About the Project
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.
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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