Berlin 2012 – wissenschaftliches Programm

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BP: Fachverband Biologische Physik

BP 26: Posters: Membranes and Vesicles

BP 26.7: Poster

Donnerstag, 29. März 2012, 17:30–19:30, Poster A

Dual emission GFP as highly sensitive fluorophore for the determination of intracellular pH with fluorescence lifetime imaging microscopy (FLIM) — •Franz-Josef Schmitt, Cornelia Junghans, Marco Vitali, and Thomas Friedrich — Biophysical Chemistry, Berlin Institute of Technology, Germany

The determination of the pH in the cell cytoplasm or in intracellular organelles is of high relevance in cell biology. During infection with the influenza virus, cells produce the M2 proton channel, which is encoded by the viral genome and represents a crucial component of the viral reproduction cycle. Influenza treatment utilizes drugs like amantadine, which are targeted against the M2 channel. We present a novel multi-parameter FLIM setup that permits the simultaneous imaging of the fluorescence amplitude ratios and lifetimes of a pH-sensitive dual-emission GFP (deGFP) enabling the determination of the activity of a fusion protein of an membrane intrinsic influenza M2 channel with tagRFP (M2-RFP) monitored by deGFP fluorescence. Both proteins (M2-RFP and deGFP) were expressed in chinese hamster ovary cells (CHO-K1) and monitored with spatial resolution of 500 nm in 2 color channels with time resolution of < 100 ps. It was shown that the presence of M2 leads to an acceleration of proton transfer across the cell membrane that is blocked by amantadine. The time-course of M2-dependent intracellular acidification can be described by a general diffusion equation for the intracellular pH in a buffered medium, thus enabling the determination of transversal proton diffusion coefficients in cell membranes.