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

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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.