Probing the Structure and Contraction Mechanism of the E. coli FtsZ-ring.

描述(由申请人提供)：这项申请的长期目标是解开细菌细胞分裂的精确分子控制的潜在机制。这里描述的以FtsZ蛋白为中心的项目是迈向这一目标的第一步。FtsZ在细菌物种中高度保守，已被确定为抗菌治疗的新药物靶点。在体内，FtsZ在内陷膜的前沿组装成超分子环状结构(因此被称为Z环)来驱动细胞分裂。本项目的目标是确定Z环的结构，特别是Z环内原细丝的排列(目标1)，并确定Z环的收缩机制(目标2)。在目标1中，使用基于单分子的超分辨率成像技术，以30纳米的空间分辨率对Z环进行成像。提出了用FtsZ原丝的3D束状排列代替2D扁平带状排列的方法。这样的安排模型将通过检验由该模型产生的三个预测来进一步验证。这一目标的最终产品是一个高分辨率的Z环结构模型，它将有助于理解Z环的收缩机制。在目标2中，将研究两种相互竞争的Z环收缩机制--拆卸和冷凝。使用超分辨率成像技术将测量Z环在收缩期间的收缩速度。然后，将这个速度与基于单分子测量的Z环的解体速度和密度变化的计算速度进行比较。还将使用FtsZ GTP酶突变体来研究GTP水解在Z环收缩中的作用。这些实验，结合将在目标1中建立的结构模型，将为Z环收缩的分子机制提供重要的见解。拟议工作的成功完成将提供：(1)高分辨率Z-环结构模型；(2)该结构如何收缩的机制；(3)一套创新的在体单分子分析。 与公共卫生相关：这项研究的目标是了解细菌细胞是如何使用中心超分子组装Z环进行分裂的。由于Z-环对于整个细菌界的生存和保守是必不可少的，更好的理解将促进更有效地开发新的抗生素来对抗由病原菌引起的传染病。

DESCRIPTION (provided by applicant): The long-term goal of this application is to unravel mechanisms underlying the precise molecular controls of bacterial cell division. The project described here, centered on the FtsZ protein, is the first step toward this goal. FtsZ is highly conserved across bacterial species and has been identified as a novel drug target for antimicrobial therapy. In vivo FtsZ assembles into a supramolecular ring-like structure (hence named the Z-ring) at the leading edge of the invaginating membrane to drive cell division. The objective of this project is to determine the structure of the Z-ring, in particular the arrangement of protofilaments inside the Z-ring (Aim 1), and to identify the contraction mechanism of the Z-ring (Aim 2). In Aim 1, using a single-molecule based superresolution imaging technique the Z-ring was imaged with a 30-nm spatial resolution. A 3D bundle arrangement of FtsZ protofilaments instead of a 2D flat ribbon was proposed. Such an arrangement model will be further verified by examining three predictions stemmed from the model. The final product of this aim is a high resolution Z-ring structural model that will be instrumental in understanding the contraction mechanism of the Z-ring. In Aim 2, two competing Z-ring contraction mechanisms-disassembly vs. condensation-will be examined. The contraction speed of the Z-ring during the constriction period will be measured using the superresolution imaging technique. This speed will then be compared with the calculated speed based on single-molecule measurements of the disassembly rate and density change of the Z-ring. The role of GTP hydrolysis in Z-ring contraction will also be examined using an FtsZ GTPase mutant. These experiments, combined with the structural model that will be established in Aim 1, will provide important insight into the molecular mechanism of Z-ring contraction. Successful completion of the proposed work will provide: (1) a high-resolution Z- ring structural model; (2) a mechanism of how this structure contracts and (3) a set of innovative in vivo single-molecule assays. PUBLIC HEALTH RELEVANCE: The goal of this study is to understand how bacterial cells divide using a central supramolecular assembly, the Z-ring. As the Z-ring is essential for survival and conserved across the bacterial kingdom, a better understanding will promote more effective development of new antibiotics to combat infectious diseases caused by pathogenic bacteria.