Caulobacter cell shape and cytoskeletal regulation

柄杆菌细胞形状和细胞骨架调节

基本信息

批准号：
8723866
负责人：
Zemer Gitai
金额：
$ 36.89万
依托单位：
PRINCETON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-01 至 2017-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8723866
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 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 public health relevance response retinal rods small molecule tool

项目摘要

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）：所有细胞都具有确保它们形成具有选择性适应性优势的特征形状的分子机制。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，形成促进运动和宿主入侵的杆状形状。介导细菌细胞形状决定的蛋白质已广为人知，并且有大量证据表明这些肽聚糖组装和图案化蛋白质必须受到高度调控。例如，不同的物种调节相同的保守形状决定因素来建立不同的形状，例如弯曲或螺旋杆。即使在单个细胞内，细胞形状决定机制在细胞周期的不同阶段和不同的代谢状态下也有不同的功能。尽管调节细胞形状至关重要，但人们对不同环境下调节肽聚糖组装蛋白的机制知之甚少。为了了解细胞形状蛋白的调节，我们正在利用新月柄杆菌的独特特征，新月柄杆菌是一种革兰氏阴性弯曲杆，具有易于同步的细胞周期和丰富的遗传和基因组工具包。我们提出了三个独立的目标来剖析细胞形状调节。首先，我们将仔细定量 MreBCD、PBP1、PBP2 和 RodAZ 细胞延伸蛋白的动态。我们将确定这些动态如何相互调节、通过细胞周期进展以及通过已知的细胞周期调节剂进行调节。我们还将对曲率突变体中的伸长蛋白动力学进行成像，以了解伸长和曲率机制如何相互关联。其次，我们将使用我们之前开发的高通量筛选工具以及 MreB 组装的体内和体外测定来识别和表征新型 MreB 调节剂。这些工作将包括表征我们最近鉴定的两种 MreB 调节因子 MbiA 和 AimB 的作用机制，以及鉴定调节 MreB 的新型蛋白质。第三，我们将利用最近发现的 CtpS 作为双功能代谢酶和细胞形状调节剂来定义形状和代谢如何共同调节。具体来说，我们将确定代谢物水平如何影响 CtpS 组装和形状、CtpS 如何与另一种决定曲率的聚合物新月蛋白相互作用，以及形状与代谢状态耦合的选择性益处。我们的努力将首次全面了解细胞生长如何在不同状态下受到调节以及它如何与其他细胞过程（例如分裂和代谢）耦合。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，决定形状的细菌细胞壁是抗生素药物最常见的靶标之一。因此，我们的工作将有助于开发下一代抗生素，以补充因现有药物耐药性增加而迅速耗尽的治疗库。