Co-workers:

We study the eukaryotic microtubule cytoskeleton, using fission yeast Schizosaccharomyces pombe as a model system, combining classical and molecular genetics with microscopy and biochemistry/proteomics.

Microtubule nucleation depends on the g-tubulin complex (g-TuC), a multi-protein complex enriched at microtubule organizing centers such as the centrosome. Differentiated cells often have multiple, diverse microtubule organizing centers, but the mechanisms that target the g-TuC to diverse sites are mostly unknown. The fission yeast proteins Mto1 and Mto2 form a complex (Mto1/2 complex) that binds the g-TuC and targets it to different sites during the cell cycle. Mutations in the human homolog of Mto1 lead to the brain disease microcephaly. We have identified modules within Mto1 involved in targeting Mto1/2 to different subcellular sites during the cell cycle; these appear to interact with different partners at different sites. Further experiments have suggested that Mto1/2 not only localizes the g-TuC but also activates it, possibly by inducing conformational changes upon binding. Current work also suggests that the molecular architecture of the Mto1/2 complex is regulated by phosphorylation during the cell cycle. One important biological function of microtubules is to direct positioning of cell-polarity factors. In fission yeast, the protein Tea1 associates with growing ends of microtubules until they reach the cell tips, whereupon Tea1 is deposited at the cell cortex. Using a combination of genetics, FRAP microscopy, and mathematical modelling we identified a novel mechanism in which the membrane protein Mod5 plays a catalytic role in promoting cortical anchoring of Tea1. Proteomic analysis suggests that Tea1 turnover at cell tips may be regulated by phosphorylation. In a separate project identifying novel Tea1-interacting proteins, we have unexpectedly found that proteins associating with Tea1 contribute to exocytosis.

An important element of our research involves making new tools for fission yeast genetics, microscopy, biochemistry and proteomics. Recently we developed a robust platform for differential proteomics in fission yeast, using Stable Isotope Labeling by Amino Acids in Culture (SILAC), as well as a novel matrix for rapid largescale purification of tagged proteins from yeast.

Selected publications:

Bicho, C.C., de Lima Alves, F., Chen, Z.A., Rappsilber, J., and Sawin, K.E. (2010). A genetic-engineering solution to the “arginine-conversion problem” in stable isotope labeling by amino acids in culture (SILAC). Mol Cell Proteomics 9, 1567- 1577.
Sawin, K.E., Bicho, C.C., and Snaith, H.A. (2010). Inexpensive synthetic-based matrix for both conventional and rapid purification of protein A- and tandem affinity purification-tagged proteins. Anal Biochem 397, 241-243.
Samejima, I., Miller, V.J., Groocock, L.M., and Sawin, K.E. (2008). Two distinct regions of Mto1 are required for normal microtubule nucleation and efficient association with the gamma-tubulin complex in vivo. J Cell Sci 121, 3971-3980.
Anders, A., Watt, S., Bahler, J., and Sawin, K.E. (2008). Improved tools for efficient mapping of fission yeast genes: identification of microtubule nucleation modifier mod22-1 as an allele of chromatin-remodeling factor gene swr1. Yeast 25, 913-925.