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Methods Mol Biol. 2009;571:417-35. doi: 10.1007/978-1-60761-198-1_28.

Single-molecule imaging techniques to visualize chemotactic signaling events on the membrane of living Dictyostelium cells.

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Laboratories for Nanobiology, Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan.


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.

[Indexed for MEDLINE]

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