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

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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.