Structural and mechanistic studies of cell cycle regulatory multi-subunit complexes
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Research in the group is focussed on understanding the structures and mechanisms of multi-subunit protein complexes that regulate the eukaryotic cell cycle. Of particular interest are complexes and cellular structures that control sister chromatid separation in mitosis, for example the anaphase-promoting complex/cyclosome (APC/C) and components of the mitotic spindle and kinetochore. Progression through mitosis is regulated by the spindle assembly checkpoint (SAC), a surveillance mechanism that ensures that sister chromatids only separate when all chromatids have attained bi-polar attachment to the mitotic spindle apparatus. Chromosome-mitotic spindle attachment is achieved through kinetochores, large protein complexes that assemble on centromeres during mitosis. The SAC functions by detecting the lack of kinetochore-microtubule attachment and subsequently generating a signal that inhibits the APC/C, the E3 ubiquitin ligase that triggers sister chromatid separation.
The aim of the PhD project will be to understand the molecular mechanisms underlying the functions of specific cell cycle regulatory complexes responsible for these processes. The approach will be to apply structural techniques (mainly single particle electron cryo-microscopy (cryo-EM) with the potential for protein crystallography) and biochemical and biophysical approaches to understand the structures and mechanisms of multi-subunit complexes that mediate these processes. The project will also apply time-resolved approaches to understand the dynamics of the assembly and disassembly of multi-subunit complexes. Ultimately we seek to combine molecular information from single particle cryo-EM with cellular structural data obtained using cryo electron tomographic methods.
The project will involve reconstitution of protein complexes using the insect cell/baculovirus expression system, protein purification, single particle electron microscopy, protein crystallography and biochemical and biophysical approaches such as SPR and ITC.
Sivakumar, S. and Gorbsky, G..J (2015).
Spatiotemporal regulation of the anaphase-promoting complex in mitosis.
Nat Rev Mol Cell Biol. 16, 82-94.
Foley, E.A., and Kapoor, T.M. (2013).
Microtubule attachment and spindle assembly checkpoint signalling at the kinetochore.
Nat Rev Mol Cell Biol 14, 25-37.
Jia, L., Kim, S., and Yu, H. (2013).
Tracking spindle checkpoint signals from kinetochores to APC/C.
Trends Biochem Sci 38, 302-311.
Lara-Gonzalez, P., Westhorpe, F.G., and Taylor, S.S. (2012).
The spindle assembly checkpoint.
Curr Biol 22, R966-980.
Primorac, I., and Musacchio, A. (2013).
Panta rhei: the APC/C at steady state.
J Cell Biol 201, 177-189.
Westhorpe, F.G. and Straight, A.F. (2014).
The centromere: epigentic control of chromsome segregation during mitosis.
Cold Spring Harb Perspect Biol. 7(1):a015818
Wieser, S., and Pines, J. (2015).
The biochemistry of mitosis.
Cold Spring Harb Perspect Biol. 7(3):a015776
Alfieri, C., Chang, L., Zhang, Z., Yang, Y., Maslen, S., Skehel, M. and Barford, D.
Molecular basis of APC/C regulation by the spindle assembly checkpoint.
Nature (2016) (AOP 10th August).
Zhang, S., Chang, L., Alfieri, C., Yang, J., Zhang, Z., Maslen, S., Skehel, M. and Barford, D.
Molecular mechanism of APC/C activation by mitotic phosphorylation.
Nature, 533, 260-264 (2016).
Chang, L., Zhang, Z., Yang, J., McLaughlin, S.H. & Barford, D. (2015).
Atomic structure of the APC/C and its mechanism of protein ubiquitination
Nature 522: 450-454.
Chang, L., Zhang, Z., Yang, J., McLaughlin, S.H. & Barford, D. (2014).
Molecular architecture and mechanism of the anaphase-promoting complex
Nature 513: 388-393.
Chao, W.C., Kulkarni, K., Zhang, Z., Kong, E.H., and Barford, D. (2012).
Structure of the mitotic checkpoint complex.
Nature 484, 208-213.