Molecular and cellular structural studies of chromosome segregation
Funder: Cancer Research UK

Abstract: Background: Accurate chromosome segregation during mitosis is critical to ensure each daughter cell inherits the correct complement of chromosomes. Failure to correctly segregate chromosomes results in aneuploidy, a hallmark of cancerous cells. The process of chromosome segregation is mediated by kinetochores, large protein complexes that attach centromeric chromatin to the mitotic spindle, and harness the energy of microtubule depolymerization to move chromosomes to the centrosomes. Additionally, kinetochores ensure the fidelity of chromosome segregation through two inter-dependent correction mechanisms; the tension sensing error correction mechanism, in which aberrant kinetochore-microtubule interactions are resolved, and the spindle assembly checkpoint. Mutations and/or the aberrant expression of proteins that coordinate these mechanisms are associated with a range of cancers. Aims: To understand these processes, we plan a programme to determine the molecular and cellular structure and mechanism of the kinetochore. We will focus on reconstituting the entire kinetochore onto centromeric chromatin and microtubules for structural, biochemical and biophysical analysis. This will result in reconstitution of sister chromatid segregation in vitro to simulate the error correction and spindle assembly checkpoint processes. We will use cryo-electron tomography to visualize the human and yeast kinetochore-centromere assemblies, and conclude our studies of the yeast spindle pole body to understand the mechanism of microtubule nucleation at centrosomes. Methods: We will determine atomic-resolution structures of reconstituted recombinant human and budding yeast kinetochore complexes using single particle cryo-electron microscopy. Kinetochores will be assembled onto centromeric chromatin, and attached to microtubules. We aim to reconstitute kinetochores with and without applied tension, the latter, simulating the tension of a properly attached sister chromatid pair at metaphase. This will provide insights into how kinetochores sense microtubule attachment and bi-orientation, thereby controlling the spindle assembly checkpoint and error correction mechanism. These approaches will allow us to determine how the kinetochore changes its structure under conditions of applied tension. We will also use cryo-electron tomography to visualize the kinetochore-centromere assembles in chromosomes using purified metaphase chromosomes, and in an in situ context using FIB-milled metaphase arrested HeLa cells and yeast nuclei.. How the results of the research will be used: The outcome of our research will be a better understanding of the mechanisms and regulation of chromosome segregation, including the control checkpoints, at a molecular and cellular level. It will provide information for the rational targeting of the protein complexes involved for drug discovery, and help explain the consequence of cancer mutations in chromosome segregation genes.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE