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Natsumi Saito, Fumio Hayashi; Single Molecule Imaging Of Phosphodiesterase 6 On Disc Membrane Of Frog Rod Photoreceptor. Invest. Ophthalmol. Vis. Sci. 2011;52(14):56.
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© ARVO (1962-2015); The Authors (2016-present)
To characterize the motion and distribution of phosphodiesterase 6 (PDE6), the target enzyme for activated transducin, in native disc membrane of frog rod photoreceptor.
Using single molecule imaging technique, we visualized PDE6 on the disc membrane by probing it’s α-subunit (P α) via the fluorescently labeled-Fab’ fragment. Frog (Rana catesbeiana) intact rod outer segments (ROS) were prepared by Percoll density gradient centrifugation in the complete darkness, and truncated by mechanical sharing. The truncated ROS was incubated with a very low concentration of the fluorescently labeled Fab’ fragment against P α, and re-centrifuged in Percoll to remove unbound Fab’. HiLyte Fluor 750-maleimide was used to label the Fab’. Specimens were distributed on a glass surface on an objective type TIRF microscope, and fluorescent spots moving on the disc membrane exposed at the bottom of truncated ROS standing on the glass surface can be observed. The effective diffusion coefficient within 100 ms (D100ms) was calculated from 2-dimensional trajectories of fluorescent spots.
In the dark, one third of PDE6 forms aggregates at the periphery of the disc membrane. The rest of PDE6 undergoes diffusion with the D100ms of wide spectrum from 0.01 to 1 µm2/s. The median of the D100ms of mobile PDE6 is ~ 0.1 µm2/s. Light exposure of the disc membrane induces the dissociation of the aggregates, and increase in the number of mobile molecules of which median value is ~0.2 µm2/s. Upon addition of unhydrolyzable GTP analogue, GTPγS, the D100ms of PDE6 increases drastically to the median value of ~0.6 µm2/s. At the same time, activated PDE6 shows tendency to form aggregates at the periphery of the disc membrane again.
PDE6 changes distribution and diffusivity in the disc membrane dependently on both the light-mediated formation of rhodopsin/transducin-complex (R*/Gt-complex) and PDE6’s own activation induced by GTP-bound (activated) transducin α subunit. Light- and activation-dependent changes of distribution and diffusivity of PDE6 suggest complexity of the regulatory mechanism of PDE6 in vertebrate phototransduction system.
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