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

发生在细胞周围的亚细胞分子事件对器官发育、维持细胞和组织以及愈合至关重要。细胞内蛋白质“正常”功能的失常常常导致疾病的表现。细胞骨架（细胞骨架）是基于丝状蛋白结构的动态分组。在大多数细胞中，这些成分中研究最突出的是肌动蛋白丝。这些纤维是关键的亚细胞元件，参与整个细胞的运动，细胞内颗粒的运输，以及分子从细胞外到细胞内的内化。在细菌与细胞相互作用的过程中，这些生物经常为了自己的目的而征用细胞。其中一种细菌，肠致病性大肠杆菌（EPEC），留在宿主细胞外，注入细菌蛋白来控制宿主细胞的功能。这些细菌不断与细胞周围相互作用，并在细菌/宿主细胞接触点产生富含肌动蛋白的结构（称为“基座”）。在这些位点鉴定的蛋白质是正常细胞功能的必要因素。为了识别细胞周围的新蛋白质，我们将检查EPEC产生的基座，并将其作为模型系统加以利用。由于基座的长度均匀且相对较大，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。新发现的蛋白质将广泛表征基座和细胞外围的运动和不运动的细胞。通过了解细胞周围的分子成分，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，并将为各种疾病的检查提供靶点。

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.