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

• 批准号: 7477335
• 负责人: Christine Jacobs-Wagner
• 金额: $ 28.03万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 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/7477335
• 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

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，已经在人类中鉴定了67个中间丝（IF）基因。它们的突变导致了30多种人类疾病，但由于功能冗余和缺乏用于中频研究的简单单细胞遗传模型系统，人们对中频蛋白的体内特性知之甚少。我们最近发现的月桂素是一种与IF蛋白高度相似的细菌细胞骨架元素，它为理解IF功能提供了一个高度可处理的细菌系统。本项目旨在阐明月桂素引起月桂茎杆菌细胞弯曲的机制。具体来说，这将通过三种方法来实现。1)大量月桂素突变体的构建及其在体外和体内特性的表征将决定制造具有IF特性的细胞骨架元件的结构要求。2)荧光显微镜技术与定量免疫印迹技术相结合，可以分析月桂素在细胞内的结构、定位和动态。在细胞周期中发生的变化将通过使用同步细胞周期种群来确定。此外，我们将分析月桂素对细菌细胞壁形状（类似于动物细胞的细胞外基质）的影响，方法是确定存在和不存在月桂素时细胞壁生长的模式，并通过快速破坏活细胞内的月桂素结构来观察任何直接的形态变化。3)高度保守的分子伴侣DnaK （Hsp70）和肌动蛋白同源物MreB在月牙素组装和功能中的作用，将在DnaK或MreB失去功能的情况下，通过荧光显微镜和代谢标记观察细胞内月牙素的结构、合成和稳定性来确定。共定位、下拉和体外实验将检验DnaK和MreB与月牙素的相互作用。相关性：许多人类疾病，从皮肤起泡和肌肉萎缩到脂肪营养不良和早衰，都是由中间丝蛋白异常引起的。月桂素是一种细菌版本的中间丝蛋白，这项研究可能会让我们深入了解其人类同类蛋白在细胞内的组装和功能。了解中间丝功能的基础可能揭示中间丝为基础的疾病是如何发展的，并提出治疗策略。