The Role of Peptidoglycan-binding Factors in Cytokinetic Ring Stabilization

描述(申请人提供)：对我们国家和世界健康的一个日益增长的威胁是多重耐药细菌感染的增加。为了促进新治疗方法的设计和开发，我们需要更好地了解细菌中的基本细胞生物学过程。本项目旨在确定以大肠杆菌为模式生物组装和稳定细菌细胞动环的机制。我们希望，这将导致发现新的方法来扰乱这一基本结构的功能。FtsZ是真核微管蛋白的结构同源物。FtsZ和微管蛋白都形成动态聚合物，是各种重要细胞功能所必需的。在细菌细胞中，FtsZ在中间细胞形成环状结构(Z环)。这一结构是招聘师机械所有其他下游部件所必需的。在大肠杆菌中，Z-环的形成被广泛表征的Min系统引导到中间细胞区。然而，除了这种远程的空间调控系统外，Z环形成的许多方面仍然是个谜。例如，目前尚不清楚FtsZ聚合物是如何结合成Z环的，也不清楚是什么使这种动态聚合物的环状聚集在一起。为了开始解决这些问题，我们启动了一项使用时间推移荧光显微镜对活细胞中Z环动力学的研究。我们注意到，在非收缩细胞和收缩细胞中，中细胞Z环偶尔都会破裂。在变得不稳定后不久，Z环就在原来的位置上进行了改革，并恢复了划分过程。基于这些观察，我们建议必须建立机制来：i)一旦Z环形成，就将其稳定在中间细胞；ii)标记Z环的位置，以便在环图案离域的情况下，它可以精确地在它最初组装的地方进行改革。我们假设具有细胞壁结合活性的分裂组分在这些过程中发挥关键作用，并且它们是通过将FtsZ聚合的正调控因子锚定在细胞壁上来做到这一点的。原则上，这将在有利于FtsZ聚合的中间细胞区内创建一个紧密区，从而有助于稳定动态结构并引导其重新组装。这项提议的目标是检验这一假说，并揭示其潜在的分子机制。初步证据表明，EnvC细胞壁结合蛋白在这一过程中发挥了作用。我们将通过研究EnvC和EnvC细胞中的Z环动力学来进一步研究这一点。由于EnvC完全是周质的，为使其发挥所提出的功能，需要额外的因子将其与细胞质中的Z环连接起来。我们将使用遗传和生化相结合的方法来寻找这些因素。最后，由于EnvC不是本质的，我们假设存在冗余的Z-环镇定系统。我们将使用无偏见的遗传筛选和候选方法来研究这一点，该方法侧重于具有细胞壁结合活性的分裂机制的其他已知组件。总体而言，我们预计这个项目将为细菌的胞质分裂过程提供重要的新线索，并揭示扰乱分裂装置功能的新方法。

DESCRIPTION (provided by applicant): An ever-growing threat to the health of our nation and the world is the increase in multi-drug resistant bacterial infections. To facilitate the design and development of new treatments, we need a better understanding of fundamental cell biological processes in bacteria. This project is designed to determine the mechanism by which the bacterial cytokinetic ring is assembled and stabilized using Escherichia coli as a model organism. Our hope is that this will lead to the discovery of new ways to disrupt the function of this essential structure. FtsZ is a structural homologue of the eukaryotic tubulin protein. Both FtsZ and tubulin form dynamic polymers that are required for a variety of vital cellular functions. In bacterial cells, FtsZ forms a ring-like structure (the Z- ring) at midcell. This structure is required for the recruitment of all other downstream components of the division machinery. In E. coli, Z-ring formation is directed to the midcell zone by the extensively characterized Min system. However, aside from this long-range, spatial regulatory system, many aspects of Z-ring formation remain mysterious. For example, it is not clear how FtsZ polymers coalesce into the Z-ring or what keeps this ring-like assemblage of dynamic polymers together once it forms. To begin addressing these issues, we initiated a study of Z-ring dynamics in live cells using time-lapse fluorescence microscopy. We noticed that midcell Z-rings occasionally fall apart in both non-constricting and constricting cells. Shortly after becoming destabilized, Z-rings reform exactly at their original position and resume the division process. Based on these observations we propose that mechanisms must be in place to: i) stabilize the Z-ring at midcell once it is formed and ii) mark the location of the Z-ring such that in instances when the ring pattern delocalizes, it can reform precisely where it was initially assembled. We hypothesize that divisome components with cell wall binding activity play key roles in these processes and that they do so by anchoring positive regulators of FtsZ polymerization to the cell wall. This would, in principle, create a tight zone within the midcell region where FtsZ polymerization is favored and thus serve to stabilize the dynamic structure and guide its reassembly. The goal of this proposal is to test this hypothesis and uncover the underlying molecular mechanisms. Preliminary evidence suggests a role for the EnvC cell wall binding protein in this process. We will investigate this further by studying Z-ring dynamics in EnvC+ and EnvC- cells. Since EnvC is entirely periplasmic, for it to function as proposed, additional factors are required to connect it with the Z-ring in the cytoplasm. We will search for these factors using a combined genetic and biochemical approach. Finally, since EnvC is not essential, we hypothesize that redundant Z-ring stabilization systems exist. We will investigate this using an unbiased genetic screen and a candidate approach focusing on other known components of the division machinery with cell wall binding activity. Overall, we anticipate that this project will shed significant new light on the process of cytokinesis in bacteria and reveal novel ways to disrupt the function of the division apparatus.