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P.D. Calvert, J.A. Peet, E.N. Pugh, Jr; Through the Connecting Cilium: Protein Equilibration Between the Inner and Outer Segments of Rods Observed in Real Time With Photoactivatable–GFP . Invest. Ophthalmol. Vis. Sci. 2005;46(13):4782.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To measure the mobility of a moderate sized soluble protein in the chief compartments (IS and OS) of rod photoreceptors, and the movement of the protein between the two segments. Methods: Photo–activatable GFP (31 kD) expressed in Xenopus rods was activated at a "point" by two–photon excitation with a 100 µs, 820 nm pulse produced by a Ti–Sapphire laser. A confocal microscope with a 488 nm scanning laser and photon–counting detectors was used to measure the redistribution of the photoactivated form of PA–GFP (PAd–GFP), with 100 µm line scans taken every 3 ms either parallel or transverse to the rod's long axis. Models of diffusion in 3D from an impulsive point source were developed, applicable to the isotropic conditions of the IS at early times, and to the anisotropic OS. Results: The pattern of diffusion of PAd–GFP from a "point" source in the center of the IS was independent of the axis of the scan, and thus isotropic. Radial diffusion in the OS could be clearly observed with transverse scanning, with PAd–GFP created in the OS center remaining removed from the plasma membrane boundary up to 80 ms after creation. A rapid and a slow component of diffusion were measured in both transverse and longitudinal scans of the OS: these results implicate anisotropic diffusion, and suggest radial diffusion in the OS to be about 20–fold faster than longitudinal diffusion. Estimates of the radial and longitudinal diffusion coefficients depend on details of the apparatus, including the shape of the 3D point–spread function of both the photoactivating and scanning lasers, and will be discussed. 2P photo– activation pulses of 100 ms duration in the IS produced a large bolus of PAd–GFP, which equilibrated rapidly (∼ 2–5 s) in the IS, and more slowly between the IS and OS through the connecting cilium over several minutes: the half–time to IS/OS equilibration was ∼ 3 min. Conclusions: (1) The diffusion of PAd–GFP from an "impulsive point source" created by a two–photon flash can be observed within the main subcellular compartments of living rods. (2) PAd–GFP diffuses most rapidly in the IS, with radial diffusion in the OS somewhat slower, and longitudinal diffusion in the OS more than 20–fold slower still. (3) Equilibration of PAd–GFP between the IS and OS through the connecting cilium is surprisingly fast, suggesting that under our light–exposed conditions the connecting cilium is not a major barrier to the redistribution of soluble transduction proteins, such as arrestin, which are known to redistribute between the OS and IS in a light–dependent manner on the time scale of 10 min.
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