Intermediate filament cytoskeleton and cell shape in Caulobacter crescentus

描述（由申请人提供）：迄今为止，已在人类中鉴定出67个中间丝（IF）基因。它们中的突变是导致30多种人类疾病的原因，但IF蛋白的体内特性知之甚少，部分原因是功能冗余和缺乏用于IF研究的简单单细胞遗传模型系统。我们最近发现的crescentin，细菌细胞骨架元素与IF蛋白具有高度的相似性，提供了一个非常容易处理的细菌系统作为理解IF功能的工具。本项目的目的是阐明新月菌素引起新月柄杆菌细胞弯曲的机制。具体而言，这将通过三种方法来实现。1)构建大量的新月体突变体并在体外和体内表征它们的性质，将确定制备具有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将使新月体的结构，定位和细胞内的动力学分析。细胞周期期间发生的变化将通过使用同步的细胞周期群体来确定。此外，将通过测定存在和不存在crescentin时的细胞壁生长模式，以及通过快速破坏活细胞内的crescentin结构以观察任何即时形态学变化，分析crescentin对细菌细胞壁形状（类似于动物细胞的细胞外基质）的影响。3)高度保守的分子伴侣DnaK（Hsp70）和肌动蛋白同源物MreB在新月体蛋白组装和功能中的作用将通过在DnaK或MreB功能丧失的条件下使用荧光显微镜和代谢标记观察细胞内新月体蛋白的结构、合成和稳定性来确定。共定位、下拉和体外实验将检查DnaK和MreB与新月体蛋白的相互作用。相关性：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，都是由中间丝蛋白异常引起的。这项对crescentin（一种中间丝蛋白的细菌版本）的研究可能会深入了解其人类对应物在细胞内的组装和功能。了解中间纤维功能的基础可能会揭示中间纤维性疾病是如何发展的，从而提出治疗策略。

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.