Advanced light microscopy methods to study the molecular mechanisms regulating cell adhesion and migration

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Cell migration is central to many fundamental biological processes such as tissue organization, neural development, wound healing and the immune response. During migration cells undergo a cyclic process of: 1) membrane protrusion, 2) formation and stabilization of many adhesions that link the cell to its substrate at the front 3) translocation of the cell in the forward direction, 4) disassembly of adhesions and detachment at the cell rear. Adhesions have to be strong enough to create traction for the cell to proceed forward but not so strong that the cell cannot move. Thus, there must be a tight regulation of adhesion location, formation and disassembly in space and time as the cell moves.Hundreds of proteins are involved in adhesion formation and regulation, but little is known about the molecular mechanisms at play. In this project we will used advanced light microscopy techniques to study the adhesion adaptor protein paxillin and its binding partners. We will measure cell migration, protein distributions and dynamics within adhesions across the cell and interactions between key binding partners that are known to play a role in regulating cell migration. We will focus on kinases and phosphatases that act to turn proteins "on" and "off" as master regulators. These advanced microscopy techniques will allow us to determine new details about the complex molecular mechanisms at play in regulating cell migration. The molecular details we uncover will lead to a new and more refined understanding of cell migration and the quantitative data we generate can be applied to molecular models of cell migration.To watch the cells and measure cellular and protein dynamics we express fusions of fluorescent proteins of interest. However, in the process of imaging live samples over time with fluorescence microscopy there can be photo-toxic side effects. So, we have developed procedures to watch living systems with high spatial and temporal resolution but minimal photo-toxicity. To maintain cell health we will will continue to develop, apply and refine fluorescence illumination light engineering protocols.This project will train two master's students and two summer students each year for a total of at least eight trainees over the course of the funding. These trainees will learn advanced imaging techniques, cell biology, quantitative image processing and analysis, scientific data preparation and presentation, and mentoring other students. This will prepare them for their future careers in academic, corporate or government positions. The cross-discipline nature of the research, the training environment at McGill University in the Department of Physiology and access to state-of-the-art technology in the Advanced BioImaging Facility (ABIF) will ensure these trainees well be uniquely trained and prepared to be leaders in their future positions.