Pascale G Charest
Assistant Professor, BIO5 Institute
Assistant Professor, Chemistry and Biochemistry - Med
Associate Professor
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

Publications

Charest, P. G., Scavello, M., Petlick, A. R., Thompson, V. F., Ramesh, R., & Lotfi, P. (2017). Protein Kinase A spatiotemporally controls chemoattractant signaling pathways and is critical for gradient sensing in Dictyostelium. Journal of Cell Science. doi:10.1242/jcs.177170
Hecht, I., Skoge, M. L., Charest, P. G., Ben-Jacob, E., Firtel, R. A., Loomis, W. F., Levine, H., & Rappel, W. (2011). Activated membrane patches guide chemotactic cell motility. PLoS Computational Biology, 7(6).

PMID: 21738453;PMCID: PMC3127810;Abstract:

Many eukaryotic cells are able to crawl on surfaces and guide their motility based on environmental cues. These cues are interpreted by signaling systems which couple to cell mechanics; indeed membrane protrusions in crawling cells are often accompanied by activated membrane patches, which are localized areas of increased concentration of one or more signaling components. To determine how these patches are related to cell motion, we examine the spatial localization of RasGTP in chemotaxing Dictyostelium discoideum cells under conditions where the vertical extent of the cell was restricted. Quantitative analyses of the data reveal a high degree of spatial correlation between patches of activated Ras and membrane protrusions. Based on these findings, we formulate a model for amoeboid cell motion that consists of two coupled modules. The first module utilizes a recently developed two-component reaction diffusion model that generates transient and localized areas of elevated concentration of one of the components along the membrane. The activated patches determine the location of membrane protrusions (and overall cell motion) that are computed in the second module, which also takes into account the cortical tension and the availability of protrusion resources. We show that our model is able to produce realistic amoeboid-like motion and that our numerical results are consistent with experimentally observed pseudopod dynamics. Specifically, we show that the commonly observed splitting of pseudopods can result directly from the dynamics of the signaling patches. © 2011 Hecht et al.

Charest, P. G., & Firtel, R. A. (2007). Big roles for small GTPases in the control of directed cell movement. Biochemical Journal, 401(2), 377-390.

PMID: 17173542;PMCID: PMC1820805;Abstract:

Small GTPases are involved in the control of diverse cellular behaviours, including cellular growth, differentiation and motility. In addition, recent studies have revealed new roles for small GTPases in the regulation of eukaryotic chemotaxis. Efficient chemotaxis results from co-ordinated chemoattractant gradient sensing, cell polarization and cellular motility, and accumulating data suggest that small GTPase signalling plays a central role in each of these processes as well as in signal relay. The present review summarizes these recent findings, which shed light on the molecular mechanisms by which small GTPases control directed cell migration. © 2007 Biochemical Society.

Sumita, K., Yoshino, H., Sasaki, M., Majd, N., Kahoud, E. R., Takahashi, H., Takeuchi, K., Kuroda, T., Lee, S., Charest, P. G., Takeda, K., Asara, J. M., Firtel, R. A., Anastasiou, D., & Sasaki, A. T. (2014). Degradation of activated K-ras orthologue via K-ras-specific lysine residues is required for cytokinesis. Journal of Biological Chemistry, 289(7), 3950-3959.

Abstract:

Background: Targeting oncogenic K-Ras for cancer therapy has remained challenging. Results: Ubiquitination specifically occurs on the activated K-Ras orthologue in Dictyostelium via evolutionary conserved K-Ras lysines, which promotes K-Ras protein degradation. Conclusion: Our results indicate the existence of GTP-loaded K-Ras orthologue-specific degradation system in Dictyostelium. Significance: This work reveals a novel negative feedback regulation for the K-Ras isoform, which is critical for cytokinesis in Dictyostelium. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc..

Charest, P. G., Shen, Z., Lakoduk, A., Sasaki, A. T., Briggs, S. P., & Firtel, R. A. (2010). A ras signaling complex controls the RasC-TORC2 pathway and directed cell migration. Developmental Cell, 18(5), 737-749.

PMID: 20493808;PMCID: PMC2893887;Abstract:

Ras was found to regulate Dictyostelium chemotaxis, but the mechanisms that spatially and temporally control Ras activity during chemotaxis remain largely unknown. We report the discovery of a Ras signaling complex that includes the Ras guanine exchange factor (RasGEF) Aimless, RasGEFH, protein phosphatase 2A (PP2A), and a scaffold designated Sca1. The Sca1/RasGEF/PP2A complex is recruited to the plasma membrane in a chemoattractant- and F-actin-dependent manner and is enriched at the leading edge of chemotaxing cells where it regulates F-actin dynamics and signal relay by controlling the activation of RasC and the downstream target of rapamycin complex 2 (TORC2)-Akt/protein kinase B (PKB) pathway. In addition, PKB and PKB-related PKBR1 phosphorylate Sca1 and regulate the membrane localization of the Sca1/RasGEF/PP2A complex, and thereby RasC activity, in a negative feedback fashion. Thus, our study uncovered a molecular mechanism whereby RasC activity and the spatiotemporal activation of TORC2 are tightly controlled at the leading edge of chemotaxing cells. © 2010 Elsevier Inc.