Temporal and Spatial Control of the Cell Cycle in Caulobacter crescentus

描述（由申请人提供）：细菌是极化的，它们利用这种特性进行广泛的细胞过程，从信号转导到细胞周期进程和毒力。我们的目标是在分子水平上了解细胞极性是如何内在地建立和维持的，以及蛋白质和蛋白质复合物在细胞两极的定位如何影响和调节细胞功能。我们选择的细菌模型是遗传上易于处理的二型细菌新月柄杆菌，细胞极性的形态和分子表现已被充分证明。In C.新月形，每次分裂是不对称的，产生不同大小，形态和命运的子细胞。染色体分离和细胞分裂依赖于蛋白质和蛋白质复合物的极化。此外，外部细胞器如鞭毛、皮利和柄在细胞周期的离散时期在特定的细胞极形成。极性形态发生和细胞周期进程的时间和空间调节是通过复杂的信号转导网络实现的。该网络的一个主要组成部分是CckA途径，其组分在细胞周期的不同阶段显示极性定位。这种时空调节的生理功能还不清楚。因此，我们的第一个目标将是确定如何的本地化和时间调节的CckA途径组件影响蛋白质功能和磷酰基基团的流动的途径。这将使用定量荧光显微镜、诱变和磷酸化测定来实现。我们的第二个目标是阐明极性蛋白定位是如何实现的。极性因子PopZ和TipN广泛影响参与不同细胞功能的蛋白质和蛋白质复合物的极性定位，这表明C。crescentus在两极形成组织中心，以调解，也许协调，多重极化功能。作为一个切入点，我们将研究的极化功能的PopZ和PopZ积累在极点的机制，通过进行显微镜实验（在各种遗传和条件背景）和生化试验在体外和体内。我们的第三个目标是揭示不对称分裂和细胞区分细胞两极的能力背后的破坏细胞分裂的机制。由于TipN影响极的身份和极化的细胞分裂，TipN的功能和可能的连接肌动蛋白样MreB细胞骨架将使用诱变，定量荧光显微镜和生化分析进行研究。公共卫生相关性细菌依赖于细胞极化的各种各样的过程，从基本的细胞周期事件的发病机制的重要方面。该项目将深入了解细菌实现和维持细胞极化的分子机制，这可能会导致新的广谱手段来控制细菌细胞生长。

DESCRIPTION (provided by applicant): Bacteria are polarized and they exploit this property for a wide array of cellular processes ranging from signal transduction to cell cycle progression and virulence. Our goal is to understand, at the molecular level, how cell polarity is inherently established and maintained, and how the localization of protein and protein complexes at the cell poles impacts and regulates cell function. Our bacterial model of choice is the genetically tractable dimorphic bacterium Caulobacter crescentus, for which morphological and molecular manifestations of cell polarity have been well documented. In C. crescentus, each division is asymmetric, yielding daughter cells of different sizes, morphologies and fates. Chromosome segregation and cell division depend on the polarization of proteins and protein complexes. Furthermore, external organelles such as the flagellum, pili and stalk form at a specific cell pole during discrete periods of the cell cycle. The temporal and spatial regulation of polar morphogenesis and cell cycle progression is achieved by an intricate signal transduction network. A major component of this network is the CckA pathway whose components display polar localization at distinct stages of the cell cycle. The physiological function of this spatio-temporal regulation is not well understood. Thus, our first objective will be to determine how the localization and temporal regulation of the CckA pathway components affect protein function and the flow of phosphoryl groups in the pathway. This will be achieved using quantitative fluorescence microscopy, mutagenesis and phosphorylation assays. Our second objective will be to elucidate how polar protein localization is achieved. The identification of the polarity factors PopZ and TipN, which broadly affect the polar localization of proteins and protein complexes involved in different cellular functions, suggests that C. crescentus forms organizing centers at the poles to mediate, and perhaps coordinate, multiple polarized functions. As an entry point, we will study the polarizing function of PopZ and the mechanisms by which PopZ accumulates at the poles by performing microscopy experiments (in various genetic and conditional backgrounds) and biochemical assays in vitro and in vivo. Our third objective will be to uncover the symmetry-breaking mechanisms that underlie asymmetric division and the cell's ability to distinguish between the two cell poles. Since TipN affects pole identity and the polarization of cell division, the function of TipN and its possible connection with the actin-like MreB cytoskeleton will be investigated using mutagenesis, quantitative fluorescence microscopy and biochemical assays. PUBLIC HEALTH RELEVANCE Bacteria rely on cell polarization for a wide variety of processes, from essential cell cycle events to important aspects of pathogenesis. This project will provide insights into the molecular mechanisms by which bacteria can achieve and maintain cell polarization, which may lead to new, broad spectrum means to control bacterial cell growth.