Abstract
The rod outer segment (OS), comprised of tightly stacked disk membranes packed with rhodopsin, is in a dynamic equilibrium governed by a diurnal rhythm with newly synthesized membrane inserted at the OS base balancing membrane loss from the distal tip via disk shedding. Using transgenic Xenopus and live cell confocal imaging, we found OS axial variation of fluorescence intensity in cells expressing a fluorescently tagged rhodopsin transgene. There was a light synchronized fluctuation in intensity, with higher intensity in disks formed at night and lower intensity for those formed during the day. This fluctuation was absent in constant light or dark conditions. There was also a slow modulation of the overall expression level that was not synchronized with the lighting cycle or between cells in the same retina. The axial variations of other membrane-associated fluorescent proteins, eGFP-containing two geranylgeranyl acceptor sites and eGFP fused to the transmembrane domain of syntaxin, were greatly reduced or not detectable, respectively. In acutely light-adapted rods, an arrestin-eGFP fusion protein also exhibited axial variation. Both the light-sensitive Rho-eGFP and arrestin-eGFP banding were in phase with the previously characterized birefringence banding (Kaplan, Invest. Ophthalmol. Vis. Sci. 21, 395–402 1981). In contrast, endogenous rhodopsin did not exhibit such axial variation. Thus, there is an axial inhomogeneity in membrane composition or structure, detectable by the rhodopsin transgene density distribution and regulated by the light cycle, implying a light-regulated step for disk assembly in the OS. The impact of these results on the use of chimeric proteins with rhodopsin fused to fluorescent proteins at the carboxyl terminus is discussed.
Highlights
The vertebrate photoreceptor is a highly polarized neuron with a modified cilium specialized for light detection
We have previously reported that the expression levels of rhodopsin transgenes under control of the XOP promoter are substantially lower (,5%) than endogenous rhodopsin [31] and do not represent a significant overexpression of this membrane protein
One type is a slow modulation of the overall expression level that was not synchronized with the light cycle or between cells in the same retina
Summary
The vertebrate photoreceptor is a highly polarized neuron with a modified cilium specialized for light detection. In Xenopus, the birefringence banding pattern has a spatial period of about 1.0 –1.6 mm, which corresponds to ,35 – 60 disks [16]. They are more pronounced at the base of the OS. The spatial periodicity is modulated by the length of the light-dark cycle, and is abolished under constant illumination conditions [17]. While these observations suggest an underlying variation in the disks produced in light compared to dark [18,19,20], the origin of the light-cycle dependent banding remains unexplained
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