Molecular mechanisms of Tea1 cortical anchoring in Schizosaccharomyces pombe
Bicho, Cláudia do Céu Afonso
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Establishment and maintenacne of a polarized axis is essential for all organisms. Cells can either change their shape in response to extracellular cues or maintain a stable polarity axis via landmarks defined in relation to internal cues. In the fission yeast Schizosaccharomyces pombe,microtubules regulate cortical cell polarity together with the landmark protein Tea1. Tea1 is transported to cell tips on microtubule plus-‐ends and deposited upon microtubule contact with the membrane. Although Tea1 has been shown to interact with several binding-partners, Tea1 anchoring at the cell tip depends mostly on the membrane-associated protein, Mod5. Tea1 and Mod5 accumulate in clusters at the cell tip in a mutually dependent manner. I used a combination of live-‐cell imaging, FRAP (Fluorescence Recovery After Photobleaching) and computational modeling to dissect the dynamics of the Tea1-‐Mod5 interaction. I have shown that although Tea1 is stably associated with the cell tip, Mod5 is mobile within the cell tip. I proposed a model in which Tea1 is stable at the cell tip due to self-‐polymerization and association in the form of a cluster-‐network. In the model, the role of Mod5 in the cluster-‐network is to facilitate the formation of Tea1-‐Tea1 interactions. Moreover, in the model, Mod5 is restricted to the cell tip due to iterative binding to and release from the Tea1 cluster-‐network. The properties of the proposed Tea1 cluster-‐ network might contribute to the behavior of Tea1 as a polarity landmark. I hypothesized that Tea1 transfer from the microtubules to the cell tip was regulated by phosphorylation. Tea1 phosphorylated residues were mapped using mass spectroscopy (MS), and identified to be mostly enriched within a central region of the protein. Using a combination of mutagenic analysis and live-‐cell imaging I demonstrate that Tea1 phosphorylation might be required for its dissociation from the cluster-‐network at the cell tip. This suggests that Tea1 interactions within the cluster network are phospho-‐regulated by one of the several tip-‐localized kinases. It has been shown in other organisms and in this thesis that comparison among MS samples requires quantitative MS methodologies. Thus, I developed a robust SILAC (Stable Isotope Labeling in Cell Culture) method to perform quantitative MS in S. pombe. As a proof-‐of-‐principle of the method I performed a proteome-‐wide comparison between the late G2 and the G1/S transition of the cell cycle. The cell cycle proteome-‐wide analysis not only quantified variation in expression levels of cell cycle regulated proteins but also identified novel cell cycle regulated proteins.It has been previously shown that Tea1, Tea3 and Mod5 can interact simultaneously, with each pair interacting independently of the third protein. I describe here a Mod5 mutagenic analysis screen designed to separate Tea1 and Tea3 binding site on Mod5. The Mod5-‐mutants obtained from this analysis indicate that the Tea3-‐Mod5 interaction may play a role in cell polarity establishment. Moreover, although Tea3 is non-‐essential for the cluster-‐network formation, Tea3 might be important for its compaction, which may be particularly important during de novo formation of cell polarity.