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M. Najafi, M. Haeri, T. Gill, N. Maza, B. E. Knox, P. D. Calvert; Variable Confinement of Rhodopsin to Rod Photoreceptor Disc Lobules Revealed by High Resolution FRAP. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3487.
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© ARVO (1962-2015); The Authors (2016-present)
We have employed high spatial resolution imaging to examine rhodopsin mobility within individual outer segment disc lobules of live photoreceptors.
Transgenic Xenopus laevis expressing rhodopsin with PAGFP fused to the c-terminus were generated using restriction enzyme mediated integration. Retina from late stage tadpoles or juvenile frogs adapted to darkness for at least two hours were sliced and placed into the live tissue imaging chamber of our custom built confocal/multiphoton microscope. Rhodopsin-PAGFP was photoactivated with 0.2 ms pulses from a titanium:sapphire laser tuned to 820 nm and focused to the diffraction limit with a 60x, 1.2 NA, water immersion objective. This allowed placement of a field of brightly fluorescent molecules within individual "lobules" (regions of the discs circumscribed by incisures). Redistribution of the photoactivated molecules was subsequently monitored with rapid, confocal, x-y scanning at 488nm. Diffusion models were employed to estimate diffusion coefficients.
Disc lobules varied significantly in geometry. Rhodopsin redistribution after photoactivation was biphasic in all lobules with similar fast phases and variable slow phases. The average rhodopsin diffusion coefficient estimated from the fast phase was 1.21 ± 0.07 micron2s-1. The slow phase, clearly representing rhodopsin equilibration with the bulk disc, varied widely both in amplitude and time course depending on the overall disc/incisure geometry, in some cases lasting more than ten minutes.
We have, for the first time, measured rhodopsin diffusion in individual live photoreceptor disc lobules. The fast phase of equilibration represents rhodopsin mobility in the lobe prior to incisure encounter, and thus represents pure membrane diffusion. The high degree of variability in lobule geometry and narrowness of lobe connections to the bulk disc account for the variable slow phase, which in turn explains previous, lower estimates of rhodopsin diffusion coefficients found using models that do not adequately take into consideration the details of this high variability.
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