Purchase this article with an account.
Mohammad Haeri, Barry E. Knox; Observation of Opsin Transport By Live Cell Imaging of Photoreceptors. Invest. Ophthalmol. Vis. Sci. 2011;52(14):909.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
To study the biogenesis of rod disks and rhodopsin biosynthesis, we utilized live cell confocal imaging to quantify the transport rate of rhodopsin to the outer segment in rods.
We prepared transgenic Xenopus that expressed rhodopsin fused to a fluorescent protein in photoreceptors using the Xenopus rhodopsin promoter. Live retinas were examined by high-resolution confocal microscopy and time-lapse image acquisition to determine the rate of rhodopsin transport to the outer segment. A stack of 5 optical slices 0.3 µ in thickness focused in the inner segment were collected over 3.5 s. Repeat scans were obtained every 5 s for a total duration of 120 s. Sample drift was corrected manually (~5-10 s) and time-lapse image collection continued as described. Stacks were reconstructed in three dimensions, and movement was determined from the projection in the XY plane. Velocities were determined as averages of individual fluorescent foci (n=8).
We generated three dimensional images of live photoreceptors and successfully visualized fluorescently labeled opsin within the inner segment in transit to the outer segment. We observed some rhodopsin that was immobile over 120 s. However, there was a significant fraction of the fluorescent protein that exhibited movement in the inner segment. One fraction had non-directional movement with apparent coalescence of fluorescent foci. A second fraction showed directed movement toward the outer segment. We measured the rate of this fast population at the apical end of the inner segment to be 42 ± 7 nm/s. This rate would suggest a transit time from site of synthesis to the outer segment (~15 µ) of approximately 6 min if the movement were continuous in a straight path.
The 3D visualization of the rate of opsin transport within photoreceptors revealed fast dynamics of protein transport in photoreceptors. This is the first report of visualizing and measuring vesicular transport in live photoreceptors.
This PDF is available to Subscribers Only