About the Project
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 a large cellular apparatus, the bi-polar mitotic spindle. The spindle is organized from centrosomes, located at opposite poles of the cell. 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 bi-polar 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, a process powered by microtubule depolymerization.
http://www2.mrc-lmb.cam.ac.uk/group-leaders/a-to-g/david-barford/
The PhD project will involve joining a team that is reconstituting mitosis in vitro in order to understand the structures of the kinetochore, how it attaches chromosomes to the mitotic spindle, how tension is exerted, and how it activates the spindle assembly checkpoint in response to loss of attachment to microtubules and lack of tension. The project will include a variety of techniques including single particle cryo-electron microscopy, super-resolution fluorescence microscopy and in vitro reconstitution approaches.
The specific aim of the project is to determine which microtubule associated proteins (MAPs) directly interact with the kinetochore using Saccharomyces cerevisiae as a model system. Although the main microtubule binding sites on kinetochores are known, additional factors are necessary for establishment of tension and bi-orientation in vivo. This suggests that other protein(s) perform essential functions in establishment of the mitotic spindle. To identify which proteins are involved, known factors implicated in kinetochore function will be purified and tested for binding to kinetochores and microtubules. Their functions will be tested in biochemical assays for chromosome segregation (TIRF microscopy), and their structural mechanisms determined using cryo-EM, cryo-ET and protein crystallography.
References
Yan, K., Yang J, Zhang Z, McLaughlin SH, Chang L, Fasci D, Ehrenhofer-Murray AE, Heck AJR and Barford, D. Structure of the inner kinetochore CCAN complex assembled onto a centromeric nucleosome. Nature, 574, 278-282 (2019).
Dewar, H., Tanaka, K., Nasmyth, K. and Tanaka, T. U. Tension between two kinetochores suffices for their bi-orientation on the mitotic spindle. Nature, 428, 93-97 (2004).
- Initial observations of the KT capture and bi-orientation establishment.
Tanaka, K., Mukae, N., Dewar, H., van Breugel, M., James, E. K., Prescott, A. R., Antony, C. and Tanaka T. U. Molecular mechanisms of kinetochore capture by spindle microtubules. Nature 434, 987-94, (2005).
Jenni, S., & Harrison, S. C. (2018). Structure of the DASH/Dam1 complex shows its role at the yeast kinetochore-microtubule interface. Science, 360, 552-558.
Alushin, G. M., Ramey, V. H., Pasqualato, S., Ball, D. A., Grigorieff, N., Musacchio, A., & Nogales, E. (2010). The Ndc80 kinetochore complex forms oligomeric arrays along microtubules. Nature, 467, 805-810.
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