The Centrosome as a Compass for Asymmetric Cells

目标和目的：这项NSERC研究计划的长期目标是定量描述中期与中心体定位和与细胞极性轴对齐有关的Kar9对称性破缺过程。这一目标将通过研究计划的两个目标来实现。1)我们将定量表征Kar9不对称断裂，以确定其是否不可逆，是否足以用于中心体定位和排列，并测试Kar9- myo2相互作用调节的工作模型2)我们将确定如何控制Kar9对称断裂的时间。科学方法概述：本研究项目结合实验生物物理学和细胞生物学方法，利用强大的遗传工具和芽殖酵母细胞的不对称形态。本研究的核心方法是定量活细胞显微镜。显微镜是基于快速，三维共聚焦成像，同时两个或更多的荧光报告蛋白。为了研究几个细胞周期的过程，我们使用了微流体室。我们已经开发了定制的分析软件来测量细胞内Kar9不对称断裂和中心体运动之间的关系。对自然科学和工程的新颖性和预期意义：使用经典的遗传和细胞生物学方法研究了细胞中的对称性破缺。本研究采用生物物理概念和新的定量方法，探索不对称细胞中中心体定位的动态系统。这个过程发生在一个嘈杂、复杂的系统中，必须使用新的定量方法来增加我们对对称性破缺时间是如何控制的理解，以及它是否以及为什么是不可逆的。由于这是细胞生物学的基础问题，我们的工作将对细胞生物学领域产生重大影响。同样，学员在生物物理学和细胞生物学方面获得的STEM专业知识将有利于加拿大的科学。

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.