Structure and Assembly Dynamics of FtsZ

FtsZ 的结构和装配动力学

基本信息

批准号：
7912090
负责人：
HAROLD P ERICKSON
金额：
$ 12.52万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-11 至 2010-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7912090
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（由申请人提供）：FTSZ是微管蛋白的同源物，是细菌细胞分裂中的主要细胞骨架蛋白。它组装成〜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，该Z环将细胞以中点包围，并最终收缩以分裂细胞。我们最近表明，FTSZ在体内和体外的半个时间为8秒，是非常动态的。我们在这里提出了4个新项目，以探索组装动态和分裂的机制。（1）我们将使用TIRF显微镜对单个FTSZ PFS进行映像，并跟随生长和收缩。我们将首先研究结核分枝杆菌FTSZ的PF，该PF形成长5 um，在光学显微镜中易于可见的PFS。一旦开发了该技术，我们将其应用于大肠杆菌的更具挑战性的FTSZ，其PF比光学显微镜的分辨率短。我们期望基于动态不稳定性的装配机理，而FTSZ的结果应阐明微管中GTP盖的机理。（2）我们将尝试重建线粒体的FTSZ分区机器。线粒体应该是这样的理想囊泡，因为它们的尺寸和形状是正确的，并且最初使用FTSZ进行除法。我们将在CHO细胞和酵母的线粒体中共表达FTSZ和FTSA，并期望它们足以组装Z环。我们将检验以下假设：FTSZ足以发展收缩力。（3）我们将应用我们最近为大肠杆菌FTSZ开发的荧光技术到结核分枝杆菌，其FTSZ PFS在结构上非常不同。我们将在体外和体内获得初始组装动力学和周转率的完整表征，以与大肠杆菌相比。（4）我们已经开始研究新发现的细菌小管，Btuba和Btubb，并将其组装成PF对。我们将开发与FTSZ相似的溶液荧光测定，以确定Btuba/B原丝的组装动力学。从BTUBA/B组装的PF对将是单分子TIRF显微镜的理想受试者。同样，我们认为该机制将与微管动态不稳定性有关。（5）我们将追求我们的追求，以了解单链FTSZ PFS如何显示合作组装。我们将使用高浓度的独立折叠n和c末端结构域来确定它们如何毒化PF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。