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Structure and Assembly Dynamics of FtsZ

FtsZ 的结构和装配动力学

基本信息

批准号：
7648030
负责人：
HAROLD P ERICKSON
金额：
$ 44.6万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
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/7648030
关键词：
Actins Address Bacteria Biochemical Process Biological Assay Biophysics C-terminal Cell Nucleus Cell division Cells Chinese Hamster Ovary Cell Complex Cytokinesis Cytoskeletal Proteins Data Dominant-Negative Mutation Encapsulated Escherichia coli Exhibits Filament Fluorescence Fluorescence Microscopy Fluorescence Resonance Energy Transfer Goals Growth Guanosine Triphosphate Guanosine Triphosphate Phosphohydrolases Homologous Gene Hydrolysis Image In Vitro Kinetics Lead Life Cycle Stages Light Light Microscope Lipids Measurement Measures Membrane Microfilaments Microtubules Mind Mitochondria Modeling Molecular Mycobacterium tuberculosis Nucleotides Pathway interactions Physiology Poison Polymers Proteins Reporting Resolution Shapes Solutions Spottings Structure System Techniques Technology Testing Theoretical model Thick Time Tubulin Vesicle Work Yeasts base constriction dimer in vivo insight mutant reconstitution research study single molecule z-ring

项目摘要

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（由申请方提供）：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pf进一步组装成Z环，其在中点处包围细胞，并最终收缩以分裂细胞。我们最近已经表明，FtsZ是非常动态的营业额与8秒的半时间在体内和体外。我们在这里提出了4个新的项目来探索组装动力学和分裂机制。(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。我们首先研究M的pfs。结核FtsZ，其形成约5 μ m长的pfs，在光学显微镜下容易看到。一旦我们开发出这项技术，我们将把它应用到更具挑战性的E。coli，其pfs小于光学显微镜的分辨率。我们期待一个组装机制的基础上动态不稳定性，和FtsZ的结果应该阐明的机制的GTP帽在微管。(2)我们将尝试重建线粒体中的FtsZ分裂机器。线粒体应该是一个理想的囊泡，因为它们的大小和形状都很合适，而且他们最初使用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)我们将应用荧光技术，我们最近开发的E。coli FtsZ转化为M.结核病，其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E。杆菌(4)我们已经开始了新发现的细菌微管蛋白，BtubA和BtubB的研究，其特征在于它们的组装成pf对。我们将开发类似于FtsZ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将与微管动态不稳定性有关。(5)我们将继续我们的追求，以了解如何单链FtsZ pfs可以显示合作组装。我们将使用高浓度的独立折叠的N-和C-末端结构域，以确定它们如何毒害组装和动力学。我们还将尝试从这些结构域重新创建二聚体核。