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Mehdi Najafi, Peter Calvert; Steric Volume Exclusion Leads to Size-dependent Protein Distribution in Mouse Ciliary Photoreceptors. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2454.
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We recently demonstrated that ‘steric volume exclusion’ mediates size-dependent soluble protein distribution in Xenopus laevis rod photoreceptors 1. However, the large variation in the morphology of photoreceptors among species raises the question of whether the steric volume exclusion effect is significant for all photoreceptors. We therefore examined the subcellular distributions of soluble molecules of different sizes in mouse photoreceptors using live cell confocal imaging.
Microslices of retina from dark-adapted mice expressing EGFP in rod cells under the NRL promoter were prepared under infrared illumination, placed in an imaging chamber containing mouse ringer solution, and imaged using our custom confocal microscope. Distribution of EGFP in rods was compared to that of the small fluorescent probe Calcein introduced into WT retinas that were similarly prepared. Corrections were made to account for the fact that mouse rods are below the resolution limit of confocal microcopy.
The distribution of Calcein significantly differed from that of EGFP in mouse photoreceptors. The ratio of the mean fluorescence in the outer segment to that in the myoid region (FOS/FMY) was 0.91±0.03 in photoreceptors containing Calcein, and dropped to 0.44 ±0.06 in EGFP expressing rods. A size-dependent distribution was evident in other areas of the cell as well. While Calcein uniformly filled the nucleus, EGFP was excluded from the central areas of this compartment where heterochromatin is located.
Our results indicate that steric volume exclusion governs protein distribution in mammalian photoreceptors in much the same manner as in Xenopus. The lower FOS/FMY ratio in EGFP expressing rods compared to Calcein demonstrates a size-dependent exclusion from the outer segment due to the higher packing density of disk membranes in this compartment. We project a molecular size cut-off of ~4.0nm for protein access to the outer segment in mouse which is consistent with our previous estimation in Xenopus photoreceptors (4.2 nm). Our experiments indicate that despite significant variation in photoreceptor morphology among species, steric volume exclusion remains a general mechanism for regulation of protein access to the ciliary outersegment compartment.<br/><br/> 1. Najafi et al. PNAS, 012 Jan 3;109(1):203-8
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