Microtubules (MTs) are found in nearly all eukaryotic cells and have critical roles in many fundamental biological functions, such as cell division, intracellular transport, cell motility, and neuronal function. The importance of MTs in cell biology is underscored by the success of tubulin-targeting drugs such as paclitaxel (Taxol) and vinca alkaloids in cancer therapy, as well as the use of MT-depolymerising drugs such as colchicine, which has been used therapeutically since antiquity.
To perform their functions in cells, MTs are nucleated from specific structures known as MT-organising centres (MTOCs). MT nucleation at MTOCs depends on a multiprotein, megadalton-scale complex known as the gamma-tubulin complex. The gamma-tubulin complex is conserved from yeast to humans. Our laboratory studies the gamma-tubulin complex and its regulation in the fission yeast Schizosaccharomyces pombe, a unicellular model eukaryote that is easily manipulated by both classical and molecular genetic methods and is also ideal for sophisticated microscopy and biochemistry approaches. We identified the multiprotein Mto1/2 complex as a critical regulator of the gamma-tubulin complex. The Mto1/2 complex, which is made of multiple copies of the proteins Mto1 and Mto2, helps the gamma-tubulin complex assemble into higher-order structures and also localizes the gamma-tubulin complex to MTOCs in vivo.
The Mto1 protein in particular is conserved in humans, and mutations in the human homolog of Mto1 lead to the neurodevelopmental disorder microcephaly. In order to understand the molecular mechanisms controlling MT nucleation by the gamma-tubulin complex, we have very recently reconstituted the gamma-tubulin complex, the Mto1/2 complex and associated proteins in vitro, through purification of recombinant proteins expressed in baculovirus. We have begun to characterise these complexes via biophysical and structural methods, including X-ray crystallography and cross-linking mass spectrometry, and we have reconstituted MT nucleation in vitro with the purified proteins.
The PhD project will focus on understanding novel aspects of how the Mto1/2 complex interacts with the gamma-tubulin complex. This will involve purification and structural determination of components of the gamma-tubulin complex and/or the Mto1/2 complex, in isolation and also in complex with other proteins, in order to understand how assembly into higher-order structures is driven by cooperative protein-protein interactions. Models of interactions derived from structural analysis will then be tested in vivo by structure-based mutagenesis in yeast, with analysis of phenotypes by live-cell fluorescence microscopy and co-immunoprecipitation experiments.
In spite of the demonstrated value of tubulin-targeting drugs in cancer therapy, such drugs are nevertheless far from perfect, because they also have significant side effects such as neuropathy, due to the fact that all MTs in the body can be affected. In the longer term, a deeper understanding of the mechanisms regulating MT nucleation by the gamma-tubulin complex may lead to identification of new small-molecule targets for disrupting the MT cytoskeleton in a therapeutic context, with fewer side effects.
Techniques acquired during the PhD project include: design of synthetic genes and molecular cloning, protein expression and purification by chromatography, protein crystallisation and structural analysis, protein cross-linking and mass spectrometry, classical and reverse-genetic approaches in yeast, live-cell fluorescence microscopy.
Borek, W. E., Groocock, L. M., Samejima, I., Zou, J., de Lima Alves, F., Rappsilber, J., & Sawin, K. E. (2015). Mto2 multisite phosphorylation inactivates non-spindle microtubule nucleation complexes during mitosis. Nature Communications 6, 7929. http://doi.org/10.1038/ncomms8929.
Lynch, E. M., Groocock, L. M., Borek, W. E., & Sawin, K. E. (2014). Activation of the γ-Tubulin Complex by the Mto1/2 Complex. Current Biology 24, 896–903. http://doi.org/10.1016/j.cub.2014.03.006.
Wu, J., and Akhmanova, A. (2017). Microtubule-Organizing Centers. Annu Rev Cell Dev Biol. http://doi.org/10.1146/annurev-cellbio-100616-060615.