Single-molecule imaging techniques to visualize chemotactic signaling events on the membrane of living Dictyostelium cells.
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.
Miyanaga
Yukihiro
Y
Laboratories for Nanobiology, Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan.
Matsuoka
Satomi
S
Ueda
Masahiro
M
D000818:Animals; D002633:Chemotaxis / Q000502:physiology*; D004023:Dictyostelium / Q000378:metabolism* / Q000502:physiology*; D008856:Microscopy, Fluorescence / Q000379:methods*; D015800:Protozoan Proteins / Q000378:metabolism; D011953:Receptors, Cyclic AMP / Q000378:metabolism*
D015800:Protozoan Proteins; D011953:Receptors, Cyclic AMP; C068125:cyclic AMP receptor cAR1