Structure and Assembly Dynamics of FtsZ

FtsZ 的结构和装配动力学

• 批准号: 7100484
• 负责人: HAROLD P ERICKSON
• 金额: $ 43.15万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7100484
• 关键词: CHO cells; Escherichia coli; Mycobacterium tuberculosis; analog; bacterial genetics; cell cycle; chemical kinetics; fluorescence microscopy; fluorescence resonance energy transfer; guanosine triphosphate; hydrolysis; method development; microfilaments; mitochondria; molecular assembly /self assembly; molecular dynamics; nanotechnology; protein structure function; solutions; transfection; tubulin; vesicle /vacuole; yeasts

DESCRIPTION (provided by applicant): FtsZ, a homolog of tubulin, is the major cytoskeletal protein in bacterial cell division. It assembles into protofilaments (pfs) that are~30 subunits (120 nm) long. In vivo these pfs are further assembled into a Z ring, which encircles the cell at mid-point, and eventually constricts to divide the cell. We have recently shown that FtsZ is extremely dynamic-turnover with a half time of 8 seconds both in vivo and in vitro. We propose here 4 new projects to explore assembly dynamics and the mechanism of division. (1) We will use TIRF microscopy to image single FtsZ pfs and follow growth and shrinking. We will first study pfs of M. tuberculosis FtsZ, which form pfs that are~5 um long, easily visible in the light microscope. Once we have developed the technology we will apply it to the much more challenging FtsZ of E. coli, whose pfs are shorter than the resolution of the light microscope. We expect an assembly mechanism based on dynamic instability, and the results with FtsZ should shed light on the mechanism of the GTP cap in microtubules. (2) We will attempt to reconstitute the FtsZ division machine in mitochondria. Mitochondria should be an ideal vesicle for this because they are the right size and shape, and they originally used FtsZ for division. We will co-express FtsZ and FtsA in mitochondria of CHO cells and yeast, and expect that these should be sufficient to assemble a Z ring. We will test the hypothesis that FtsZ is sufficient to develop the constriction force. (3) We will apply the fluorescence techniques that we have recently developed for E. coli FtsZ to M. tuberculosis, whose FtsZ pfs are structurally very different. We will obtain a complete characterization of initial assembly kinetics and turnover in vitro and in vivo, to compare with those of E. coli. (4) We have initiated studies of the newly discovered bacterial tubulins, BtubA and BtubB, and characterized their assembly into pf pairs. We will develop solution fluorescence assays similar to those used for FtsZ, to determine the assembly dynamics of BtubA/B protofilaments. The pf pairs assembled from BtubA/B will be ideal subjects for single molecule TIRF microscopy. Again we believe the mechanism will be related to microtubule dynamic instability. (5) We will pursue our quest to understand how the single-stranded FtsZ pfs can show cooperative assembly. We will use high concentrations of the independently folding N-and C-terminal domains, to determine how they poison pf assembly and dynamics. We will also attempt to re-create the dimer nucleus from these domains.

描述（由申请人提供）：FtsZ 是微管蛋白的同系物，是细菌细胞分裂中的主要细胞骨架蛋白。它组装成约 30 个亚基 (120 nm) 长的原丝 (pfs)。在体内，这些 pfs 进一步组装成 Z 环，在中点环绕细胞，并最终收缩以分裂细胞。我们最近证明 FtsZ 具有极高的动态周转率，在体内和体外的半衰期均为 8 秒。我们在此提出 4 个新项目来探索装配动力学和分裂机制。 (1) 我们将使用 TIRF 显微镜对单个 FtsZ pfs 进行成像并跟踪生长和收缩。我们将首先研究结核分枝杆菌 FtsZ 的 pfs，其形成约 5 um 长的 pfs，在光学显微镜下容易可见。一旦我们开发出这项技术，我们将把它应用于更具挑战性的大肠杆菌 FtsZ，它的 pfs 比光学显微镜的分辨率短。我们期望基于动态不稳定性的组装机制，并且 FtsZ 的结果应该能够阐明微管中 GTP 帽的机制。 (2)我们将尝试在线粒体中重建FtsZ分裂机。线粒体应该是一个理想的囊泡，因为它们具有合适的大小和形状，并且它们最初使用 FtsZ 进行分裂。我们将在 CHO 细胞和酵母的线粒体中共表达 FtsZ 和 FtsA，并期望这些应该足以组装 Z 环。我们将检验 FtsZ 足以产生收缩力的假设。 (3) 我们将把最近为大肠杆菌 FtsZ 开发的荧光技术应用于结核分枝杆菌，其 FtsZ pfs 在结构上非常不同。我们将获得体外和体内初始组装动力学和周转的完整表征，以与大肠杆菌进行比较。 (4)我们启动了对新发现的细菌微管蛋白BtubA和BtubB的研究，并表征了它们组装成pf对的特征。我们将开发类似于 FtsZ 所用的溶液荧光测定法，以确定 BtubA/B 原丝的组装动力学。由 BtubA/B 组装的 pf 对将是单分子 TIRF 显微镜的理想对象。我们再次相信该机制与微管动态不稳定性有关。 (5) 我们将继续探索单链 FtsZ pfs 如何表现出合作组装。我们将使用高浓度的独立折叠 N 端和 C 端结构域，以确定它们如何毒害组装和动力学。我们还将尝试从这些域重新创建二聚体核。