Structure and Assembly Dynamics of FtsZ

描述（由申请人提供）：FtsZ是微管蛋白的同源物，是细菌细胞分裂的主要细胞骨架蛋白。它组装成约30个亚基（120纳米）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。我们最近的研究表明，FtsZ在体内和体外都是非常动态的，半周期为8秒。在此，我们提出了四个新的项目来探索装配动力学和划分机制。(1)我们将使用TIRF显微镜对单个FtsZ pfs进行成像，并跟踪其生长和萎缩。我们将首先研究结核分枝杆菌FtsZ的pfs，它形成的pfs长约5 μ m，在光学显微镜下很容易看到。一旦我们开发了这项技术，我们将把它应用到大肠杆菌更具挑战性的FtsZ上，它的pfs比光学显微镜的分辨率还要短。我们期待一种基于动态不稳定性的组装机制，FtsZ的结果将有助于揭示GTP帽在微管中的机制。(2)我们将尝试重建线粒体中的FtsZ分裂机。线粒体应该是一个理想的囊泡，因为它们有合适的大小和形状，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同表达FtsZ和FtsA，并期望这些应该足以组装Z环。我们将检验自贸区足以发展收缩力的假设。(3)我们将把我们最近为大肠杆菌FtsZ开发的荧光技术应用于结核分枝杆菌，结核分枝杆菌的FtsZ pfs结构非常不同。我们将在体外和体内获得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)启动了新发现的细菌微管蛋白BtubA和BtubB的研究，并表征了它们成对的组装。我们将开发类似于用于FtsZ的溶液荧光分析，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。我们再次相信其机制将与微管动力学不稳定性有关。(5)我们将继续探索单链FtsZ pfs如何表现出协同组装。我们将使用高浓度的独立折叠n和c端结构域，来确定它们是如何影响组装和动力学的。我们还将尝试从这些结构域重建二聚体核。

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.