Identification and functional characterization of novel molecular components involved in cell motility: Taking advantage of bacterial pathogens

向公众提出的建议摘要***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，这些产生力的作用使细胞能够移动。由于迁移细胞前端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进入细胞的微生物，并在感染过程中劫持这些细胞的肌动蛋白细胞骨架。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进行自身运动。其中一种微生物，即致病性大肠杆菌，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”的小型结构。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蛋白质。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，并使用质谱法来鉴定超过 90 种从未在这些位点鉴定过的蛋白质，我们已经开始表征这些蛋白质的过程，以确定它们在各种富含肌动蛋白的结构和全细胞运动过程中的功能。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。

Summary of proposal to the public ***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 actions enable the cell to move. Because of the complexity of the events occurring at the front 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 enter 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 movement. One of these microbes, pathogenic E. coli, 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 new proteins used during actin-based cellular motility. Using the funds from our previous NSERC grant we developed a strategy to concentrate E. coli pedestals and used mass spectrometry to identify >90 proteins that had never been identified at these sites, we have begun the process of characterizing these proteins to determine their functions at various actin-rich structures 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.