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Chemotaxis pp 417-435 | Cite as

Single-Molecule Imaging Techniques to Visualize Chemotactic Signaling Events on the Membrane of Living Dictyostelium Cells

  • Yukihiro Miyanaga
  • Satomi Matsuoka
  • Masahiro Ueda
Protocol
Part of the Methods in Molecular Biology™ book series (MIMB, volume 571)

Summary

In this chapter, we describe methods to monitor signaling events at the single-molecule level on the membrane of living cells by using total internal reflection fluorescence microscopy (TIRFM). The techniques provide a powerful tool for elucidating the stochastic properties of signaling molecules involved in chemotaxis of the cellular slime mold Dictyostelium discoideum. Taking cAMP receptor 1 (cAR1) as an example of a target protein for single-molecule imaging, we describe the experimental setup of TIRFM, a method for labeling cAR1 with a fluorescent dye, and a method for investigating the receptor’s lateral mobility. We discuss how the developmental progression of cells modulates both cAR1 behavior and the phenotypic variability in cAR1 mobility for different cell populations.

Key words:

TIRFM Single molecule cAR1 Lifetime Diffusion Lateral mobility 

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Copyright information

© Humana Press 2009

Authors and Affiliations

  • Yukihiro Miyanaga
    • 1
    • 2
  • Satomi Matsuoka
    • 1
    • 2
  • Masahiro Ueda
    • 1
    • 2
  1. 1.Laboratories for Nanobiology, Graduate School of Frontier BiosciencesOsaka UniversityOsakaJapan
  2. 2.Japan Science and Technology Agency, CRESTOsakaJapan

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