Assembly dynamics and cellular function of Actin-like proteins in bacteria

细菌中肌动蛋白样蛋白的组装动力学和细胞功能

基本信息

批准号：
8134382
负责人：
R DYCHE MULLINS
金额：
$ 37.51万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-09-01 至 2014-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8134382
关键词：
Actins Anisotropy Antibiotic Therapy Archaea Architecture Attention Bacillus (bacterium)Bacillus subtilis Bacteria Biochemical Biological Assay Cell Shape Cell Wall Cell division Cell physiology Cells Cellular biology Complex Cytoplasm Cytoskeletal Proteins Cytoskeleton DNA DNA Sequence DNA-Binding Proteins Drug resistance Electron Microscopy Elements Energy Transfer Eubacterium Family Filament Fluorescence Fluorescence Microscopy Fluorescence Polarization Goals Growth In Vitro Intermediate Filaments Label Laboratories Life Microscopy Mitosis Molecular Motor Natto Open Reading Frames Organelles Organism Plasmids Play Polymers Process Prokaryotic Cells Property Proteins Public Health Role Sequence Analysis Shapes Structure System Techniques Tubulin Virulence Factors Work base cell growth cellular imaging enteric pathogen frontier in vivo innovation light microscopy light scattering pathogen photoactivation polymerization public health relevance reconstitution research study retinal rods scaffold segregation self assembly single molecule tool

项目摘要

DESCRIPTION (provided by applicant): Eubacteria and archaea use cytoskeletal elements including, actin-like filaments, tubulin- related polymers, and even intermediate filaments, to: (1) control their shape; (2) to divide; (3) to establish order in the cytoplasm; and (4) to move intracellular cargo. A long-term goal of my laboratory is to chart the structural and biochemical diversity of bacterial cytoskeletal proteins (especially actin-like proteins) and to understand how the unique properties of each are adapted to its function. In this project we focus on a gram positive actin-like protein, called AlfA, which stabilizes plasmids in Bacillus subtilis during both vegetative growth and sporulation. We focus on AlfA for three reasons: (1) It provides the first opportunity to study a cargo-hauling actin from a gram positive organism. (2) It is involved in segregating a stable plasmid in a commercially important strain of B. subtilis (natto) and may be related to systems that maintain virulence factors in gram positive pathogens. (3) Preliminary experiments reveal that the structure and assembly dynamics of AlfA are dramatically different from those of any previously characterized actin. Namely, in preliminary experiments we found that AlfA: (i) lacks the dynamic instability which is key to the cellular function of other DNA-segregating polymers and (ii) assembles into two-stranded helical filaments, which spontaneously associate into stable, mixed-polarity bundles. Live-cell imaging suggests that these stable bundles are the functional form of AlfA and reveals that AlfA filaments simultaneously assemble and disassemble (treadmill) inside these bundles. Together these observations rule out the possibility that AlfA segregates DNA by any previously proposed mechanism and suggest that AlfA forms a bi-directional treadmill that continuously carries plasmids to the poles of Bacillus cells (and into the forespore during sporulation). The present proposal is aimed at uncovering the mechanism by which AlfA segregates and stabilizes plasmids and determining how the unique properties of AlfA enable it to carry out this task. PUBLIC HEALTH RELEVANCE: We are just beginning to understand the details of how bacteria control their shapes, organize their insides, and divide. These processes all require the assembly of complex molecular scaffolds, called cytoskeletal networks. Understanding the assembly and function of these networks will enable us to better understand how pathogens acquire and maintain drug resistance (segregation of drug resistance plasmids) and provide new targets for antibiotic therapy (e.g. cytoskeletal proteins that control cell growth and division).

描述（由申请人提供）：EUBACTERIA和古细菌使用细胞骨架元素，包括肌动蛋白样细丝，微管蛋白相关的聚合物，甚至中间丝，用于：（1）控制其形状；（2）分裂；（3）在细胞质中建立顺序；（4）移动细胞内货物。我的实验室的一个长期目标是绘制细菌细胞骨架蛋白（尤其是类似肌动蛋白样蛋白）的结构和生化多样性，并了解每个蛋白的独特性能如何适应其功能。在这个项目中，我们着重于革兰氏阳性肌动蛋白样蛋白，称为ALFA，该蛋白在营养生长和孢子形成过程中稳定枯草芽孢杆菌的质粒。我们专注于阿尔法的原因有三个：（1）它提供了从革兰氏阳性生物体研究货物肌动蛋白的第一个机会。（2）它参与了在枯草芽孢杆菌（Natto）的商业菌株中隔离稳定的质粒，并且可能与维持革兰氏阳性病原体中毒力因子的系统有关。（3）初步实验表明，阿尔法的结构和组装动力学与任何先前表征的肌动蛋白的结构和组装动力学大不相同。也就是说，在初步实验中，我们发现Alfa：（i）缺乏动态不稳定性，这是其他DNA分离聚合物的细胞功能的关键，并且（ii）组装成两链的螺旋细丝，它们自发地将其与稳定的混合偏置套件自发地关联。活细胞成像表明，这些稳定的束是阿尔法的功能形式，并揭示了阿尔法丝同时在这些捆绑中组装并拆卸（跑步机）。这些观察结果共同规定了Alfa通过任何先前提出的机制将DNA隔离的可能性，并表明ALFA形成了双向跑步机，该跑步机连续将质粒携带到杆菌细胞的杆子（并在孢子虫期间进入前孔）。本提案旨在揭示Alfa隔离和稳定质粒的机制，并确定Alfa的独特特性如何使其能够执行此任务。公共卫生相关性：我们才刚刚开始了解细菌如何控制其形状，组织内部和分裂的细节。这些过程都需要组装复杂的分子支架，称为细胞骨架网络。了解这些网络的组装和功能将使我们能够更好地了解病原体如何获得和维持耐药性（耐药性质粒的隔离），并为抗生素治疗提供新的靶标（例如，控制细胞生长和分裂的细胞骨架蛋白质）。