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

描述（由申请人提供）：真细菌和古细菌使用细胞骨架元件，包括肌动蛋白样细丝、微管蛋白相关聚合物、甚至中间细丝，以：（1）控制它们的形状;（2）分裂;（3）在细胞质中建立秩序;和（4）移动细胞内货物。我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，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，我们专注于一个革兰氏阳性肌动蛋白样蛋白，称为AlfA，它稳定枯草芽孢杆菌在营养生长和孢子形成过程中的质粒。我们专注于AlfA有三个原因：（1）它提供了第一个机会来研究来自革兰氏阳性生物体的货物运输肌动蛋白。(2)它参与分离商业上重要的B菌株中的稳定质粒。枯草芽孢杆菌（纳豆）的致病性，并且可能与在革兰氏阳性病原体中维持毒力因子的系统有关。(3)初步的实验表明，结构和组装动力学的AlfA是显着不同的任何先前表征的肌动蛋白。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，（ii）组装成双链螺旋丝，自发地结合成稳定的混合极性束。活细胞成像表明，这些稳定的束是AlfA的功能形式，并揭示了AlfA丝在这些束内同时组装和拆卸（跑步机）。总之，这些观察结果排除了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的独特性质如何使其能够执行这一任务。 公共卫生相关性：我们刚刚开始了解细菌如何控制它们的形状，组织它们的内部和分裂的细节。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).