Caulobacter cell shape and cytoskeletal regulation

柄杆菌细胞形状和细胞骨架调节

• 批准号: 8860201
• 负责人: Zemer Gitai
• 金额: $ 36.89万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8860201
• 关键词: Actins; Address; Antibiotics; Bacteria; Biochemistry; Biological Assay; Biophysics; Bundling; CTP synthase; Caulobacter; Caulobacter crescentus; Cell Cycle; Cell Cycle Progression; Cell Cycle Stage; Cell Shape; Cell Wall; Cell physiology; Cells; Characteristics; Coupled; Coupling; Cytoskeletal Proteins; Development; Ensure; Enzymes; Escherichia coli; Filament; Future Generations; Genes; Genetic; Genome; Genomics; Goals; Growth; Health; Homologous Gene; Image; Image Analysis; In Vitro; Learning; Length; Mediating; Metabolic; Metabolism; Molecular; Motion; Pattern; Peptidoglycan; Pharmaceutical Preparations; Polymers; Property; Protein Dynamics; Proteins; Regulation; Regulation of Cell Shape; Resistance; Resolution; Shapes; Speed; System; Therapeutic; Work; cell growth; cell motility; fitness; high throughput screening; in vitro Assay; in vivo; mutant; novel; overexpression; polymerization; protein function; protein structure; response; retinal rods; small molecule; tool

DESCRIPTION (provided by applicant): All cells have molecular mechanisms that ensure that they develop characteristic shapes with selective fitness advantages. For example, many bacteria assemble peptidoglycan cell walls in specific spatial and temporal patterns to take on rod-like shapes that promote motility and host invasion. The proteins that mediate bacterial cell-shape determination are largely known and there is substantial evidence that these peptidoglycan assembly and patterning proteins must be highly regulated. For example, different species modulate the same conserved shape determinants to establish different shapes, such as curved or helical rods. Even within a single cell, the cell shape determination machinery functions differently at different stages of the cell cycle and in different metabolic states. Despite the central importance of regulating cell shape, very little is known about the mechanisms that regulate peptidoglycan assembly proteins in different contexts. To understand the regulation of cell shape proteins, we are harnessing the unique features of Caulobacter crescentus, a gram-negative curved rod with a readily synchronized cell cycle and rich genetic and genomic toolkit. We propose three independent aims to dissect cell shape regulation. First, we will carefully quantitate the dynamics of the MreBCD, PBP1, PBP2, and RodAZ cell elongation proteins. We will determine how these dynamics are regulated by one another, by cell cycle progression, and by known cell cycle regulators. We will also image elongation protein dynamics in curvature mutants to understand how the elongation and curvature machineries inter-relate. Second, we will use our previously developed high-throughput screening tools and both in vivo and in vitro assays for MreB assembly to identify and characterize novel MreB regulators. These efforts will include characterizing the mechanisms of action of two MreB regulators that we recently identified, MbiA and AimB, as well as identifying novel proteins that modulate MreB. Third, we will exploit our recent discovery of CtpS as a bifunctional metabolic enzyme and cell shape regulator to define how shape and metabolism are co-regulated. Specifically, we will determine how metabolite levels influence both CtpS assembly and shape, how CtpS interacts with another curvature-determining polymer, crescentin, and the selective benefits of coupling shape to metabolic state. Our efforts will establish the first comprehensive understanding of how cell growth is regulated in different states and how it is coupled to other cellular processes such as division and metabolism. These studies will provide a roadmap for cell shape studies in other systems. Furthermore, the shape-determining bacterial cell wall represents one of the most common targets for antibiotic drugs. Thus, our work will aid in the development of future generations of antibiotics to replenish the therapeutic arsenal that is being rapidly depleted by the rise of resistance to existing drugs.

描述（由申请人提供）：所有细胞均具有分子机制，可确保它们具有具有选择性适应性优势的特征形状。例如，许多细菌以特定的空间和时间模式组装肽聚糖细胞壁，以采用杆状形状，从而促进运动能力并促进宿主侵袭。介导细菌细胞形状测定的蛋白质在很大程度上是已知的，并且有大量证据表明这些肽聚糖的组装和构图蛋白必须高度调节。例如，不同的物种调节相同的保守形状决定因素，以建立不同的形状，例如弯曲或螺旋杆。即使在单个细胞中，细胞形状的测定机制在细胞周期的不同阶段和不同代谢状态下的功能也有所不同。尽管调节细胞形状的重要性，但对在不同情况下调节肽聚糖蛋白的机制知之甚少。为了了解细胞形状蛋白的调节，我们正在利用Caulobacter Crescentus的独特特征，Carobacter Crescentus，一种具有易于同步的细胞周期以及丰富的遗传和基因组工具包的革兰氏阴性弯曲棒。我们提出了三个独立的目标，以剖析细胞形状调节。首先，我们将仔细量化MREBCD，PBP1，PBP2和Rodaz细胞伸长蛋白的动力学。我们将通过细胞周期进程和已知的细胞周期调节剂来确定这些动力学如何相互调节。我们还将在曲率突变体中对伸长蛋白动力学进行图像，以了解伸长率和曲率机器如何相互融合。其次，我们将使用先前开发的高通量筛选工具以及体内和体外测定法，以识别和表征新型MREB调节剂。这些努力将包括表征我们最近确定的两个MREB调节剂的作用机理，以及鉴定调节MREB的新型蛋白质。第三，我们将利用最近发现CTP作为双功能代谢酶和细胞形状调节剂的发现来定义形状和代谢如何共同调节。具体而言，我们将确定代谢物水平如何影响CTPS组装和形状，CTP与另一种曲率确定的聚合物，新月形素以及耦合形状与代谢状态的选择性益处。我们的努力将建立对在不同状态中如何调节细胞生长以及如何与其他细胞过程（例如分裂和代谢等其他细胞过程结合在一起）的第一个全面理解。这些研究将为其他系统中的细胞形状研究提供路线图。此外，确定形状的细菌细胞壁代表了抗生素药物最常见的靶标之一。因此，我们的工作将有助于发展子孙后代的抗生素，以补充因对现有药物的抗药性而迅速消耗的治疗库。