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.
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Miyanaga, Y., Matsuoka, S., Ueda, M. (2009). Single-Molecule Imaging Techniques to Visualize Chemotactic Signaling Events on the Membrane of Living Dictyostelium Cells. 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_28
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DOI: https://doi.org/10.1007/978-1-60761-198-1_28
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