The Role of Peptidoglycan-binding Factors in Cytokinetic Ring Stabilization

肽聚糖结合因子在细胞动力学环稳定中的作用

• 批准号: 8253883
• 负责人: Nicholas Thomas Peters
• 金额: $ 4.92万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-01-31 至 2014-01-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8253883
• 关键词: Address; Animal Model; Antibiotics; Bacteria; Bacterial Infections; Bacterial Proteins; Binding; Binding Proteins; Biochemical; Biological Models; Biological Process; Cell Wall; Cell division; Cell physiology; Cells; Cytokinesis; Cytoplasm; Defect; Development; Diffuse; Escherichia coli; Event; Fluorescence Microscopy; Genetic; Genetic Screening; Goals; Health; Homologous Gene; Knowledge; Lead; Life; Light; Location; Mediating; Molecular; Monitor; Multi-Drug Resistance; Mutation; Newborn Infant; Organism; Pathway interactions; Pattern; Peptidoglycan; Periplasmic Proteins; Play; Polymers; Positioning Attribute; Process; Protein Binding; Proteins; Role; Shapes; Site; Structure; System; Testing; Tubulin; Work; base; cell envelope; daughter cell; design; enhancing factor; falls; interest; mutant; novel; periplasm; polymerization; prevent; prospective; scaffold; time use

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.

多重耐药细菌感染的增加对我们国家和世界的健康构成日益严重的威胁。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。该项目旨在使用大肠杆菌作为模型生物来确定细菌细胞因子环的组装和稳定机制。我们希望这将导致发现破坏这一基本结构功能的新方法。 FtsZ 是真核微管蛋白的结构同源物。 FtsZ 和微管蛋白均形成多种重要细胞功能所需的动态聚合物。在细菌细胞中，FtsZ 在细胞中部形成环状结构（Z 环）。该结构对于招聘该部门机器的所有其他下游组件是必需的。在大肠杆菌中，Z 环的形成通过广泛表征的 Min 系统引导至细胞中部区域。然而，除了这种远程空间调控系统之外，Z 环形成的许多方面仍然是神秘的。例如，目前尚不清楚 FtsZ 聚合物如何聚结成 Z 环，也不清楚这种动态聚合物的环状组合一旦形成，是什么将其保持在一起。为了开始解决这些问题，我们使用延时荧光显微镜启动了活细胞中 Z 环动力学的研究。我们注意到，中细胞 Z 形环在非收缩细胞和收缩细胞中偶尔会破裂。在变得不稳定后不久，Z 形环会完全恢复到原来的位置并恢复分裂过程。 基于这些观察，我们建议必须采取适当的机制来：i）Z 形环形成后将其稳定在中间单元，ii）标记 Z 形环的位置，以便在环图案离域的情况下，它可以精确地重新形成其最初组装的位置。我们假设具有细胞壁结合活性的分裂成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到细胞壁来实现这一点。原则上，这将在细胞中部区域内创建一个紧密区域，其中有利于 FtsZ 聚合，从而有助于稳定动态结构并引导其重新组装。该提案的目的是检验这一假设并揭示潜在的分子机制。初步证据表明 EnvC 细胞壁结合蛋白在此过程中发挥作用。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，为了使其发挥所提出的功能，需要额外的因素将其与细胞质中的 Z 环连接。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。我们将使用无偏见的遗传筛选和候选方法来研究这一问题，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。总的来说，我们预计该项目将为细菌胞质分裂过程提供重要的新线索，并揭示破坏分裂装置功能的新方法。