Live cell imaging based study into the role of phosphorylation in regulating the activity and function class I and V myosins

Class I and V myosins are highly-conserved motor proteins that exert force against actin, in order to undertake diverse cellular functions. Myosin I (Myo1) interacts with cellular membranes and actin, with functions in endocytosis and the cell cycle. Myosin V (Myo52) similarly binds to vesicles for transport. The Mulvihill lab has been investigating conserved phosphorylation events in Schizosaccharomyces pombe Myo1 and Myo52, which play a role in modulating their activity. Phosphorylation of the phospho-serines is hypothesised to elicit domain-specific changes in conformation and ligand binding to regulate function. The purpose of these phosphorylation sites is yet to be reported in the literature.

This thesis presents the latest findings from fluorescence live cell imaging observing dynamics of myosins and associated proteins in myosin phospho-mutant S. pombe strains. It also presents latest models to explain myosin regulation by phosphorylation. Serine-to-alanine substitutions in the phospho-mutants prevent phosphorylation of the sites of interest. TORC2 dependent phosphorylation of these sites modifies myosin conformation and can impact interactions with calmodulin light chains and other associated proteins. The myo1.S742A phospho-mutant strain displayed greater monopolar growth in the vegetative cycle. Endocytic actin activity was also significantly different at the ends of the cells during bipolar growth in G2. This indicated Myo1 S742 phosphorylation to have regulatory impact on the switch from monopolar growth to bipolar growth. During the meiotic cycle in these cells, Cam2-GFP foci lifetimes were significantly extended and endocytosis was further minimised, indicating Myo1 S742 phosphorylation is required for Cam2 binding and correct Myo1 function in endocytosis. Myo52 phospho-mutants displayed various changes in polarised cell growth, delocalised actin patches and reduced Myo52 motility. These observations indicate the Myo52 phosphorylation sites have an impact the regulation of cell growth, actin localisation and Myo52 motility. The observations of these myosin phosphorylation sites highlight them as regulators of Myo1 and Myo52 function.

To develop methods of fluorescence imaging, conditions that do not induce phototoxic effects on contractile actin ring constriction were determined and used to study CAR defects in temperature-sensitive tropomoyosin Cdc8 mutant strains, cdc8.27 and cdc8.110. CAR defects revealed cdc8.27 and cdc8.110 to impact the actin stabilising function of Cdc8. A novel simultaneous two-channel spinning-disk imaging system, developed by Cairn Research, was also tested with live cells to demonstrate successful imaging of real-time co-movement of fluorescently labelled proteins in the red and green channel, without channel-to-channel bleed. This confirmed the system is able to capture the dynamics of proteins and reduce the time lag in time-lapses acquired by standard confocal imaging systems.