Pascale G Charest
Associate Professor
Associate Professor, BIO5 Institute
Associate Professor, Chemistry and Biochemistry-Sci
Primary Department
(520) 626-2916
Research Interest
Our research focuses on the signal transduction pathways and molecular mechanisms controlling directed cell migration, or chemotaxis, in eukaryotic cells. 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 and Dictyostelium, transmit the chemoattractant signal to other cells by themselves secreting chemoattractants, which increases the number of cells reaching the chemoattractant source.To investigate key mechanisms of signal transduction underlying chemotaxis, we are 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 aim is to understand the molecular foundation of directed cell migration, which is expected to guide the design of efficient anti-metastatic treatments.Our approach is interdisciplinary, in which we combine molecular genetics and proteomics to identify new signaling proteins and pathways involved in the control of chemotaxis, 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


Hamdan, F. F., Rochdi, M. D., Breton, B., Fessart, D., Michaud, D. E., Charest, P. G., Laporte, S. A., & Bouvier, M. (2007). Unraveling G protein-coupled receptor endocytosis pathways using real-time monitoring of agonist-promoted interaction between β-arrestins and AP-2. Journal of Biological Chemistry, 282(40), 29089-29100.

PMID: 17675294;Abstract:

The most widely studied pathway underlying agonist-promoted internalization of G protein-coupled receptors (GPCRs) involves β-arrestin and clathrin-coated pits. However, both β-arrestin- and clathrin-independent processes have also been reported. Classically, the endocytic routes are characterized using pharmacological inhibitors and various dominant negative mutants, resulting sometimes in conflicting results and interpretational difficulties. Here, taking advantage of the fact that β-arrestin binding to the β2 subunit of the clathrin adaptor AP-2 (β2-adaptin) is needed for the β-arrestin-mediated targeting of GPCRs to clathrin-coated pits, we developed a bioluminescence resonance energy transfer-based approach directly assessing the molecular steps involved in the endocytosis of GPCRs in living cells. For 10 of the 12 receptors tested, including some that were previously suggested to internalize via clathrin-independent pathways, agonist stimulation promoted β-arrestin 1 and 2 interaction with β2-adaptin, indicating a β-arrestin-and clathrin-dependent endocytic process. Detailed analyses of β-arrestin interactions with both the receptor and β2-adaptin also allowed us to demonstrate that recruitment of β-arrestins to the receptor and the ensuing conformational changes are the leading events preceding AP-2 engagement and subsequent clathrin-mediated endocytosis. Among the receptors tested, only the endothelin A and B receptors failed to promote interaction between β-arrestins and β2-adaptin. However, both receptors recruited β-arrestins upon agonist stimulation, suggesting a β-arrestin- dependent but clathrin-independent route of internalization for these two receptors. In addition to providing a new tool to dissect the molecular events involved in GPCR endocytosis, the bioluminescence resonance energy transfer-based β-arrestin/β2-adaptin interaction assay represents a novel biosensor to assess receptor activation. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

Takeda, K., Shao, D., Adler, M., Charest, P. G., Loomis, W. F., Levine, H., Groisman, A., Rappel, W., & Firtel, R. A. (2012). Cell biology: Incoherent feedforward control governs adaptation of activated ras in a eukaryotic chemotaxis pathway. Science Signaling, 5(205).

PMID: 22215733;PMCID: PMC3928814;Abstract:

Adaptation in signaling systems, during which the output returns to a fixed baseline after a change in the input, often involves negative feedback loops and plays a crucial role in eukaryotic chemotaxis. We determined the dynamical response to a uniform change in chemoattractant concentration of a eukaryotic chemotaxis pathway immediately downstream from G protein - coupled receptors. The response of an activated Ras showed near-perfect adaptation, leading us to attempt to fit the results using mathematical models for the two possible simple network topologies that can provide perfect adaptation. Only the incoherent feedforward network accurately described the experimental results. This analysis revealed that adaptation in this Ras pathway is achieved through the proportional activation of upstream components and not through negative feedback loops. Furthermore, these results are consistent with a local excitation, global inhibition mechanism for gradient sensing, possibly with a Ras guanosine triphosphatase - activating protein acting as a global inhibitor.

Charest, P. G., & Firtel, R. A. (2010). TORCing Neutrophil Chemotaxis. Developmental Cell, 19(6), 795-796.

PMID: 21145496;PMCID: PMC3033560;Abstract:

During cell migration, chemoattractant-induced signaling pathways determine the direction of movement by controlling the spatiotemporal dynamics of cytoskeletal components. In this issue of Developmental Cell, Liu et al. report that the target of rapamycin complex 2 (TORC2) controls cell polarity and chemotaxis through regulation of both F-actin and myosin II in migrating neutrophils. © 2010 Elsevier Inc.

Sasaki, A. T., Janetopoulos, C., Lee, S., Charest, P. G., Takeda, K., Sundheimer, L. W., Meili, R., Devreotes, P. N., & Firtel, R. A. (2007). G protein-independent Ras/PI3K/F-actin circuit regulates basic cell motility. Journal of Cell Biology, 178(2), 185-191.

PMID: 17635933;PMCID: PMC2064438;Abstract:

Phosphoinositide 3-kinase (PI3K)γ and Dictyostelium PI3K are activated via G protein-coupled receptors through binding to the Gβγ subunit and Ras. However, the mechanistic role(s) of Gβγ and Ras in PI3K activation remains elusive. Furthermore, the dynamics and function of PI3K activation in the absence of extracellular stimuli have not been fully investigated. We report that gβ null cells display PI3K and Ras activation, as well as the reciprocal localization of PI3K and PTEN, which lead to local accumulation of PI(3,4,5)P3. Simultaneous imaging analysis reveals that in the absence of extracellular stimuli, autonomous PI3K and Ras activation occur, concurrently, at the same sites where F-actin projection emerges. The loss of PI3K binding to Ras - guanosine triphosphate abolishes this PI3K activation, whereas prevention of PI3K activity suppresses autonomous Ras activation, suggesting that PI3K and Ras form a positive feedback circuit. This circuit is associated with both random cell migration and cytokinesis and may have initially evolved to control stochastic changes in the cytoskeleton. © The Rockefeller University Press.

Charest, P. G., Oligny-Longpré, G., Bonin, H., Azzi, M., & Bouvier, M. (2007). The V2 vasopressin receptor stimulates ERK1/2 activity independently of heterotrimeric G protein signalling. Cellular Signalling, 19(1), 32-41.

PMID: 16857342;Abstract:

The V2 vasopressin receptor (V2R) activates the mitogen activated protein kinases (MAPK) ERK1/2 through a mechanism involving the scaffolding protein βarrestin. Here we report that this activating pathway is independent of Gαs, Gαi, Gαq or Gβγ and that the V2R-mediated activation of Gαs inhibits ERK1/2 activity in a cAMP/PKA-dependent manner. In the HEK293 cells studied, the βarrestin-promoted activation was found to dominate over the PKA-mediated inhibition of the pathway, leading to a strong vasopressin-stimulated ERK1/2 activation. Despite the strong MAPK activation and in contrast with other GPCR, V2R did not induce any significant increase in DNA synthesis, consistent with the notion that the stable interaction between V2R and βarrestin prevents signal propagation to the nucleus. βarrestin was found to be essential for the ERK1/2 activation, indicating that the recruitment of the scaffolding protein is necessary and sufficient to initiate the signal in the absence of any other stimulatory cues. Based on the use of selective pharmacological inhibitors, dominant negative mutants and siRNA, we conclude that the βarrestin-dependent activation of ERK1/2 by the V2R involves c-Src and a metalloproteinase-dependent trans-activation event. These findings demonstrate that βarrestin is a genuine signalling initiator that can, on its own, engage a MAPK activation machinery upon stimulation of a GPCR by its natural ligand. © 2006 Elsevier Inc. All rights reserved.