Identification of novel molecular components at the cell periphery: Taking advantage of EPEC pedestals

细胞运动对于有机体发育、伤口愈合和组织维护至关重要。当细胞迁移时，它们以动态方式使用称为细胞骨架的内部骨架。细胞骨架由许多组件组成，但迄今为止对细胞运动最重要的是肌动蛋白细胞骨架。肌动蛋白形成可组装和分解的细丝。当与细胞膜上的细胞外围协调时，这些力产生事件使细胞能够移动。由于迁移细胞前沿发生的事件的复杂性，研究人员已经确定了其他基于肌动蛋白的运动系统，可以模仿整个细胞运动过程中发生的动作。一些最常用的模型系统利用侵入细胞并在感染过程中劫持这些细胞的肌动蛋白细胞骨架的微生物。肌动蛋白细胞骨架的控制导致细菌或病毒利用宿主内的肌动蛋白来实现其在靶细胞内的运动。我的实验室已经意识到，有一种细菌比其他细菌能更好地模仿细胞运动过程中发生的事件。该系统利用致病性大肠杆菌。这些细菌停靠在它们感染的细胞外部，并在大肠杆菌附着区域产生称为“肌动蛋白基座”的小结构。这些基座使细菌能够在受感染的细胞顶部“冲浪”，并提供一个理想的系统来识别基于肌动蛋白的细胞运动过程中使用的新型蛋白质。与其他侵入性微生物系统不同，大肠杆菌位于细胞外，迫使宿主细胞膜成为基座运动事件中不可或缺的参与者，就像整个细胞运动期间发生的那样。利用我们之前 NSERC 资助的资金，我们制定了一种浓缩大肠杆菌基座的策略，并使用质谱法鉴定了样品中的 121 种蛋白质。现在我们准备确认这一鉴定并确定大肠杆菌底座和全细胞运动过程中的蛋白质功能。我们期望我们的工作将提供参与肌动蛋白/细胞膜相互作用的新细胞蛋白，以及与肌动蛋白本身相关并调节其功能的蛋白质。

Cellular movement is crucial for organism development, wound healing and tissue maintenance. When cells migrate, they use their internal skeleton, called the cytoskeleton, in dynamic ways. The cytoskeleton is made-up of a number of components, but by far the most important for cell motility is the actin cytoskeleton. Actin proteins form filaments that assemble and disassemble. When coordinated with the periphery of the cell at the cell membrane these force generating events enable the cell to move. Because of the complexity of the events occurring at the leading edge of a migrating cell, researchers have identified other actin-based motility systems that mimic the actions occurring during whole cell movement. Some of the most often used model systems exploit microbes that invade cells and hijack the actin cytoskeleton of those cells during their infectious processes. The control of the actin cytoskeleton causes the bacteria or viruses to use the actin within their hosts for their own motility within their target cells. My lab has realized that there is a bacterium that better mimics the events occurring during cell motility over others. This system utilizes pathogenic E. coli. These bacteria dock onto the outside of the cells that they infect and generate small structures referred to as "actin pedestals" at regions of E. coli attachment. These pedestals enable the bacteria to "surf" atop the infected cells and provide an ideal system to identify novel proteins used during actin-based cellular motility. Unlike the other invasive microbial systems, the location of E. coli outide of the cells forces the host cell membrane to be an integral player in the pedestal motility events, as occurs during whole cell movement. Using the funds from our previous NSERC grant we developed a strategy to concentrate E. coli pedestals and used mass spectrometry to identify 121 proteins within our samples. Now we are poised to confirm this identification and determine the protein functions at E. coli pedestals and during whole cell motility. We expect that our work will provide new cellular proteins involved in actin/cell membrane interactions as well as proteins that associate with actin itself and regulate its functions.