The Charest Lab's research interests
Signalling pathways and molecular mechanisms controlling directed cell migration.
|
Our research focuses on the signal transduction pathways and molecular mechanisms controlling cell migration, and particularly chemotaxis. Chemotaxis is central to many biological processes, including the embryonic development, wound healing, the migration of white blood cells (leukocytes) to sites of inflammation or bacterial infection, as well as the metastasis of cancer cells. Cells can sense chemical gradients that are as shallow as a 2% difference in concentration across the cell, and migrate towards the source of the signal, the chemoattractant. This is achieved through an intricate network of intracellular signaling pathways that are triggered by the chemoattractant signal. These pathways ultimately translate the detected chemoattractant gradient into changes in the cytoskeleton that lead to cell polarization and forward movement. In addition, many cells such as leukocytes, cancer cells, and Dictyostelium, transmit the chemoattractant signal to other cells by themselves secreting chemoattractants, which increases the number of cells reaching the chemoattractant source.
|
Dictyostelium cells migrating towards the chemoattractant cAMP released from a micropipette (bottom right).
|
Our aim is to understand the molecular foundation of directed cell migration to guide the development of efficient anti-metastatic treatments. In particular, we are interested in understanding how chemoattractant receptors promote the polarized activation of chemotactic signaling pathways and remodeling of the cytoskeleton, as well as how cells adapt to spatially and temporally changing concentrations of chemoattractants. Current investigations focus on the role and regulation of key chemoattractant signal transduction pathways, particularly those involving Ras and Rap1 small GTPases, as well as the Target of Rapamycin Complex 2 (TORC2), using the social amoeba Dictyostelium discoideum as well as human cancer cell models. Cell motility and chemotaxis of Dictyostelium cells is very similar to that of leukocytes and cancer cells, using the same underlying cellular processes as these higher eukaryotic cells. Dictyostelium is amenable to cell biological, biochemical, and genetic approaches that are unavailable in more complex systems. The discoveries we make using Dictyostelium are then confirmed in human cells and, in particular, in the context of directed cancer cell migration and metastasis.
Our approach is interdisciplinary, in which we combine molecular genetics and proteomics to identify new signaling proteins and pathways involved in the control of directed cell migration, with live cell imaging using fluorescent reporters to understand the spatiotemporal dynamics of the signaling events, as well as biochemical analyses and proximity assays [including Bioluminescence Resonance Energy Transfer (BRET) and FRET] to understand how proteins interact and function within the signaling network. In addition, in collaboration with Dr. Wouter-Jan Rappel at UC San Diego, we generate quantitative models of the chemotactic signaling networks to help identify key regulatory mechanisms and link them to whole cell behavior.
Our approach is interdisciplinary, in which we combine molecular genetics and proteomics to identify new signaling proteins and pathways involved in the control of directed cell migration, with live cell imaging using fluorescent reporters to understand the spatiotemporal dynamics of the signaling events, as well as biochemical analyses and proximity assays [including Bioluminescence Resonance Energy Transfer (BRET) and FRET] to understand how proteins interact and function within the signaling network. In addition, in collaboration with Dr. Wouter-Jan Rappel at UC San Diego, we generate quantitative models of the chemotactic signaling networks to help identify key regulatory mechanisms and link them to whole cell behavior.
|
4D imaging of a migrating Dictyostelium cell expressing a fluorescent marker (Lifeact-GFP) that labels the cytoskeletal protein F-actin at the leading edge. We use diverse fluorescent markers and activity reporters to analyze the spatiotemporal dynamics of signaling pathways during chemotaxis.
|
BRET between a bioluminescent energy donor (Renilla luciferase; RLuc) and an energy acceptor (variant of the green fluorescent protein; FP) fused to proteins of interests allows detecting protein-protein interactions in live cells with high temporal resolution. We use BRET to study the dynamics of chemoattractant signal transduction pathways.
|
Research Funding
|
Current funding NSF-MCB 2426002 NIH-NIGMS R01 GM131200 NIH-NIGMS T32 GM008804 NIH-NIGMS T34 GM008718 Arnold and Mabel Beckman Foundation Past funding American Cancer Society (127940-RSG-15-024-01-CSM) NIH-T32 GM136536 University of Arizona Foundation UA Technology Research Initiative Funds |
|