The Role of Peptidoglycan-binding Factors in Cytokinetic Ring Stabilization

描述（由申请人提供）：对我们国家和世界健康日益增长的威胁是多重耐药细菌感染的增加。为了促进新疗法的设计和开发，我们需要更好地了解细菌的基本细胞生物学过程。本项目旨在以大肠杆菌为模式生物，确定细菌细胞动力学环组装和稳定的机制。我们的希望是，这将导致发现新的方法来破坏这一基本结构的功能。FtsZ是真核微管蛋白的结构同源物。FtsZ和微管蛋白形成的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在细胞中间形成环状结构（Z-环）。这种结构是需要招聘所有其他下游部件的部门机械。在大肠杆菌中，z环的形成是由广泛表征的Min系统引导到中细胞区。然而，除了这种远距离的空间调控系统之外，z环形成的许多方面仍然是神秘的。例如，目前尚不清楚FtsZ聚合物是如何结合成z环的，或者是什么使这种动态聚合物的环状组合在形成后保持在一起。为了开始解决这些问题，我们使用延时荧光显微镜对活细胞中的z环动力学进行了研究。我们注意到在非收缩细胞和收缩细胞中，中间细胞的z环偶尔会破裂。在变得不稳定后不久，z环精确地在其原始位置进行改革并恢复分割过程。基于这些观察，我们建议必须有适当的机制来：i)一旦z -环形成，就将z -环稳定在中间单元，ii)标记z -环的位置，以便在环图案脱域的情况下，它可以精确地在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，并且它们通过将FtsZ聚合的正调节因子锚定在细胞壁上来实现这一点。原则上，这将在细胞中间区域内创建一个紧密的区域，在这里FtsZ聚合是有利的，从而有助于稳定动态结构并指导其重组。本提案的目标是验证这一假设，并揭示潜在的分子机制。初步证据表明EnvC细胞壁结合蛋白在这一过程中起作用。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完全是周质性的，为了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不是必需的，我们假设存在冗余z环稳定系统。我们将使用无偏遗传筛选和候选方法来研究这一点，重点关注具有细胞壁结合活性的分裂机制的其他已知组分。总的来说，我们预计该项目将为细菌细胞分裂过程提供重要的新见解，并揭示破坏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.