The Centrosome as a Compass for Asymmetric Cells

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Objective and aims: The long-term objective of this NSERC research program is to quantitatively describe the process of Kar9 symmetry breaking in relation to centrosome positioning and alignment with the polarity axis of the cell during metaphase. This objective will be achieved by the two aims of the research plan. 1) we will quantitatively characterize Kar9 asymmetry breaking to determine whether it is irreversible and sufficient for centrosome positioning and alignment and test a working model for regulation of the Kar9-Myo2 interaction 2) we will determine how the timing of Kar9 symmetry breaking is controlled. Summary of Scientific Approach: This research program uses a combination of experimental biophysics and cell biology methods and utilizes the powerful genetic tools and the asymmetric morphology of budding yeast cells. The core method used for the research is quantitative live cell microscopy. The microscopy is based in fast, 3-dimensional confocal imaging that simultaneously two or more fluorescent reporter proteins. To study the process over several cell cycles, we use a microfluidics chamber. We have developed custom analysis software to measure the relationship between Kar9 asymmetry breaking and centrosome movements within the cell. Novelty and expected significance to natural sciences and engineering: Symmetry breaking in cells has been studied using classical genetic and cell biological approaches. The proposed research uses biophysical concepts and new quantitative methods that probe the dynamic system of centrosome positioning in asymmetric cells. This process occurs within a noisy, complex system and new, quantitative approaches must be used to increase our understanding of how the timing of symmetry breaking is controlled, and if and why it is irreversible. As this is fundamental question in cell biology, our work will have significant impact on the cell biology field. Equally, the STEM expertise in both biophysics and cell biology gained by trainees will benefit Canadian science.