Summary
Many eukaryotic cells can elicit intracellular signaling relays to produce pseudopodia and move up to the chemoattractant gradient (chemotaxis) or move randomly in the absence of extracellular stimuli and nutrients (random movement). A precise spatiotemporal regulation of Ras-GTPases, such as Ras and Rap, is crucial to induce pseudopodia formation and cellular adhesion during the chemotaxis and random movement. Here, we describe biochemical and real-time imaging methods for using Dictyostelium to understand the signaling events important for chemotaxis and random cell movement. The chapter includes (1) a biochemical method to assess Ras and Rap1 activation in response to chemoattractant, (2) an imaging method to detect endogenous Ras and Rap1 activation in moving cells, and (3) a simultaneous imaging method to decipher the precise order and localization of these signaling events. With a combination of powerful Dictyostelium genetics, these methods will facilitate to elucidate a dynamic activation of Ras proteins and their inter relay with other signaling molecules during chemotaxis and random movement.
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References
Sasaki, A. T., and Firtel, R. A. (2006) Regulation of chemotaxis by the orchestrated activation of Ras, PI3K, and TOR. Eur. J. Cell Biol. 85, 873–895.
Janetopoulos, C., and Firtel, R. A. (2008) Directional sensing during chemotaxis. FEBS Lett. 582, 2075–2085.
Pollard, T. D., and Borisy, G. G. (2003) Cellular motility driven by assembly and disassembly of actin filaments. Cell 112, 453–465.
Sasaki, A. T., and Firtel, R. A. (2005) Finding the way: directional sensing and cell polarization through Ras signalling. Novartis Found. Symp. 269, 73–87.
Charest, P. G., and Firtel, R. A. (2007) Big roles for small GTPases in the control of directed cell movement. Biochem. J. 401, 377–390.
Mahadeo, D. C., and Parent, C. A. (2006) Signal relay during the life cycle of Dictyostelium. Curr. Top. Dev. Biol. 73, 115–140.
Willard, S. S., and Devreotes, P. N. (2006) Signaling pathways mediating chemotaxis in the social amoeba, Dictyostelium discoideum. Eur. J. Cell Biol. 85, 897–904.
Cernuda-Morollon, E., and Ridley, A. J. (2006) Rho GTPases and leukocyte adhesion receptor expression and function in endothelial cells. Circ. Res. 98, 757–767.
Van Haastert, P. J., and Devreotes, P. N. (2004) Chemotaxis: signalling the way forward. Nat. Rev. Mol. Cell Biol. 5, 626–634.
Sasaki, A. T., Janetopoulos, C., Lee, S., Charest, P. G., Takeda, K., Sundheimer, L. W., et al. (2007) G protein-independent Ras/PI3K/F-actin circuit regulates basic cell motility. J. Cell Biol. 178, 185–191.
Wessels, D., Soll, D. R., Knecht, D., Loomis, W. F., De Lozanne, A., and Spudich, J. (1988) Cell motility and chemotaxis in Dictyostelium amoebae lacking myosin heavy chain. Dev. Biol. 128, 164–177.
Condeelis, J., Singer, R. H., and Segall, J. E. (2005) The great escape: when cancer cells hijack the genes for chemotaxis and motility. Annu. Rev. Cell Dev. Biol. 21, 695–718.
Mendoza, M. C., and Firtel, R. A. (2006) Assaying chemotaxis of Dictyostelium cells. Methods Mol. Biol. 346, 393–405.
Wicki, A., and Niggli, V. (2001) The Rho/Rho-kinase and the phosphatidylinositol 3-kinase pathways are essential for spontaneous locomotion of Walker 256 carcinosarcoma cells. Int. J. Cancer 91, 763–771.
Hancock, J. F. (2003) Ras proteins: different signals from different locations. Nat. Rev. Mol. Cell Biol. 4, 373–384.
Ehrhardt, A., Ehrhardt, G. R., Guo, X., and Schrader, J. W. (2002) Ras and relatives – job sharing and networking keep an old family together. Exp. Hematol. 30, 1089–1106.
Bos, J. L., Rehmann, H., and Wittinghofer, A. (2007) GEFs and GAPs: critical elements in the control of small G proteins. Cell 129, 865–877.
Downward, J. (2003) Targeting RAS signalling pathways in cancer therapy. Nat. Rev. Cancer 3, 11–22.
Vetter, I. R., and Wittinghofer, A. (2001) The guanine nucleotide-binding switch in three dimensions. Science 294, 1299–1304.
Spoerner, M., Nuehs, A., Ganser, P., Herrmann, C., Wittinghofer, A., and Kalbitzer, H. R. (2005) Conformational states of Ras complexed with the GTP analogue GppNHp or GppCH2p: implications for the interaction with effector proteins. Biochemistry 44, 2225–2236.
Sasaki, A. T., Chun, C., Takeda, K., and Firtel, R. A. (2004) Localized Ras signaling at the leading edge regulates PI3K, cell polarity, and directional cell movement. J. Cell Biol. 167, 505–518.
Bos, J. L. (2005) Linking Rap to cell adhesion. Curr. Opin. Cell Biol. 17, 123–128.
Caron, E. (2003) Cellular functions of the Rap1 GTP-binding protein: a pattern emerges. J. Cell Sci. 116, 435–440.
Bivona, T. G., Wiener, H. H., Ahearn, I. M., Silletti, J., Chiu, V. K., and Philips, M. R. (2004) Rap1 up-regulation and activation on plasma membrane regulates T cell adhesion. J. Cell Biol. 164, 461–470.
Jeon, T. J., Lee, D. J., Merlot, S., Weeks, G., and Firtel, R. A. (2007) Rap1 controls cell adhesion and cell motility through the regulation of myosin II. J. Cell Biol. 176, 1021–1033.
Jeon, T. J., Lee, D. J., Lee, S., Weeks, G., and Firtel, R. A. (2007) Regulation of Rap1 activity by RapGAP1 controls cell adhesion at the front of chemotaxing cells. J. Cell Biol. 179, 833–843.
Taylor, S. J., and Shalloway, D. (1996) Cell cycle-dependent activation of Ras. Curr. Biol. 6, 1621–1627.
Taylor, S. J., Resnick, R. J., and Shalloway, D. (2001) Nonradioactive determination of Ras-GTP levels using activated ras interaction assay. Methods Enzymol. 333, 333–342.
Herrmann, C., Martin, G. A., and Wittinghofer, A. (1995) Quantitative analysis of the complex between p21ras and the Ras-binding domain of the human Raf-1 protein kinase. J. Biol. Chem. 270, 2901–2905.
Chiu, V. K., Bivona, T., Hach, A., Sajous, J. B., Silletti, J., Wiener, H., et al. (2002) Ras signalling on the endoplasmic reticulum and the Golgi. Nat. Cell Biol. 4, 343–350.
Nakamura, T., Aoki, K., and Matsuda, M. (2005) Monitoring spatio-temporal regulation of Ras and Rho GTPase with GFP-based FRET probes. Methods 37, 146–153.
Pertz, O., and Hahn, K. M. (2004) Designing biosensors for Rho family proteins – deciphering the dynamics of Rho family GTPase activation in living cells. J. Cell Sci. 117, 1313–1318.
Kinosita, K., Jr., Itoh, H., Ishiwata, S., Hirano, K., Nishizaka, T., and Hayakawa, T. (1991) Dual-view microscopy with a single camera: real-time imaging of molecular orientations and calcium. J. Cell Biol. 115, 67–73.
Fridman, M., Maruta, H., Gonez, J., Walker, F., Treutlein, H., Zeng, J., and Burgess, A. (2000) Point mutants of c-raf-1 RBD with elevated binding to v-Ha-Ras. J. Biol. Chem. 275, 30363–30371.
Takeda, K., Sasaki, A. T., Ha, H., Seung, H. A., and Firtel, R. A. (2007) Role of phosphatidylinositol 3-kinases in chemotaxis in Dictyostelium. J. Biol. Chem. 282, 11874–11884.
Foukas, L. C., Daniele, N., Ktori, C., Anderson, K. E., Jensen, J., and Shepherd, P. R. (2002) Direct effects of caffeine and theophylline on p110δ and other phosphoinositide 3-kinases. Differential effects on lipid kinase and protein kinase activities. J. Biol. Chem. 277, 37124–37130.
Sarkaria, J. N., Busby, E. C., Tibbetts, R. S., Roos, P., Taya, Y., Karnitz, L. M., and Abraham, R. T. (1999) Inhibition of ATM and ATR kinase activities by the radiosensitizing agent, caffeine. Cancer Res. 59, 4375–4382.
Acknowledgments
We gratefully thank Ms. Jennifer Roth and Mr. Sasson Haviv for the excellent help in preparing this manuscript. This work was supported, in part, by a Japanese Society for the Promotion of Science Research Abroad, a Kanae Foundation Fellowship, and a Genentech Fellowship to A.T. Sasaki and by the grants from the U.S. Public Health Service to the USPHS to R.A. Firtel.
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Sasaki, A.T., Firtel, R.A. (2009). Spatiotemporal Regulation of Ras-GTPases During Chemotaxis. In: Jin, T., Hereld, D. (eds) Chemotaxis. Methods in Molecular Biology™, vol 571. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-198-1_23
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DOI: https://doi.org/10.1007/978-1-60761-198-1_23
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