The Centrosome as a Compass for Asymmetric Cells

目的和目的：本NSERC研究计划的长期目标是定量描述中期Kar9对称性破坏与中心体定位和与细胞极性轴对齐的过程。这一目标将通过研究计划的两个目标来实现。1)我们将对Kar9不对称断裂进行定量表征，以确定它是否是不可逆的，是否足以进行中心体定位和比对，并测试一个调节Kar9-Myo2相互作用的工作模型。2)我们将确定Kar9对称断裂的时间是如何控制的。科学方法概述：本研究项目采用了实验生物物理学和细胞生物学相结合的方法，并利用了强大的遗传工具和发芽酵母细胞的不对称形态。这项研究的核心方法是定量活细胞显微镜。这种显微镜是基于快速的三维共聚焦成像，同时成像两个或更多个荧光报告蛋白。为了在几个细胞周期内研究这一过程，我们使用了一个微流体室。我们已经开发了定制的分析软件来测量Kar9不对称断裂和细胞内中心体运动之间的关系。新颖性和对自然科学和工程的预期意义：已经使用经典的遗传学和细胞生物学方法研究了细胞中的对称性破坏。这项研究使用生物物理学的概念和新的定量方法来探索不对称细胞中中心体定位的动态系统。这一过程发生在一个嘈杂、复杂的系统中，必须使用新的量化方法来增加我们对对称破缺的时间是如何控制的，以及它是否以及为什么是不可逆转的理解。由于这是细胞生物学中的基本问题，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专业知识也将使加拿大的科学受益。

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.