Circadian gating of cell division by the cyanobacterial oscillator

描述(申请人提供)：细胞分裂的时间与细胞生命中其他周期的其他周期事件相协调。从细菌、藻类到哺乳动物的各种CLL类型，昼夜生物钟控制着一天中细胞分裂发生的时间。细胞分裂的这种时间限制或门控的机制和功能在任何系统中都知之甚少，哺乳动物中的这种调节检查点是发育程序和癌症进展的重要因素。这个项目的总体目标是了解昼夜节律和细胞分裂是如何以及为什么相互关联的。在蓝藻中，细胞分裂的昼夜控制提供了一个优雅的系统，在其中可以解决探索时钟、细胞质分裂机制和分离染色体之间的相互作用的问题。这个项目试图回答：细胞质分裂的昼夜检查点的生物学作用是什么？连接生物钟和细胞分裂的机械部件是什么？在细胞周期和昼夜周期中，时钟振荡器组件在哪里？它们是如何遗传的？而且，染色体的分裂是否与细胞分裂的门控有关？具体目标是：(1)确定生物钟调节细胞分裂的成分，并确定绕过细胞分裂门的后果；(2)辨别生物钟蛋白在昼夜节律和细胞分裂周期中的细胞内定位和动态；以及(3)阐明时钟、胞质分裂和染色体分离之间的关系。现有的绕过昼夜细胞分裂检查点的突变体将被用来测试假说，这些假说解决了门在保护昼夜节律精确度、染色体完整性和/或基因表达中细胞间变异方面的作用。质谱学将识别与细胞相关的因素 在门禁检查站期间的分区机械。对被困在微流体室中的细胞进行延时成像将能够同时监测细胞分裂和昼夜节律，以评估在单个野生型细胞和绕过昼夜节律的突变体中门控的后果。超分辨率结构照明成像将提供足够的灵敏度和分辨率来跟踪昼夜节律示波蛋白和标记染色体在整个昼夜节律和细胞分裂周期中在野生型和突变细胞中的定位和动态。将使用分类和成像流式细胞术方法来评估野生型和突变菌株的倍性变化。这个项目将揭示生物钟如何以及为什么控制细胞分裂，这是发生在哺乳动物细胞和蓝藻中的计时电路的耦合，并将为细胞如何继承时间感提供新的见解。

DESCRIPTION (provided by applicant): The timing of cell division is coordinated with other cyclic events, of other periodicities, in the lives of cells. From bacteria, to algae, to diverse cll types in mammals, the circadian biological clock controls the time of day during which cell division can occur. The mechanism and function of this time restriction, or gating, of cell divisio is poorly understood in any system, and such regulatory checkpoints in mammals are important factors in developmental programs and the progression of cancer. The overall goal of this project is to understand how and why circadian rhythms and cell division are interlocked. The circadian control of cell division in the cyanobacterium Synechococcus elongatus provides an elegant system in which to address questions that probe the interactions of the clock, the cytokinesis machinery, and the segregating chromosomes. This project seeks to answer: What is the biological role of the circadian checkpoint of cytokinesis? What are the components of the machinery that connect the circadian clock to cell division? Where are the clock oscillator components during the cell and circadian cycles and how are they inherited? And, is the partitioning of chromosomes in S. elongatus, whose ploidy levels oscillate with circadian rhythmicity, related to the gating of cell division? The specific aims will: (1) Define the components, through which the circadian clock regulates cell division, and determine the consequences of bypassing the cell division gate; (2) Discern the intracellular localization and dynamics of clock proteins during the circadian and cell division cycles; and (3) Elucidate the relationships among the clock, cytokinesis, and chromosome segregation. Existing mutants that bypass the circadian cell division checkpoint will be used to test hypotheses that address the role of the gate in protecting circadian precision, chromosome integrity, and/or cell-to-cell variations in gene expression. Mass spectrometry will identify factors that associate with the cell division machinery during the gating checkpoint. Time-lapse imaging of cells trapped in microfluidics chambers will enable simultaneous monitoring of cell division and circadian rhythms to assess the consequences of gating in individual wild-type cells and in mutants that bypass the circadian gate. Super-resolution Structured Illumination imaging will provide sufficient sensitivity and resolution to track the localization and dynamics of circadian oscillato proteins and tagged chromosomes in wild-type and mutant cells throughout the circadian and cell division cycles. Sorting and imaging flow cytometry methods will be used to assess ploidy changes in wild-type and mutant strains. This project will reveal how and why a circadian clock controls cell division, a coupling of timing circuits that occurs in mammalian cells as well as in cyanobacteria, and will provide novel insight into how cells inherit a sense of time.