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F. Hayashi; Single–Molecule Tracking of Rhodopsin in Disc Membrane of Frog Rod Photoreceptor . Invest. Ophthalmol. Vis. Sci. 2005;46(13):4780.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To characterize the motion of rhodopsin in the disc membrane of frog rod photoreceptor outer segment (ROS). Methods: I used a total internal reflection (TIR) fluorescence microscope for single molecule observation of rhodopsin on a disc membrane. Fab’ fragment of anti–rhodopsin monoclonal antibody (Rho1D4; against C–terminal 1'∼8') was labeled with a near infra–red dye Cy7–maleimide. A low concentration of Cy7–Fab’ was applied to the fragments of ROS (stack of 20∼40 discs) prepared in the darkness. Specimens were illuminated with an Evanescence field of 690 nm in wave length on a cover glass placed on an objective lens (Plan Apo TIRF 100x), and images were acquired in a HD through an impactron–CCD camera at video rate. Experiments were done in a pseudo–intracellular medium containing GTP, and oxygen–scavenger system. Single fluorophore tracking (SFT) experiments were done on a surface of disc membrane exposed at a bottom of ROS fragment standing perpedicularly to the cover glass. When IgG was used for observation of rhodopsin dimer, Zenon Alexa Fluor 700 IgG–labeling system was employed. Results: Both Cy7–Fab’ and Alexa Fluor 700–labeled IgG were observed exclusively on the disc membrane, and they were moving laterally in the disc membrane in an apparently random manner. So far, the effect of visible light to the motion of rhodopsin has not been confirmed. In case of Fab’, the mean square displacement (MSD) vs. time interval plots often showed an initial straight line (0–500 msec) and successive saturation, indicating that rhodopsin behaves as a Brownian–particle in a confined region in the disc membrane. However, some populations of fluorophore showed initial straight lines in longer time ranges. Microscopic diffusion coefficient (Dmicro) obtained from initial slope of MSD vs. time interval plots at 20°C distributed in a wide range from 0.02 – 0.4 µm2/sec, and the mean was 0.2 µm2/sec. In contrast to Fab’, labeled IgG showed much smaller Dmicro; the mean was 0.04 µm2/sec. Conclusions: Single molecular motion of rhodopsin on the disc membrane could be monitored in terms of TIR–fluorescence microscopy. The size of the average Dmicro of rhodopsin in SFT experiments was comparable with the macroscopic diffusion coefficient deduced from classical FRAP or microspectrophotometric experiments. However, MSD vs. time interval plots of individual rhodopsin indicated that there are wide varieties in mode of motion of rhodopsin in the disc membrane. In contrast to Fab’, labeled IgG revealed very low Dmicro, suggesting that the dimerization of rhodopsin reduces the mobility of rhodopsin in the disc membrane that has been thought to be highly congested with themselves.
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