Intermediate filament cytoskeleton and cell shape in Caulobacter crescentus

描述(申请人提供)：到目前为止，已在人类中鉴定出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简单的单细胞遗传模型系统用于IF研究，人们对IF蛋白在体内的特性知之甚少。我们最近发现的新月体蛋白是一种与IF蛋白高度相似的细菌细胞骨架元件，它提供了一个高度可处理的细菌系统，作为了解IF功能的工具。本项目的目的是阐明新月球菌素导致新月桂杆菌细胞弯曲的机制。具体地说，这将使用三种方法来实现。1)构建一大组新月体蛋白突变体，并对其体内外性质进行表征，将决定制造具有IF性质的细胞骨架元件的结构要求。2)荧光显微镜技术结合定量免疫印迹技术将使新月体蛋白在细胞内的结构、定位和动力学分析成为可能。在细胞周期中发生的变化将通过使用同步的细胞周期群体来确定。此外，新月菌素对细菌细胞壁形状(类似于动物细胞的细胞外基质)的影响将通过确定在新月菌素存在和不存在的情况下的细胞壁生长模式以及通过快速破坏活细胞内的新月球素结构来观察任何即时的形态变化来分析。3)高度保守的分子伴侣DNAK(Hsp70)和肌动蛋白同源物MreB在新月体的组装和功能中的作用将通过在DNAK或MreB丧失功能的情况下通过荧光显微镜和代谢标记观察细胞内新月体的结构、合成和稳定性来确定。共定位、下拉和体外实验将检测DNAK和MreB与新月体蛋白的相互作用。相关：许多人类疾病，从皮肤起泡和肌肉营养不良到脂肪营养不良和过早衰老，都是由中间丝蛋白的异常引起的。新月体蛋白是一种细菌版本的中间丝蛋白，这项研究很可能为深入了解它的人类同行在细胞内组装和功能的方式提供帮助。了解中间纤维功能的基础可能会揭示中间纤维疾病是如何发展的，并提出治疗策略。

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.