Assaying Chemotaxis of Dictyostelium Cells

  • Michelle C. Mendoza
  • Richard A. Firtel
Part of the Methods in Molecular Biology™ book series (MIMB, volume 346)

Abstract

Both prokaryote and eukaryote cells can sense and move up chemical concentration gradients (chemotax). As a means of finding food sources during vegetative growth, Dictyostelium discoideum naturally chemotaxes toward chemicals released by bacteria. As part of its developmental life cycle, D. discoideum chemotaxes towards cAMP. This chapter describes protocols for using Dictyostelium to understand the cell biology behind and the signaling events necessary for eukaryotic amoeboid chemotaxis. The chapter includes analyses of random cell motility, directed motility up chemical gradients, cellular responses to uniform chemoattractant exposure, and the utility of fluorescent probes for chemotaxis signaling events. Random cell motility in the absence of chemoattractant is analyzed to decipher the properties of self-organizing pseudopodia extension and retraction. Monitoring chemotaxis toward cAMP and folate allows the determination of signaling events required for sensing a chemical gradient and moving in a directed, persistent manner up the gradient. Uniform chemoattractant exposure is employed to elucidate the immediate intracellular responses to chemoattractant stimulation. Finally, analyzing cells expressing fluorescent fusion proteins is vital to elucidating the location of signaling events during chemotaxis.

Key Words

cAMP amoeba chemotaxis Dictyostelium folate fluorescent probes 

References

  1. 1.
    Parent, C. A. (2004) Making all the right moves: chemotaxis in neutrophils and Dictyostelium. Curr. Opin. Cell Biol. 16, 4–13.PubMedCrossRefGoogle Scholar
  2. 2.
    Friedl, P., Borgmann, S., and Brocker, E. B. (2001) Amoeboid leukocyte crawling through extracellular matrix: lessons from the Dictyostelium paradigm of cell movement. J. Leukoc. Biol. 70, 491–509.PubMedGoogle Scholar
  3. 3.
    Soll, D. R., Wessels, D., Heid, P. J., and Zhang, H. (2002) A contextual framework for characterizing motility and chemotaxis mutants in Dictyostelium discoideum. J. Muscle Res. Cell. Motil. 23, 659–672.PubMedCrossRefGoogle Scholar
  4. 4.
    Funamoto, S., Milan, K., Meili, R., and Firtel, R. A. (2001) Role of phosphatidylinositol 3′ kinase and a downstream pleckstrin homology domain-containing protein in controlling chemotaxis in Dictyostelium. J. Cell Biol. 153, 795–809.PubMedCrossRefGoogle Scholar
  5. 5.
    Funamoto, S., Meili, R., Lee, S., Parry, L., and Firtel, R. A. (2002) Spatial and temporal regulation of 3-phosphoinositides by PI 3-kinase and PTEN mediates chemotaxis. Cell 109, 611–623.PubMedCrossRefGoogle Scholar
  6. 6.
    Meili, R., Ellsworth, C., Lee, S., Reddy, T. B., Ma, H., and Firtel, R. A. (1999) Chemoattractant-mediated transient activation and membrane localization of Akt/PKB is required for efficient chemotaxis to cAMP in Dictyostelium. EMBO J. 18, 2092–2105.PubMedCrossRefGoogle Scholar
  7. 7.
    Iijima, M. and Devreotes, P. (2002) Tumor suppressor PTEN mediates sensing of chemoattractant gradients. Cell 109, 599–610.PubMedCrossRefGoogle Scholar
  8. 8.
    Parent, C. A., Blacklock, B. J., Froehlich, W. M., Murphy, D. B., and Devreotes, P. N. (1998) G protein signaling events are activated at the leading edge of chemotactic cells. Cell 95, 81–91.PubMedCrossRefGoogle Scholar
  9. 9.
    Aubry, L. and Firtel, R. (1999) Integration of signaling networks that regulate Dictyostelium differentiation. Annu. Rev. Cell. Dev. Biol. 15, 469–517.PubMedCrossRefGoogle Scholar
  10. 10.
    Manahan, C. L., Iglesias, P. A., Long, Y., and Devreotes, P. N. (2004) Chemoattractant signaling in Dictyostelium discoideum. Annu. Rev. Cell. Dev. Biol. 20, 223–253.PubMedCrossRefGoogle Scholar
  11. 11.
    Insall, R. H., Soede, R. D., Schaap, P., and Devreotes, P. N. (1994) Two cAMP receptors activate common signaling pathways in Dictyostelium. Mol. Biol. Cell 5, 703–711.PubMedGoogle Scholar
  12. 12.
    Lim, C. J., Zawadzki, K. A., Khosla, M., Secko, D. M., Spiegelman, G. B., and Weeks, G. (2005) Loss of the Dictyostelium RasC protein alters vegetative cell size, motility and endocytosis. Exp. Cell Res. 306, 47–55.PubMedCrossRefGoogle Scholar
  13. 13.
    Wessels, D. and Soll, D. R. (1998) Computer-assisted characterization of the behavioral defects of cytoskeletal mutants of Dictyostelium discoideum, in Motion Analysis of Living Cells (Soll, D. R. and Wessels, D., eds.), Wiley-Liss, New York: pp. 101–140.Google Scholar
  14. 14.
    Postma, M., Roelofs, J., Goedhart, J., Gadella, T. W., Visser, A. J., and Van Haastert, P. J. (2003) Uniform cAMP stimulation of Dictyostelium cells induces localized patches of signal transduction and pseudopodia. Mol. Biol. Cell 14, 5019–5027.PubMedCrossRefGoogle Scholar
  15. 15.
    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.PubMedCrossRefGoogle Scholar
  16. 16.
    Jin, T., Zhang, N., Long, Y., Parent, C. A., and Devreotes, P. N. (2000) Localization of the G protein betagamma complex in living cells during chemotaxis. Science 287, 1034–1036.PubMedCrossRefGoogle Scholar
  17. 17.
    Robinson, D. N., Girard, K. D., Octtaviani, E., and Reichl, E. M. (2002) Dictyostelium cytokinesis: from molecules to mechanics. J. Muscle Res. Cell. Motil. 23, 719–727.PubMedCrossRefGoogle Scholar
  18. 18.
    de la Roche, M. A., Smith, J. L., Betapudi, V., Egelhoff, T. T., and Cote, G. P. (2002) Signaling pathways regulating Dictyostelium myosin II. J. Muscle Res. Cell. Motil. 23, 703–718.PubMedCrossRefGoogle Scholar
  19. 19.
    Wessels, D., Schroeder, N. A., Voss, E., Hall, A. L., Condeelis, J., and Soll, D. R. (1989) cAMP-mediated inhibition of intracellular particle movement and actin reorganization in Dictyostelium. J. Cell Biol. 109, 2841–2851.PubMedCrossRefGoogle Scholar
  20. 20.
    Sarbassov, D. D., Guertin, D. A., Ali, S. M., and Sabatini, D. M. (2005) Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science 307, 1098–1101.PubMedCrossRefGoogle Scholar
  21. 21.
    Lee, S., Comer, F. I., Sasaki, A., McLeod, I. X., Duong, Y., Okumura, K., Yates Iii, J. R., Parent, C. A., and Firtel, R. A. (2005) TOR complex 2 integrates cell movement during chemotaxis and signal relay in Dictyostelium. Mol. Biol. Cell 16, 4572–4583.PubMedCrossRefGoogle Scholar
  22. 22.
    Potma, E., de Boeij, W. P., van Haastert, P. J., and Wiersma, D. A. (2001) Realtime visualization of intracellular hydrodynamics in single living cells. Proc. Natl. Acad. Sci. USA 98, 1577–1582.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2006

Authors and Affiliations

  • Michelle C. Mendoza
    • 1
  • Richard A. Firtel
    • 1
  1. 1.Section of Cell and Developmental Biology, Division of Biological Sciences and Center for Molecular GeneticsUniversity of CaliforniaSan Diego, La JollaUSA

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