March 2012
Volume 53, Issue 14
Free
ARVO Annual Meeting Abstract  |   March 2012
Mechanisms Of Protein Retention Within The Cone Photoreceptor Sensory Cilium
Author Affiliations & Notes
  • Ivayla I. Geneva
    Biochemistry and Molecular Biology, Ophthalmology,
    State University of New York - Upstate, Syracuse, New York
  • Cherry G. Ignacio
    Biochemistry and Molecular Biology,
    State University of New York - Upstate, Syracuse, New York
  • Barry E. Knox
    Neuroscience and Physiology, SUNY Eye Institute,
    State University of New York - Upstate, Syracuse, New York
  • Peter D. Calvert
    Ophthalmology, Biochemistry and Molecular Biology, Neuroscience and Physiology, SUNY Eye Institute,
    State University of New York - Upstate, Syracuse, New York
  • Footnotes
    Commercial Relationships  Ivayla I. Geneva, None; Cherry G. Ignacio, None; Barry E. Knox, None; Peter D. Calvert, None
  • Footnotes
    Support  NIH Grant EY018421, EY012975, Research To Prevent Blindness
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 1586. doi:
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      Ivayla I. Geneva, Cherry G. Ignacio, Barry E. Knox, Peter D. Calvert; Mechanisms Of Protein Retention Within The Cone Photoreceptor Sensory Cilium. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1586.

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

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Abstract

Purpose: : In rod outer segments (OS), localization and retention of transduction proteins is thought to be mediated by targeted, active transport and association with OS disc membranes that are physically separate from the plasma membrane. Targeting and retention of signaling proteins within cone OSs is not understood. Unlike rods, cone OS discs are contiguous with the plasma membrane. Therefore retention of membrane-associated proteins appears to require diffusion barriers either at the cilium base, as has been suggested for primary cilia, or at the disc rims. We examined a putative cone opsin OS targeting signal that is analogous to that on rod opsin, and the possibility of diffusion barriers at the ciliary transition zone and disc rims, as possible mechanisms for protein localization and retention in the cone OS.

Methods: : Transgenic X. laevis that express EGFP fused with lipidation motifs, red cone opsin (XRCOP), or a putative XRCOP OS localization sequence, in cones under the Xenopus red cone opsin (XRCOP) promoter, were created using the REMI method. Confocal and multiphoton imaging of live cones in retinal slices were used to study protein localization and dynamics.

Results: : Lipidated-EGFP was found in both of the major photoreceptor compartments, while XRCOP-GFP was localized to the OS. EGFP fused to the C-terminus of XRCOP failed to localize to the OS. Although both integral and peripheral membrane proteins diffused laterally and axially in the OS, diffusion of opsin-GFP between discs was significantly retarded relative to lipidated-EGFP.

Conclusions: : Our results are consistent with cone opsin retention in the OS being mediated by a diffusion barrier at the ciliary transition zone. There appears to be a sieving mechanism at the disc rims that impedes the movement of integral membranes proteins between discs to a greater extent than peripheral membrane proteins. The fact that lipidated GFPs are found in both IS and OS compartments suggests either that the barrier at the ciliary transition zone is selective for intrinsic membrane proteins, or that lipidated GFPs dissociate from membranes and equilibrate between these compartments. Unlike rod opsin, addition of the C-terminal 40 amino acids of XRCOP was not sufficient to localize EGFP to the OS, possibly due to the absence of palmitoylation sites in the cone opsin sequence.

Keywords: opsins • photoreceptors • microscopy: confocal/tunneling 
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