Regulation and Function of a Bacterial Cytoskeleton

细菌细胞骨架的调节和功能

• 批准号: 8448080
• 负责人: R DYCHE MULLINS
• 金额: $ 24.45万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-25 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8448080
• 关键词: Actins; Antibiotic Resistance; Architecture; Attention; Bacteria; Binding; Binding Sites; Biochemical; Biological; Biological Assay; Cell Shape; Cell division; Cells; Chemicals; Clinical; Complex; Coupled; Cryoelectron Microscopy; Cytoplasm; Cytoskeleton; DNA; Detection; Drug resistance; Ensure; Eukaryotic Cell; Filament; Genes; Goals; In Vitro; Inherited; Label; Laboratories; Life; Maps; Measures; Mechanics; Mediating; Microfilaments; Microtubules; Molecular; Motor; Movement; Mutagenesis; Operon; Pharmaceutical Preparations; Plasmids; Polymers; Population; Positioning Attribute; Property; Proteins; Regulation; Repressor Proteins; Resolution; Shapes; Stretching; Structure; Subcellular structure; System; Techniques; Testing; Work; base; crosslink; daughter cell; dimer; enteric pathogen; improved; in vivo; innovation; insight; interest; light microscopy; macromolecule; mathematical theory; migration; optical traps; particle; plasmid DNA; polymerization; programs; public health relevance; reconstitution; research study; retinal rods; segregation; single molecule; theories

DESCRIPTION (provided by applicant): A major goal of my laboratory is to understand how cytoskeletal polymers establish long-range order in the cytoplasm and help convert a rowdy mob of macromolecules into a living cell. Initially, we focused on actin filament networks that drive migration of eukaryotic cells but, in 2004, our interests expanded to include bacterial polymers (Garner, 2004). Until recently, cytoskeletal polymers like actin filaments and microtubules were thought to be eukaryotic innovations, not present in bacteria. Recent work, however, has identified a number of cytoskeletal systems in bacteria, including actin-like proteins required to maintain cell shape (Jones, 2001); transport cargo through cytoplasm (Moller-Jensen, 2003; Kruse, 2003); and organize intracellular compartments (Komeili, 2006). My laboratory has focused particular attention on the actin-like protein, ParM, which forms dynamic filaments that push cargo to opposite poles of rod-shaped bacteria. The ParM gene is part of a partitioning locus (the par operon) found on many low-copy plasmids (e.g. clinically important R1 and R100 drug-resistance plasmids). To ensure plasmid inheritance the par operon constructs a DNA-segregating spindle from three components: (1) a stretch of centromeric DNA called parC (Dam, 1994); (2) a repressor protein, ParR, that binds the parC locus (van den Ent, 2002); and (3) the actin-like protein ParM. The ParR/parC complex harnesses the energy of ParM polymerization to produce forces that push pairs of plasmids in opposite directions through the cytoplasm (Moller-Jensen, 2002; Moller-Jensen, 2003). We previously characterized the assembly dynamics of ParM filaments (Garner, 2004) and reconstituted ParM-based DNA segregation in vitro using purified components (Garner, 2007). The goal of the present proposal is to understand ParM-mediated plasmid movement in molecular detail. We pursue these studies for several reasons, including: (1) understanding ParM-dependent DNA segregation provides insight into related systems that work to organize prokaryotic cytoplasm. (2) ParM filaments have a simpler architecture than microtubules, making them the best system for studying the molecular basis of dynamic instability. (3) Understanding mechanisms that maintain drug resistance plasmids in bacterial populations can help us deal with the emergence of clinical drug resistance. We will perform quantitative studies at three size scales: (i) single molecule and bulk biochemical studies; (ii) biophysical and microscopical studies of reconstituted ParM spindles; and (iii) cell biological studies of ParM in living cells.

描述（由申请人提供）： 我实验室的一个主要目标是了解细胞骨架聚合物如何在细胞质中建立长程有序，并帮助将一群吵闹的大分子转化为活细胞。最初，我们专注于驱动真核细胞迁移的肌动蛋白丝网络，但在2004年，我们的兴趣扩展到包括细菌聚合物（Garner，2004）。直到最近，像肌动蛋白丝和微管这样的细胞骨架聚合物被认为是真核生物的创新，而不存在于细菌中。然而，最近的工作已经确定了细菌中的许多细胞骨架系统，包括维持细胞形状所需的肌动蛋白样蛋白（Jones，2001）;通过细胞质运输货物（Moller-Jensen，2003; Kruse，2003）;以及组织细胞内区室（Komeili，2006）。我的实验室特别关注肌动蛋白样蛋白ParM，它形成动态细丝，将货物推向杆状细菌的两极。ParM基因是在许多低拷贝质粒（例如临床上重要的R1和R100耐药质粒）上发现的分配位点（par操纵子）的一部分。为了确保质粒遗传，par操纵子从三个组分构建DNA分离纺锤体：（1）一段称为parC的着丝粒DNA（Dam，1994）;（2）结合parC基因座的阻遏蛋白ParR（货车den Ent，2002）;和（3）肌动蛋白样蛋白ParM。ParR/parC复合物利用ParM聚合的能量产生力，将质粒对以相反的方向推过细胞质（Moller-Jensen，2002; Moller-Jensen，2003）。我们先前表征了ParM细丝的组装动力学（Garner，2004），并使用纯化的组分在体外重构基于ParM的DNA分离（Garner，2007）。本建议的目标是了解ParM介导的质粒运动的分子细节。我们进行这些研究有几个原因，包括：（1）理解ParM依赖的DNA分离提供了对组织原核细胞质的相关系统的深入了解。(2)ParM细丝具有比微管更简单的结构，使其成为研究动态不稳定性分子基础的最佳系统。(3)了解在细菌种群中维持耐药质粒的机制可以帮助我们处理临床耐药的出现。我们将在三个尺度上进行定量研究：（i）单分子和散装 生物化学研究;（ii）重组ParM纺锤体的生物物理学和显微镜研究;和（iii）活细胞中ParM的细胞生物学研究。