Development of single-molecule functional imaging microscopy and its applications to intracellular signal transduction

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We have developed a novel microscope to measure optical and electrical signals simultaneously from a single protein incorporated into artificial or bio membranes. Using this microscope, we measured lateral movement and ion transport activity of a single ion channel molecule and signal transduction of a G-protein coupled receptor (GPCR) in living cells. During the period of this grant, we obtained the following achievements.(1) Simultaneous measurement of optical and electrical signal of single ion channels : we have developed single channel recording apparatus that can be equipped with a single-molecule imaging microscope. The artificial membranes were formed horizontally in an aqueous environment or on thin agarose layer. Single molecules in the bilayer were observed under an epi-fluorescence or an objective-type total internal reflection fluorescence microscope. Using this microscope, lateral diffusion movement of single alamethicin, Ca-activated K channel and cardiac ryanodine receptor was observed simultaneously with their single-channel gating fluctuation.(2) Single-molecule imaging of a GPCR in living cells : A cAMP receptor for chemotaxis of Dictyostelium cells, CAR1 and a cytoplasmic protein, CRAC working downstream of CAR1 were fused with GFP and observed in living cells in single-molecules. Binding of cAMP conjugated with a fluorophore Cy3 and CAR1 was analyzed in single-molecules. After concentration jump of cAMP using flash photolysis of caged cAMP, the affinity between Cy3-cAMP and CAR1 was transiently increased suggesting a positive feedback loop of the binding of cAMP and CAR1.