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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.

Cell membranes have the ability to bend and curve, thus providing clathrine-coated pits and plasmalemma caveolae, and facilitating many cell functions such as receptor-mediated endocytosis. On the other hand all intracellular membranes are highly deformable, producing cargo vesicles destined to organelles and plasma membrane. Generation of membrane curvature is currently believed to involve the penetration of amphipathic helix into the cytosolic face of the membrane bilayer, producing an asymmetry between the two membrane leaflets and generating bending and curvature towards the cytosol. Here we show, using thin section and freeze-fracture electron microscopy, that ethidium bromide is able to produce negative curvature toward the cytosol in Candida utilis yeast cells. The curvatures were produced in grooves area, resulting in cup-shaped structures with centrally located groove or in polymorph structures with laterally located grooves; these structures were termed "nanocups". Apparently the curvatures were not able to generate vesicles and tubules, suggesting that they were not involved in intracellular trafficking. Thus besides mechanically- or biologically-produced curvature, we can add chemically-produced curvature the function of which remains to be elucidated.