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：
8499370
负责人：
R DYCHE MULLINS
金额：
$ 31.59万
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/8499370
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 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.

描述（由申请人提供）：真细菌和古细菌使用细胞骨架元件，包括肌动蛋白样丝、微管蛋白相关聚合物，甚至中间丝，以：（1）控制它们的形状； (2) 划分； (3)建立细胞质的秩序； (4)移动细胞内的货物。我实验室的长期目标是绘制细菌细胞骨架蛋白（尤其是肌动蛋白样蛋白）的结构和生化多样性图表，并了解每种蛋白的独特特性如何适应其功能。在这个项目中，我们重点研究一种革兰氏阳性肌动蛋白样蛋白，称为 AlfA，它可以在营养生长和孢子形成过程中稳定枯草芽孢杆菌中的质粒。我们关注 AlfA 的原因有三个：（1）它提供了第一个研究来自革兰氏阳性生物体的货物运输肌动蛋白的机会。 (2) 它参与在商业上重要的枯草芽孢杆菌（纳豆）菌株中分离稳定质粒，并且可能与维持革兰氏阳性病原体毒力因子的系统有关。 (3) 初步实验表明，AlfA 的结构和组装动力学与任何先前表征的肌动蛋白显着不同。也就是说，在初步实验中，我们发现 AlfA：(i) 缺乏动态不稳定性，而动态不稳定性是其他 DNA 分离聚合物的细胞功能的关键；(ii) 组装成双链螺旋丝，该丝自发地结合成稳定的混合结构。极性束。活细胞成像表明这些稳定的束是 AlfA 的功能形式，并揭示了 AlfA 细丝在这些束内同时组装和分解（跑步机）。这些观察结果共同排除了 AlfA 通过任何先前提出的机制分离 DNA 的可能性，并表明 AlfA 形成了一个双向跑步机，不断地将质粒携带到芽孢杆菌细胞的两极（并在孢子形成期间进入前孢子）。本提案旨在揭示 AlfA 分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。