Regulation and Function of a Bacterial Cytoskeleton

细菌细胞骨架的调节和功能

• 批准号: 7932461
• 负责人: R DYCHE MULLINS
• 金额: $ 7.12万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7932461
• 关键词: ATP Hydrolysis; Actins; Affect; Bacteria; Binding; Biochemical; Biological Assay; Cell Shape; Cell division; Clinical; Color; Complex; Cytoplasm; Cytoskeleton; DNA; Daughter; Drug resistance; Electron Microscopy; Ensure; Eukaryotic Cell; Filament; Fluorescence Resonance Energy Transfer; Genes; Goals; Homologous Gene; Human; Hydrolysis; In Vitro; Individual; Inherited; Intracellular Space; Kinetics; Knowledge; Measures; Mechanics; Mediating; Methods; Microfilaments; Microscopic; Microtubules; Mitotic spindle; Molecular; Movement; Mutagenesis; Nucleotides; Operon; Plasmids; Polymers; Population; Prokaryotic Cells; Property; Protein Family; Proteins; Regulation; Repressor Proteins; Research Personnel; Stretching; Structure; Structure-Activity Relationship; System; Total Internal Reflection Fluorescent; Work; base; biological systems; cell motility; detector; fluorescence imaging; in vivo; innovation; insight; monomer; particle; pathogen; polymerization; programs; reconstitution; retinal rods; segregation; tool

DESCRIPTION (provided by applicant): The goal of the present proposal is to characterize the structure and assembly dynamics of the prokaryotic actin-like protein, ParM, and to understand how the biochemical properties of ParM and its regulators affect the ability of ParM filaments to find cargo molecules and actively push them to the poles of rod-shaped cells. For these studies we will combine three approaches: (i) in vitro biochemical and biophysical assays on ParM and its regulator the ParR/parC complex, (ii) reconstituted motility assays, and (iii) in vivo studies of filament assembly and cargo movement. The parM gene is part of a partitioning locus (the par operon) found on many low-copy plasmids, such as the R1 and R100 drug-resistance plasmids. To ensure that a copy of the plasmid is inherited by each daughter during cell division, the par operon constructs a simple bipolar spindle from three components: (1) a stretch of centromeric DNA called parC (Dam et al., 1994); (2) a repressor protein, ParR, that binds the parC locus; and (3) the actin-like protein ParM (van den Ent et al., 2002). The ParR/parC complex is thought to harness the force of ParM polymerization to push plasmids through the cytoplasm (Moller-Jensen et al., 2002 and Moller-Jensen et al., 2003). We previously characterized the assembly dynamics of ParM filaments (Garner et al., 2004) and, more recently, reconstituted ParM-based DNA segregation in vitro using purified components (Preliminary Results). To our knowledge, this is the first such reconstitution of any biological system for segregating DNA and it provides a unique opportunity to understand how prokaryotes use cytoskeletal systems to organize their intracellular spaces. For the present study, we propose the following specific aims: 1. Determine the structural and biochemical bases of ParM assembly dynamics. 2. Determine the molecular mechanism by which the ParR/parC complex promotes ParM polymerization. 3. Determine the effect of perturbing the biochemical properties of ParM and the ParR/parC complex on the accuracy and efficiency of plasmid segregation in vivo and in vitro.

描述（由申请人提供）：本提案的目的是表征原核肌动蛋白样蛋白，PARM的结构和组装动力学，并了解PARM及其调节剂的生化特性如何影响Parm细丝的能力，以找到货物分子并将其推向棒状细胞的杆子。在这些研究中，我们将结合三种方法：（i）在PARM及其调节剂的体外生化和生物物理测定parr/parc复合物，（ii）重构运动性测定，以及（iii）对细丝组装和货物运动的体内研究。 PARM基因是在许多低拷贝质粒（例如R1和R100抗药性质粒）上发现的分区基因座（PAR操纵子）的一部分。为了确保每个女儿在细胞分裂期间遗传质粒的副本，PAR操纵子从三个组件中构造了一个简单的双极主轴：（1）一段称为PARC的中心透明体DNA（Dam等，1994）； （2）结合PARC基因座的阻遏蛋白Parr； （3）肌动蛋白样蛋白parm（van den et al。，2002）。 PARR/PARC复合物被认为可以利用PARM聚合的力以通过细胞质推动质粒（Moller-Jensen等，2002和Moller-Jensen等，2003）。我们先前表征了PARM细丝的组装动力学（Garner等，2004），以及最近使用纯化的成分（初步结果）在体外重构基于PARM的DNA分离。据我们所知，这是任何生物系统隔离DNA的第一个重构，它提供了一个独特的机会来了解原核生物如何使用细胞骨架系统来组织其细胞内空间。对于本研究，我们提出以下具体目的： 1。确定PARM组装动力学的结构和生化基础。 2。确定PARR/PARC复合物促进PARM聚合的分子机制。 3。确定PARM和PARR/PARC复合物的生化特性对体内和体外质粒分离的准确性和效率的影响。