Temporal and Spatial Control of the Cell Cycle in Caulobacter crescentus

描述(申请人提供)：细菌是极化的，它们利用这一特性进行广泛的细胞过程，从信号转导到细胞周期进展和毒力。我们的目标是在分子水平上了解细胞极性是如何固有地建立和维持的，以及蛋白质和蛋白质复合体在细胞极点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驯化的二相性细菌新月弯杆菌，其细胞极性的形态和分子表现已经被很好地记录下来。在Crescentus中，每一次分裂都是不对称的，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取决于蛋白质和蛋白质复合体的极化。此外，在细胞周期的不同时期，鞭毛、菌毛和柄等外部细胞器在特定的细胞极形成。极地形态发生和细胞周期进程的时空调控是通过一个复杂的信号转导网络实现的。这个网络的一个主要组成部分是Cck A通路，它的组成部分在细胞周期的不同阶段显示出极地定位。这种时空调节的生理功能还不是很清楚。因此，我们的第一个目标将是确定Cck A途径组分的定位和时间调节如何影响蛋白质功能和磷酸基团在该途径中的流动。这将使用定量荧光显微镜、诱变和磷酸化分析来实现。我们的第二个目标将是阐明极性蛋白质定位是如何实现的。对极性因子Popz和TipN的鉴定表明，新月浑藻在两极形成组织中心，以调节甚至协调多种极化功能。作为切入点，我们将通过进行显微镜实验(在不同的遗传和条件背景下)和体外和体内的生化分析来研究POPZ的极化功能和POPZ在极点积累的机制。我们的第三个目标将是揭示不对称分裂背后的对称破坏机制，以及细胞区分两个细胞极的能力。由于TipN影响细胞分裂的极点识别和极化，因此将通过突变、定量荧光显微镜和生化分析来研究TipN的功能及其与肌动蛋白样MreB细胞骨架的可能联系。与公共卫生相关的细菌依赖于细胞极化进行广泛的过程，从基本的细胞周期事件到发病的重要方面。该项目将为细菌实现和维持细胞极化的分子机制提供见解，这可能导致控制细菌细胞生长的新的、广谱的手段。

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.