Abstract
Mutations in rhodopsin cause retinitis pigmentosa in humans and retinal degeneration in a multitude of other animals. We utilized high-resolution live imaging of the large rod photoreceptors from transgenic frogs (Xenopus) to compare the properties of fluorescently tagged rhodopsin, Rho-EGFP, and RhoP23H-EGFP. The mutant was abnormally distributed both in the inner and outer segments (OS), accumulating in the OS to a concentration of ∼0.1% compared to endogenous opsin. RhoP23H-EGFP formed dense fluorescent foci, with concentrations of mutant protein up to ten times higher than other regions. Wild-type transgenic Rho-EGFP did not concentrate in OS foci when co-expressed in the same rod with RhoP23H-EGFP. Outer segment regions containing fluorescent foci were refractory to fluorescence recovery after photobleaching, while foci in the inner segment exhibited recovery kinetics similar to OS regions without foci and Rho-EGFP. The RhoP23H-EGFP foci were often in older, more distal OS disks. Electron micrographs of OS revealed abnormal disk membranes, with the regular disk bilayers broken into vesiculotubular structures. Furthermore, we observed similar OS disturbances in transgenic mice expressing RhoP23H, suggesting such structures are a general consequence of mutant expression. Together these results show that mutant opsin disrupts OS disks, destabilizing the outer segment possibly via the formation of aggregates. This may render rods susceptible to mechanical injury or compromise OS function, contributing to photoreceptor loss.
Highlights
The most common allele in North America for autosomal dominant retinitis pigmentosa, a major cause of retinal degeneration and vision loss worldwide [1], is a missense mutation (P23H) in the gene encoding rhodopsin [2]
Quantitative analysis of Rho-EGFP and RhoP23H-EGFP distributions in transgenic Xenopus rods showed a marked difference between the two fusion proteins (Fig. 1,2,3 and Fig. S1)
Rho-EGFP localized primarily in the outer segments (OS) of mature rods, while RhoP23H-EGFP was distributed in both OS and inner segments (IS), localizing around the nucleus and more diffusely in the ellipsoid and myoid areas but not in the synaptic region
Summary
The most common allele in North America for autosomal dominant retinitis pigmentosa, a major cause of retinal degeneration and vision loss worldwide [1], is a missense mutation (P23H) in the gene encoding rhodopsin [2]. We have quantified the distribution and properties of a RhoP23H-EGFP fusion protein in transgenic Xenopus, a system wellsuited for live cell imaging. Quantitative analysis of Rho-EGFP and RhoP23H-EGFP distributions in transgenic Xenopus rods showed a marked difference between the two fusion proteins (Fig. 1,2,3 and Fig. S1). Mutant opsin exhibited abnormal foci that contained high concentrations of fluorescent mutant protein in both the IS and OS (Fig. 2b).
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
