CAREER: Interaction Between Cell Division Proteins during the Caulobacter Cell Cycle.

Brun细胞分裂是一个复杂的过程，涉及许多蛋白质，其作用必须与DNA复制周期协调。很明显，要解释分裂是如何被控制的，以及它是如何与DNA复制周期协调的，就需要了解分裂机制的不同组成部分的组装时间，以及不同组成部分如何以及何时相互作用。本研究将具体研究FtsA的功能及其与FtsZ的相互作用。FtsZ是一种微管蛋白样GTdR，调节何时何地分裂的决定。FtsZ是一种高度保守的蛋白质，其在细胞分裂位点聚合成与细胞质膜相关的环状结构。FtsZ将其他细胞分裂蛋白质募集到细胞分裂的位点，并可能收缩，为分裂提供机械力。FtsA以FtsZ依赖性方式定位于分裂位点。FtsA不是胞质分裂起始所必需的，但是胞质分裂完成所必需的。该项目的长期目标是了解细胞分裂蛋白的功能，并建立细胞周期中细胞分裂机制中蛋白质-蛋白质相互作用的图像。这项工作将在新月柄杆菌中进行，其中每次细胞分裂产生两种形态和功能不同的子代细胞：能动的游动细胞和无柄柄细胞。柄杆菌属细胞可以容易地同步化，并且在分化途径期间的特定形态学变化提供了细胞周期阶段的方便指示。因为每个发育周期只有一个DNA复制周期，所以事件可以明确地分配到细胞周期的特定阶段。FtsZ和FtsA已经在柄杆菌中鉴定，并且已经使用酵母双杂交系统建立了这两种蛋白质之间的遗传相互作用。FtsA基因不同区域突变的影响将被分析，以获得更好的理解其功能和结构域的贡献，FtsZ和FtsA之间的相互作用的结构域将被定义使用酵母双杂交系统和融合谷胱甘肽S-转移酶。FtsA和FtsZ的定位及其相互作用将在细胞周期期间使用免疫荧光和体内交联进行分析。这将允许在一个细胞周期内确定这两种重要的细胞分裂蛋白的定位和相互作用的时间，以及其与染色体复制阶段和类核分离时间的相关性。细胞分裂的机制是微生物学中最基本的问题之一。这项调查将导致更好地了解细胞分裂，并将允许在细胞分裂机制中的蛋白质-蛋白质相互作用的图片，因为它们发生在细胞周期期间被建立。更好地了解细胞分裂的机制将有助于开发新的药物靶点。这项研究将在高级研究人员的研究实验室和新的分子和遗传学独立研究课程中进行，这将为大量本科生提供急需的独立研究机会。学生将通过在柄杆菌中进行细胞分裂和细胞分化的研究，并在文献，科学写作，口头陈述和研究伦理的批判性分析课程中学习科学调查的不同阶段和方面。

Brun Cell division is a complex process that involves many proteins whose action must be coordinated with the DNA replication cycle. It is clear that an understanding of the timing of assembly of the different components of the division machinery and how and when the different components interact will be required to explain how division is controlled and how it is coordinated with the DNA replication cycle. This research will specifically examine the function of FtsA and its interaction with FtsZ. FtsZ is a tubulin-like GTPase that regulates the decision of when and where to divide. FtsZ is a highly conserved protein that polymerizes into a ring structure associated with the cytoplasmic membrane at the site of cell division. FtsZ recruits other cell division proteins to the site of cell division and may constrict, providing mechanical force for division. FtsA is localized to the division site in a FtsZ-dependent manner. FtsA is not required for the initiation of cytokinesis but is required for completion of cytokinesis. The long-term goal of this project is to understand the function of cell division proteins and to build a picture of protein-protein interactions in the cell division machinery as they occur during the cell cycle. This work will be performed in Caulobacter crescentus where each cell division gives rise to two morphologically and functionally different progeny cells: a motile swarmer cell and a sessile stalked cell. Caulobacter cells can be easily synchronized and specific morphological changes during the differentiation pathway provide a convenient indication of the stage of the cell cycle. Because there is only one DNA replication cycle per developmental cycle, events can be unambiguously assigned to a specific stage of the cell cycle. FtsZ and FtsA have been identified in Caulobacter and genetic interaction between the two proteins has already been established using the yeast two-hybrid system. The effect of mutations in different regions of FtsA will be analyzed to gain a bet ter understanding of its function and the domains that contribute to it. The domains of interaction between FtsZ and FtsA will be defined using the yeast two-hybrid system and fusions to glutathlone S-transferase. The localization of FtsA and FtsZ and their interaction will be analyzed during the cell cycle using immunofluorescence and in vivo cross-linking. This will allow a determination of the timing of the localization and the interaction of these two important cell division proteins within one cell cycle and its correlation with the stage of chromosome replication and the timing of nucleoid segregation. The mechanisms of cell division constitute one of the most fundamental problems in microbiology. This investigation will lead to a better understanding of cell division and will allow a picture of protein-protein interactions in the cell division machinery as they occur during the cell cycle to be established. A better understanding of the mechanisms of cell division will help the development of novel drug targets. The research will be done both in a research laboratory by senior investigators and in a new Independent Research in Molecular and Genetics course that will provide much needed independent research opportunities for a large number of undergraduate students. Students will learn about the different stages and aspects of scientific investigation by performing research on cell division and cell differentiation in Caulobacter and in sessions on critical analysis of the literature, scientific writing, oral presentation, and research ethics.