ANALYSIS OF MEMBRANE-BINDING PROTEIN MOBILITY IN LIVING CELLS USING TOTAL INTERNAL REFLECTION FLUORESCENCE CORRELATION SPECTROSCOPY

Total internal reflection fluorescence correlation spectroscopy (TIR-FCS) is an appropriate method for measuring diffusion constants and the number of fluorescent molecules very close to the coverglass surface. Recently, we have reported the application of TIR-FCS to cell biology, measuring membrane-binding farnesylated green fluorescent proteins (EGFP-F) in living cells. In this research, we measured the signal transduction molecule, protein kinase C (PKC), fused with EGFP in living HeLa cells by using TIR-FCS. We observed two different diffusional mobilities of PKCβII-EGFP, three-dimensional faster diffusion near the plasma membrane and slower lateral diffusion on the plasma membrane after adinosine tri phosphate (ATP) activation. These results indicate that it is possible to use TIR-FCS in the study of molecular dynamics and interactions of signal transduction proteins on the plasma membrane of the living cell.