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.
During chemotaxis, cells coordinate their movements with sensing of the gradient and the paracrine release of chemoattractants (signal relay) during chemotaxis, which promotes the synchronized migration of groups of cells.
Dictyostelium cells migrating towards a chemoattractant released from a micropipette (bottom right).
M. Scavello, A.R. Petlick, R. Ramesh, V.F. Thompson, P. Lotfi, and P.G. Charest. “Protein kinase A spatiotemporally controls chemoattractant signaling pathways and is critical for gradient sensing in Dictyostelium”. J. Cell Sci., 130:1545-1558 (2017).
AFM T. Islam, B.M. Stepanski, and P.G. Charest. “Studying chemoattractant signal transduction dynamics in Dictyostelium by BRET”. Methods Mol. Biol. 1407:63-77 (2016).
A. KhannaSC*, P. LotfiT*, A.J. ChavanP, N.M. MontañoG, P. BolouraniSC, G. WeeksC, Z. ShenSC, S.P. BriggsC, H. PotsSC, P.J.M Van HaastertC, A. KortholtSC, and P.G. Charest. “The small GTPases Ras and Rap1 bind to and control TORC2 activity”. Sci. Rep. 6:25823 (2016). *equal contribution.
K. Sumita, H. Yoshino, M. Sasaki, N. Majd, E.R. Kahoud, H. Takahashi, K. Takeuchi, T. Kuroda, S. Lee, P.G. Charest, K. Takeda, J.M. Asara, R.A. Firtel, D. Anastasiou, and A.T. Sasaki. Degradation of Activated K-Ras Orthologue via K-Ras Specific Lysine Residues is Required for Cytokinesis. J Biol Chem. 289:3950-9 (2014).
V. Kolsch, Z. Shen, S. Lee, K. Plak, P. Lotfi, J. Chang, P.G. Charest, J.L. Romero, T.J. Jeon, A. Kortholt, S.P. Briggs, and R.A. Firtel. Daydreamer, a Ras effector and GSK-3 substrate, is important for directional sensing and cell motility. Mol. Biol. Cell. 24:100-14 (2013).
K. Takeda, D. Shao, M. Adler, P.G. Charest, W.F. Loomis, H. Levine, A. Groisman, W.J. Rappel, and R.A. Firtel. Incoherent feedforward control governs adaptation of activated Ras in eukaryotic chemotaxis pathway. Sci. Signal. 5, ra2 (2012).
I. Hecht, M.L. Skoge, P.G. Charest, E. Ben-Jacob, R.A. Firtel, W.F. Loomis, H. Levine, and W.J. Rappel. Activated membrane patches guide chemotactic cell motility. PLoS Comput. Biol.7(6):e1002044 (2011).
P.G. Charest and R.A. Firtel. "TORCing" neutrophil chemotaxis. Dev. Cell 19(6):795-6 (2010).
P.G. Charest, Z. Shen, A. Lakoduk, A.T. Sasaki, S.P. Briggs and R.A. Firtel. A Ras signaling complex controls the RasC-TORC2 pathway and directed cell migration. Dev. Cell. 18:737-49 (2010).
V. Kölsch, P.G. Charest and R.A. Firtel. “The regulation of cell motility and chemotaxis by phospholipid signaling”. J. Cell Sci. 121(Pt 5):551-9 (2008).
S. Zhang*, P.G. Charest*, and R.A. Firtel. Spatio-temporal Regulation of Ras Activity Provides Directional Sensing. Curr. Biol. 18(20):1587-93 (2008). * Equal authorship.
V. Kolsch, P.G. Charest and R.A. Firtel. The regulation of cell motility and chemotaxis by phospholipid signaling. J. Cell Sci. 121(Pt 5):551-9 (2008).
P.G. Charest and R.A. Firtel. Big roles for small GTPases in the control of directed cell movement.Biochem. J. 401(2):377-90 (2007).
F.F. Hamdan, M.D. Rochdi, B. Breton, D. Fessart, D.E. Michaud, P.G. Charest, S.A. Laporte and M. Bouvier. Unraveling g protein-coupled receptor endocytosis pathways using real-time monitoring of agonist-promoted interaction between beta-arrestins and AP-2. J. Biol. Chem. 282(40):29089-100 (2007).
A.T. Sasaki, C. Janetopoulos, S. Lee, P.G. Charest, K. Takeda, L.W. Sunddheimer, R. Meili, P.N. Devreotes and R.A. Firtel. G Protein-Independent Ras/PI3K/F-Actin Circuit Regulates Basic Cell Motility. J. Cell. Biol. 178(2):185-91 (2007).
P.G. Charest, G. Oligny-Longpre, H. Bonin, M. Azzi and M. Bouvier. The V2 vasopressin receptor stimulates ERK1/2 activity independently of heterotrimeric G protein signalling. Cell. Signal.19(1):32-41 (2007).
P.G. Charest and R.A. Firtel. “Feedback signaling controls leading edge formation during chemotaxis”. Curr. Opin. Genet. Dev. 16(4):339-47 (2006).
P.G. Charest, S. Terrillon and M. Bouvier. “Monitoring agonist-promoted conformational changes of β-arrestin in living cells by intramolecular BRET”. EMBO rep. 6(4): 334-40 (2005).
J. Perroy, S. Pontier, P.G. Charest, M. Aubry and M. Bouvier. “Real-time monitoring of ubiquitination in living cells by BRET”. Nat. Meth. 1(3):203-8 (2004).
P.G. Charest and M.Bouvier. “Palmitoylation of the V2 vasopressin receptor carboxyl tail facilitates βarrestin recruitment leading to efficient receptor endocytosis and ERK1/2 activation”. J. Biol. Chem. 278(42):41541-51 (2003).
M. Azzi, P.G. Charest, S. Angers, M. Bouvier and G. Pineyro. “βArrestin-mediated activation of MAPK by inverse agonists reveals distinct active conformations for GPCRs”. Proc. Natl. Acad. Sci. U S A 100(20):11406-11 (2003).