Intermediate filament cytoskeleton and cell shape in Caulobacter crescentus

新月柄杆菌的中间丝细胞骨架和细胞形状

基本信息

批准号：
7257786
负责人：
Christine Jacobs-Wagner
金额：
$ 27.17万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-08-01 至 2010-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7257786
关键词：
Actins Adopted Affect Amino Acids Animals Appendix Applications Grants Bacteria Biological Biological Assay Biological Models Biology Bulla Caulobacter crescentus Cell Cycle Cell Shape Cell Wall Cell model Cell physiology Cells Classification Co-Immunoprecipitations Communicable Diseases Condition Cytoskeletal Proteins Cytoskeleton Disease Elements Extracellular Matrix Facility Construction Funding Category Filament Fluorescence Microscopy Genetic Goals Growth Homologous Gene Human Immunoblotting In Vitro Intermediate Filament Gene Intermediate Filament Proteins Intermediate Filaments Invasive Knowledge Label Life Lipodystrophy Mammalian Cell Mechanics Metabolic Molecular Molecular Chaperones Morphogenesis Morphology Muscular Dystrophies Mutagenesis Mutation Pattern Personal Satisfaction Phenotype Population Premature aging syndrome Process Property Range Regulation Role Shapes Site Skin Structure Structure-Activity Relationship System Techniques Thinking Virulent base human disease in vitro Assay in vivo insight loss of function mutant novel pathogen research study retinal rods tool

项目摘要

DESCRIPTION (provided by applicant): So far, 67 intermediate filament (IF) genes have been identified in humans. Mutations in them are responsible for over 30 human diseases, yet the in vivo properties of IF proteins are poorly understood, due in part to functional redundancy and a lack of simple, single-cell genetic model systems for IF study. Our recent discovery of crescentin, a bacterial cytoskeletal element with a high degree of similarity with IF proteins, provides a highly tractable bacterial system as a tool for understanding IF function. The objective of this project is to elucidate the mechanism by which crescentin causes cell curvature in Caulobacter crescentus. Specifically, this will be accomplished using three approaches. 1) Construction of a large set of crescentin mutants and characterization of their properties both in vitro and in vivo will determine structural requirements for making a cytoskeletal element with IF properties. 2) Fluorescence microscopy techniques combined with quantitative immunoblotting will enable the analysis of crescentin structure, localization, and dynamics within cells. Changes occurring during the cell cycle will be determined with the use of synchronized cell cycle populations. Additionally, the effect of crescentin on the bacterial cell wall shape (analogous to the extracellular matrix of animal cells) will be analyzed by determining patterns of cell wall growth in the presence and absence of crescentin, and by rapidly disrupting crescentin structure within live cells to observe any immediate morphological changes. 3) The roles of the highly conserved molecular chaperone DnaK (Hsp70) and the actin homolog MreB in crescentin assembly and function will be determined by observing crescentin structure, synthesis and stability within cells using fluorescence microscopy and metabolic labeling under conditions of DnaK or MreB loss-of-function. Co-localization, pull- down and in vitro experiments will examine the interaction of DnaK and MreB with crescentin. RELEVANCE: Many human diseases, ranging from skin blistering and muscular dystrophy to lipodystrophy and premature aging, are caused by abnormalities in intermediate filament proteins. This study of crescentin, a bacterial version of an intermediate filament protein, is likely to provide insight into the way its human counterparts assemble and function within cells. Knowing the basis for intermediate filament function may reveal how intermediate filament-based diseases develop, suggesting treatment strategies.

描述（由申请人提供）：到目前为止，已在人类中鉴定出67个中间细丝（IF）基因。它们中的突变负责30多种人类疾病，但是如果蛋白质了解较低的IF蛋白质的体内特性，部分原因是功能冗余和缺乏简单的单细胞遗传模型系统而用于IF研究。我们最近发现的新月素是一种与IF蛋白质相似的细菌细胞骨架元素，它提供了高度可牵引的细菌系统，作为了解功能是否功能的工具。该项目的目的是阐明新月形在花椰菜菌中引起细胞曲率的机制。具体来说，这将使用三种方法来完成。 1）构建大量新月形突变体及其在体外和体内的特性表征将确定结构性要求，以使具有IF特性的细胞骨架元件。 2）与定量免疫印迹结合使用的荧光显微镜技术将使细胞内的新月形结构，定位和动力学分析。在细胞周期期间发生的变化将通过使用同步细胞周期种群来确定。此外，将通过确定在存在和不存在新月形结构的情况下确定细胞壁生长的模式，并快速破坏活细胞内的新月形结构，以观察到任何直接的形态学变化，从而分析新月素对细菌细胞壁形状的影响（类似于动物细胞的细胞外基质）。 3）高度保守的分子伴侣DNAK（HSP70）和肌动蛋白同源物MREB在新月素组装和功能中的作用将通过在DNAK或MREB损失下使用荧光显微镜和代谢标记来确定细胞内的新月素结构，合成和稳定性。共定位，拉下和体外实验将检查DNAK和MREB与新月的相互作用。相关性：许多人类疾病，从皮肤起泡和肌营养不良症到脂肪营养不良和过早衰老，都是由中间细丝蛋白异常引起的。这项针对中间细丝蛋白的细菌形式的新月素研究很可能会洞悉其人类对应物在细胞中的组装和功能的方式。了解中间细丝功能的基础可能会揭示基于中间细丝的疾病如何发展，并提出治疗策略。