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

描述(申请人提供)：真细菌和古生菌使用细胞骨架元素，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)分裂；(3)在细胞质中建立秩序；(4)移动细胞内的货物。我的实验室的一个长期目标是绘制细菌细胞骨架蛋白(特别是肌动蛋白样蛋白)的结构和生化多样性图，并了解每种蛋白的独特性质如何适应其功能。在这个项目中，我们专注于一种革兰氏阳性肌动蛋白样蛋白，称为Alfa，它在枯草芽孢杆菌的营养生长和孢子形成过程中稳定质粒。我们关注Alfa有三个原因：(1)它提供了第一次从革兰氏阳性生物中研究货物运输肌动蛋白的机会。(2)它参与分离一种商业上重要的枯草杆菌(纳豆)菌株中的稳定质粒，并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都有很大的不同。也就是说，在初步实验中，我们发现阿尔法：(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；(Ii)组装成双链螺旋细丝，这些螺旋细丝自发地结合成稳定的混合极性束。活细胞成像表明，这些稳定的纤维束是阿尔法纤维的功能形式，并揭示了阿尔法纤维在这些纤维束内同时组装和分解(跑步机)。综上所述，这些观察排除了阿尔法通过任何先前提出的机制分离DNA的可能性，并表明阿尔法形成了一个双向跑步机，不断地将质粒运送到芽孢杆菌细胞的两极(并在孢子形成过程中进入前孔)。本提案旨在揭示阿尔法分离和稳定质粒的机制，并确定阿尔法的独特性质如何使其能够完成这项任务。 与公共卫生相关：我们才刚刚开始了解细菌如何控制它们的形状、组织它们的内部以及分裂的细节。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(耐药质粒的分离)，并为抗生素治疗提供新的靶点(例如控制细胞生长和分裂的细胞骨架蛋白)。

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).