March 2012
Volume 53, Issue 14
Free
ARVO Annual Meeting Abstract  |   March 2012
Rhodopsin Mutants Destabilize Rod Outer Segment Disk Membranes
Author Affiliations & Notes
  • Mohammad Haeri
    Ophthalmology & Neurosciences & Physiology, SUNY Upstate Medical University, SUNY Eye Institute, Syracuse, New York
  • Sarah E. Reks
    Ophthalmology & Neurosciences & Physiology, SUNY Upstate Medical University, SUNY Eye Institute, Syracuse, New York
  • Barry E. Knox
    Ophthalmology & Neurosciences & Physiology, SUNY Upstate Medical University, SUNY Eye Institute, Syracuse, New York
  • Footnotes
    Commercial Relationships  Mohammad Haeri, None; Sarah E. Reks, None; Barry E. Knox, None
  • Footnotes
    Support  National Eye Institute/NIH (EY011256, EY012975, EY018421); Fight For Sight; Unrestricted grant from Research to Prevent Blindness
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 5627. doi:
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      Mohammad Haeri, Sarah E. Reks, Barry E. Knox; Rhodopsin Mutants Destabilize Rod Outer Segment Disk Membranes. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5627.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : Mutations in rhodopsin cause retinitis pigmentosa (RP) and retinal degeneration. The mechanisms that lead to photoreceptor cell death are still unclear. We have recently developed imaging methods for high resolution studies of live rods using tagged fluorescent proteins. Here, we compared the properties of five mutant opsins in both inner and outer segments with wild type protein.

Methods: : We prepared transgenic Xenopus expressing opsin-EGFP or various mutants under control of the rod opsin promoter. Live transgenic retinas were examined by high-resolution confocal microscopy. The transport and protein mobility of mutant proteins was characterized using FRAP analysis and time-lapse imaging. Ultrastructural studies were performed on fixed retina and the aggregation properties of mutant proteins were studied using western blot analysis.

Results: : Opsin mutants with deficiencies in 11-cis retinal binding were observed in both IS and OS, frequently forming dense fluorescent foci. OS regions containing fluorescent foci were refractory to fluorescence recovery after photobleaching, while those in the IS exhibited recovery kinetics similar to OS regions without foci. The foci were biased to older, more distal OS disks. EM analysis showed abnormal vesiculotubular structures, which were also observed in mouse retina expressing a RhoP23H transgene. The mutant opsins formed SDS-resistant oligomers.

Conclusions: : Our results suggest a new model for cellular pathogenesis in RP. We propose that mutant opsin initially forms oligomers that eventually become higher order aggregates in the OS. This leads to membrane disturbances and then vesiculation of photoreceptors disks. Overall, these perturbations could destabilize the OS structure and sensitize rods to mechanical injury leading to OS shortening and photoreceptor loss.

Keywords: retinal degenerations: cell biology • retinal degenerations: hereditary • photoreceptors 
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