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Chemotaxis pp 333–348Cite as

Spatiotemporal Regulation of Ras-GTPases During Chemotaxis

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Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 571))

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

  1. 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.

    Article  PubMed  CAS  Google Scholar 

  2. Janetopoulos, C., and Firtel, R. A. (2008) Directional sensing during chemotaxis. FEBS Lett. 582, 2075–2085.

    Article  PubMed  CAS  Google Scholar 

  3. Pollard, T. D., and Borisy, G. G. (2003) Cellular motility driven by assembly and disassembly of actin filaments. Cell 112, 453–465.

    Article  PubMed  CAS  Google Scholar 

  4. 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.

    Article  PubMed  CAS  Google Scholar 

  5. 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.

    Article  PubMed  CAS  Google Scholar 

  6. Mahadeo, D. C., and Parent, C. A. (2006) Signal relay during the life cycle of Dictyostelium. Curr. Top. Dev. Biol. 73, 115–140.

    Article  PubMed  CAS  Google Scholar 

  7. 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.

    Article  PubMed  CAS  Google Scholar 

  8. 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.

    Article  PubMed  CAS  Google Scholar 

  9. Van Haastert, P. J., and Devreotes, P. N. (2004) Chemotaxis: signalling the way forward. Nat. Rev. Mol. Cell Biol. 5, 626–634.

    Article  PubMed  Google Scholar 

  10. 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.

    Article  PubMed  CAS  Google Scholar 

  11. 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.

    Article  PubMed  CAS  Google Scholar 

  12. 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.

    Article  PubMed  CAS  Google Scholar 

  13. Mendoza, M. C., and Firtel, R. A. (2006) Assaying chemotaxis of Dictyostelium cells. Methods Mol. Biol. 346, 393–405.

    PubMed  CAS  Google Scholar 

  14. 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.

    Article  PubMed  CAS  Google Scholar 

  15. Hancock, J. F. (2003) Ras proteins: different signals from different locations. Nat. Rev. Mol. Cell Biol. 4, 373–384.

    Article  PubMed  CAS  Google Scholar 

  16. 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.

    Article  PubMed  CAS  Google Scholar 

  17. 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.

    Article  PubMed  CAS  Google Scholar 

  18. Downward, J. (2003) Targeting RAS signalling pathways in cancer therapy. Nat. Rev. Cancer 3, 11–22.

    Article  PubMed  CAS  Google Scholar 

  19. Vetter, I. R., and Wittinghofer, A. (2001) The guanine nucleotide-binding switch in three dimensions. Science 294, 1299–1304.

    Article  PubMed  CAS  Google Scholar 

  20. 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.

    Article  PubMed  CAS  Google Scholar 

  21. 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.

    Article  PubMed  CAS  Google Scholar 

  22. Bos, J. L. (2005) Linking Rap to cell adhesion. Curr. Opin. Cell Biol. 17, 123–128.

    Article  PubMed  CAS  Google Scholar 

  23. Caron, E. (2003) Cellular functions of the Rap1 GTP-binding protein: a pattern emerges. J. Cell Sci. 116, 435–440.

    Article  PubMed  CAS  Google Scholar 

  24. 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.

    Article  PubMed  CAS  Google Scholar 

  25. 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.

    Article  PubMed  CAS  Google Scholar 

  26. 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.

    Article  PubMed  CAS  Google Scholar 

  27. Taylor, S. J., and Shalloway, D. (1996) Cell cycle-dependent activation of Ras. Curr. Biol. 6, 1621–1627.

    Article  PubMed  CAS  Google Scholar 

  28. 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.

    Article  PubMed  CAS  Google Scholar 

  29. 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.

    Article  PubMed  CAS  Google Scholar 

  30. 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.

    PubMed  CAS  Google Scholar 

  31. 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.

    Article  PubMed  CAS  Google Scholar 

  32. 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.

    Article  PubMed  CAS  Google Scholar 

  33. 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.

    Article  PubMed  CAS  Google Scholar 

  34. 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.

    Article  PubMed  CAS  Google Scholar 

  35. 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.

    Article  PubMed  CAS  Google Scholar 

  36. 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.

    Article  PubMed  CAS  Google Scholar 

  37. 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.

    PubMed  CAS  Google Scholar 

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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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Authors and Affiliations

  1. Department of Systems Biology, Harvard Medical School and Division of Signal Transduction, Beth Israel Deaconess Medical Center, Boston, MA, USA

    Atsuo T. Sasaki & Richard A. Firtel

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  1. Atsuo T. Sasaki
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  2. Richard A. Firtel
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Editors and Affiliations

  1. Infectious Diseases (NIAID), National Institute of Allergy and, Parklawn Dr. 12441, Rockville, 20852, U.S.A.

    Tian Jin

  2. National Institute on Alcohol Abuse &, National Institute of Health, Fisher Lane 5635, Bethesda, 20892-9304, U.S.A.

    Dale Hereld

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© 2009 Humana Press

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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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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-60761-197-4

  • Online ISBN: 978-1-60761-198-1

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