Chemotaxis pp 371-383
Monitoring Dynamic GPCR Signaling Events Using Fluorescence Microscopy, FRET Imaging, and Single-Molecule Imaging
How a eukaryotic cell translates a small concentration difference of a chemoattractant across the length of its surface into highly polarized intracellular responses is a fundamental question in chemotaxis. Chemoattractants are detected by G-protein-coupled receptors (GPCRs). Binding of chemoattractants to GPCRs induces the dissociation of heterotrimeric G-proteins into Gα and Gβγ subunits, which in turn, activate downstream signaling networks. To fully understand the molecular mechanisms of chemotaxis, it is essential to quantitatively measure the dynamic changes of chemoattractant concentrations around cells, activation of heterotrimeric G-proteins, and the mobility of GPCR and G-protein subunits in the cell membrane. Here, we outline fluorescence imaging methods including Förster resonance energy transfer (FRET) imaging and a single-molecule analysis that allow us to measure the dynamic properties of GPCR signaling in single live cells.
Key words:Confocal fluorescence microscopy Förster resonance energy transfer Total internal reflection fluorescence microscopy Single-molecule imaging GPCR Heterotrimeric G-proteins Spatiotemporal dynamics