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

细胞外围新型分子成分的鉴定：利用 EPEC 基座

• 批准号: 355316-2013
• 负责人: Guttman, Julian
• 金额: $ 3.13万
• 依托单位: Simon Fraser University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2015
• 资助国家: 加拿大
• 起止时间: 2015-01-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=572560
• 关键词: Identification; novel; molecular; components; cell

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.

细胞运动对于生物体发育，伤口愈合和组织维持至关重要。 当细胞迁移时，他们会以动态方式使用其内部骨架，称为细胞骨架。细胞骨架由许多成分组成，但到目前为止，对于细胞运动，最重要的是肌动蛋白细胞骨架。肌动蛋白蛋白会形成组装和拆卸的细丝。当与细胞膜处的细胞周围协调时，这些产生事件的力使细胞移动。由于事件的复杂性发生在迁移细胞的前缘，研究人员已经确定了其他基于肌动蛋白的运动系统，这些系统模仿了整个细胞运动过程中发生的动作。一些经常使用的模型系统利用了在传染过程中侵入细胞并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会导致细菌或病毒在其宿主中使用肌动蛋白在其靶细胞中自身运动。我的实验室已经意识到，有一个细菌可以更好地模仿其他细胞运动期间发生的事件。该系统利用致病性大肠杆菌。这些细菌将它们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感染细胞上“浏览”，并提供理想的系统，以鉴定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，细胞的大肠杆菌位置迫使宿主细胞膜成为基座运动事件中不可或缺的参与者，就像整个细胞运动过程中一样。使用以前的NSERC赠款中的资金，我们制定了一种集中大肠杆菌基座的策略，并使用质谱法以识别样品中的121种蛋白质。现在，我们准备确认这种识别并确定大肠杆菌基座和全细胞运动期间的蛋白质功能。我们预计我们的工作将为肌动蛋白/细胞膜相互作用以及与肌动蛋白本身相关并调节其功能的蛋白质提供新的细胞蛋白。