*EASTBIO* Protein kinase regulation of cell polarity: a phosphoproteomics approach

Nearly all eukaryotic cells are polarised—they have an internal architecture and/or external (i.e. cell surface) organisation that gives them a front and a back, a top and a bottom, or proximal and distal regions. Cell polarity enables specific functions such as cell growth, migration, asymmetric division (e.g. stem cells), and orientation within tissues.

Our laboratory studies cell polarity in fission yeast S. pombe, a unicellular model organism most widely known for its role in elucidation of the fundamental mechanisms of cell cycle control conserved throughout all eukaryotes. Fission yeast are also extremely well suited to studies of cell polarity, as they are highly polarised rod-shaped cells. During interphase, they grow by extension, depositing new plasma membrane and cell wall at cell tips; during mitosis, they reorient polarity and growth to the middle of the cell, to form a septum that divides the cell by medial fission. Recently we have shown the importance of protein kinases in the regulation of fission yeast cell polarised growth [1,2]. Using phosphoproteomics methods, we identified novel substrates of protein kinases involved in cell polarity and showed how their phosphorylation contributes to polarised growth [2].

This interdisciplinary PhD project will investigate the role of NDR kinases in fission yeast cell polarity, combining phosphoproteomics and mass spectrometry “big data” approaches with yeast genetics and cell biology. NDR kinases are conserved in eukaryotes and are particularly important in cell differentiation as well as cell polarity during development (e.g. the “Hippo” pathway). Most organisms have a small total number (two to four) of NDR kinases, with overlapping functions. In fission yeast, the two NDR kinases are similar in sequence, but they have distinct functions in regulating cell polarity. The goal of the PhD project will be to understand how these two NDR kinases regulate distinct aspects of polarity.

Methods used in the PhD project will include: development of mutant protein kinases that can be inhibited in vivo using small-molecules; isolation, enrichment and HPLC fractionation of phosphopeptides; SILAC [3] and/or TMT mass spectrometry techniques for deep, proteome-wide analysis of changes in phosphopeptides upon inhibition of kinase activity; bioinformatic analysis of results and obtained hits; classical yeast genetics; reverse-genetics (genome mutagenesis) to generate non-phosphorylatable mutants of kinase targets in vivo; and live-cell, time-lapse fluorescence microscopy of cell-polarity phenotypes of mutated kinase targets, using GFP-tagged proteins. PhD projects combining “big-data” with yeast genetics and cell biology are an excellent way to begin a scientific research career, because a wealth of methods can be learned and applied in the context of hypothesis-driven research, and hypotheses are constantly challenged by new results that require rigorous logical evaluation, leading to new experiments.