Our research is focused on understanding the mechanisms and regulation of chromosome segregation in mitosis. During the cell cycle, accurate chromosome segregation ensures that both daughter cells inherit the correct complement of chromosomes. Errors in this process cause aneuploidy leading to cancer and developmental defects. Duplicated sister chromatids are segregated in mitosis by the mitotic spindle. At metaphase, condensed sister chromatid pairs are aligned on the metaphase plate at the centre of the mitotic spindle. Each chromatid is attached to microtubules by kinetochores, large protein complexes that specifically assemble onto centromeric chromatin. Once all chromosomes achieve bipolar orientation on the mitotic spindle, and tension is exerted at the kinetochore-microtubule attachment site, anaphase is triggered. This results in the loss of sister chromatid cohesion and the segregation of each sister chromatid to opposite poles of the cell. Kinetochores that mediate and regulate this process consist of over 100 proteins, that also function to detect and signal appropriate microtubule attachment and tension. In humans, regional centromeres are composed of thousands of a-satellite repeat sequences (171 bp in length) arranged in arrays that generate Mb-sized centromeres that assemble multiple kinetochores
We recently determined structures of the inner kinetochore (CCAN) complex from both yeast and human assembled onto the centromere-specific CENP-A nucleosome. The specific aim of the PhD project is to build on studies of the human inner kinetochore-CENP-A nucleosome complex to reconstitute multiple (eg four) a-satellite arrays each assembled with a CCAN-CENP-A nucleosome complex, and determine its structure using cryo-EM. The roles of the a-satellite array B-Box, and B-box-binding protein will also be explored, and also in collaboration with others in the group, to use cryo-electron tomography to visualise kinetochores in situ in cells.
The project will include a variety of techniques including single particle cryo-electron microscopy, cryo-electron tomography and in vitro reconstitution approaches.
References:
Yatskevich, S., Barford, D. & Muir, K.W. (2023) Conserved and divergent mechanisms of of inner kinetochore assembly onto centromeric chromatin. Curr Opin Struct Biol 81, 102638
Yatskevich, S., Yang, J., Bellini, D., Zhang, Z. & Barford, D. (2023) Structure of the human outer kinetochore KMN network complex. bioRxiv 2023.08.07.552234; doi: https://doi.org/10.1101/2023.08.07.552234
McAinsh, A.D. & Marston, A.L. (2022) The Four Causes: The function and architecture of Centromeres and Kinetochores. Annu Genet 56, 279-314
Navarro, A.P. and Cheeseman, I.M. (2021) Sem Cell Dev Biol. 117, 62-74. PMID: 33753005
Musacchio, A. & Desai, A. (2017) A Molecular View of Kinetochore Assembly and Function. Biology 6, doi:10.3390/biology6010005
Primary Literature:
Dendooven, T., Zhang, Z, Yang, J., McLaughlin, S.H., Schawb, J., Scheres, S.H.W., Yatskevich, S. & Barford, D. (2023) Cryo-EM structure of the complete inner kinetochore of the budding yeast point centromere. Science Advances.doi:10.1126/sciadv.adg7480
Yatskevich, S., Muir, K.W., Bellini, D., Zhang, Z., Yang, J., Tischer, T., Predin, M., Dendooven, T., McLaughlin, S.H., & Barford, D. (2022) Structure of the human inner kinetochore bound to a centromeric CENP-A nucleosome. Science, 376, 844-852. PMID: 35420891.
Altemose, N. et al. (2022) Complete genomic and epigenetic map of human centromeres. Science, Apr;376(6588):eabj5089. doi: 10.1126/science.abj5089. Epub 2022 Apr 1. PMID: 35357915
Logsdon, G.A. et al. (2021) The structure, function and evolution of a complete human chromosome 8. Nature, 593, 101-107. PMID: 33828295
Yan, K., Yang J, Zhang Z, McLaughlin SH, Chang L, Fasci D, Ehrenhofer-Murray AE, Heck AJR and Barford, D. (2019) Structure of the inner kinetochore CCAN complex assembled onto a centromeric nucleosome. Nature, 574, 278-282. PMID: 31578520