Microtubule nucleation, cytoskeletal organisation, and cell polarity
Our laboratory is interested in two main areas related to cellular and cytoskeletal organisation: 1) the molecular mechanisms underlying microtubule nucleation; and 2) the regulation of cell polarity, in a systems context, under both normal and stress conditions. In both areas we use the fission yeast Schizosaccharomyces pombe as a model eukaryotic organism. We combine classical and molecular genetic analysis with live-cell fluorescence microscopy, biochemistry, proteomics and computational modeling.
Microtubule nucleation in all eukaryotic cells depends on the g-tubulin complex (g-TuC), a multi-protein complex enriched at microtubule organizing centres such as the centrosome. The spatial and temporal regulation of the g-TuC remains largely a mystery. We discovered the fission yeast proteins Mto1 and Mto2, which form an oligomeric complex (the Mto1/2 complex) that targets the g-TuC to different sites in the cell and also activates g-TuC during the cell cycle. Mutations in the human homolog of Mto1 lead to the brain disease microcephaly. Our current work involves understanding the mechanism of g-TuC activation by the Mto1/2 complex, through genetic approaches in yeast, and through expression, purification and characterization of recombinant multi-protein complexes in insect cells, in vitro functional reconstitution, and structural biology analysis, including X-ray crystallography. We are also using new methods to investigate how the Mto1/2 complex is localized to different subcellular structures.
Regulation of cell polarity in fission yeast is particularly interesting because it involves multiple internal cues that cooperate and compete with each other. The Rho-family GTPase Cdc42 and its associated regulators and effectors control the actin cytoskeleton and exocytosis. Microtubules provide an additional level of control in regulating site-selection for polarised growth, through the microtubule plus-tip-associated protein Tea1, the membrane protein Mod5, and their interactors. We are currently studying how the Cdc42 system and the microtubule-based system “talk to each other" under different environmental stimuli and under stress, using a combination of mutational analysis, proteomics, FRAP microscopy, and mathematical modeling. This work has led to the discovery of new cell-polarity regulators, outside of the Cdc42- and microtubule-based systems.
An important component of our work involves developing new tools in genetics, microscopy, and proteomics. This includes a robust platform for differential proteomics in fission yeast, using Stable Isotope Labeling by Amino Acids in Culture (SILAC), which we are applying to global analysis of protein phosphorylation in cell polarity, and new methods for interrogating protein-protein interactions in complex “solid-phase” organelles.
Mutavchiev, D. R., Leda, M., and Sawin, K. E. (2016). Remodeling of the Fission Yeast Cdc42 Cell-Polarity Module via the Sty1 p38 Stress-Activated Protein Kinase Pathway. Curr. Biol 26, 2921-2928.
Borek, W.E., Groocock, L.M., Samejima, I., Zou, J., de Lima Alves, F., Rappsilber, J., and Sawin, K.E. (2015). Mto2 multisite phosphorylation inactivates non-spindle microtubule nucleation complexes during mitosis. Nat Commun 6, 7929.
Lynch, E.M., Groocock, L.M., Borek, W.E., and Sawin, K.E. (2014). Activation of the γ-tubulin complex by the Mto1/2 complex. Curr Biol 24, 896-903.