Indentification of novel molecular components at the cell periphery: taking advantage of EPEC pedestals

发生在细胞外围的亚细胞分子事件对于器官发育、维持细胞和组织以及愈合至关重要。 细胞内蛋白质“正常”功能的异常往往导致疾病的表现。细胞骨架（cytoskeleton）是一组动态的丝状蛋白质结构。 在大多数细胞中，这些成分中最突出的研究是肌动蛋白丝。 这些细丝是参与整个细胞运动、细胞内颗粒运输以及分子从细胞外内化到细胞内部的关键亚细胞元件。 在细菌与细胞的相互作用中，这些生物体经常为了自己的目的而征用细胞。 这些细菌之一，肠致病性大肠杆菌（EPEC），留在宿主细胞外，并注入细菌蛋白质来控制宿主细胞的功能。 这些细菌不断与细胞周边相互作用，并在细菌/宿主细胞接触点产生富含肌动蛋白的结构（称为“肌动蛋白”）。 在这些位点鉴定的蛋白质是正常细胞功能的必需因子。 为了在细胞外围识别新的蛋白质，我们将检查EPEC产生的蛋白质，并利用它们作为模型系统。 由于蛋白质具有均匀的长度并且相对较大，因此我们将使用先进的细胞生物学技术来识别和功能确定这些结构中的蛋白质。 新鉴定的蛋白质将在运动细胞和不运动细胞中的细胞壁和细胞周边被广泛表征。 通过了解细胞外围的分子组分，本提案中概述的工作将对正常细胞生理学产生深刻的见解，并将提供在各种疾病期间检查的目标。

The sub-cellular molecular events that occur at the cell periphery are crucial for organ development, maintaining cells and tissues, and healing. Aberrations to the "normal" functions of proteins within cells often lead to the manifestation of diseases. The cellular skeleton (the cytoskeleton) is a dynamic grouping of filamentous protein-based structures. In the majority of cells, the most prominently studied of these components are the actin filaments. These filaments are key sub-cellular elements involved in whole cell movement, transport of particles within cells, and the internalization of molecules from outside of the cell to the cell interior. During bacterial interactions with cells, these organisms often commandeer the cells for their own purposes. One of these bacteria, enteropathogenic E.coli (EPEC), remain outside of the host cells and inject bacterial proteins to control the host cell's functions. These bacteria continuously interact with the cell periphery and generate actin-rich structures (called "pedestals") at the bacteria/host cell contact points. Proteins identified at these sites are essential factors for normal cellular functions. In order to identify new proteins at the cell periphery, we will examine EPEC generated pedestals and exploit them as a model system. As pedestals are of uniform length and are relatively large, we will use advanced cell biological techniques to identify and functionally determine proteins in these structures. Newly identified proteins will be extensively characterized at pedestals and the cell periphery in motile as well as immotile cells. Through understanding the molecular components at the cell periphery, the work outlined in this proposal will generate profound insight into normal cellular physiology and will provide targets to examine during various diseases.