Abstract
Purpose:
Inherited defects in peripherin-2/rds (P/rds) cause a broad spectrum of progressive retinal degenerations. Animal models demonstrate that P/rds is required for biogenesis of rod and cone photoreceptor outer segments (OSs); however, the molecular function of this protein remains uncertain. The aim of this study was to test the hypothesis that P/rds can induce membrane curvature in support of OS disk morphogenesis.
Methods:
We investigated curvature generation by P/rds using standard membrane remodeling assays, using liposomes of defined compositions and sizes, synthetic peptides, soluble protein domains, and transmission electron microscopy. Curvature-sensing activity was measured using a liposome binding assay in conjunction with quantitative immunoblotting and fluorescence measurements. Localization of heterologously expressed P/rds in cultured cells was determined using indirect immunofluorescence and laser scanning confocal microscopy and postembedding immunogold labeling electron microscopy.
Results:
The P/rds cytoplasmic C-terminus (and a peptide mimetic of an amphipathic helix (AH) within this domain) potently increased membrane curvature of synthetic phospholipid liposomes that had a composition based on that of OS disks, and liposomes generated from natural lipids extracted directly from purified OS membranes. Binding of the P/rds cytoplasmic C-terminus to liposomes showed relatively weak curvature sensitivity, but strong dependence on phospholipid composition. Consistent with these findings, full-length P/rds expressed in AD293 cells was associated with small diameter tubulovesicular structures.
Conclusions:
The new findings suggest that P/rds employs a hydrophobic insertion mechanism to generate membrane curvature in support of OS disk rim morphology. This model provides a direct explanation of the dysmorphic OS membrane phenotypes generated by partial and complete absence of P/rds in murine retinal degeneration slow models. It also offers a novel molecular rationale for understanding normal photoreceptor OS architecture and progressive retinal degenerations associated with inherited defects in P/rds.
Keywords: 648 photoreceptors •
696 retinal degenerations: hereditary •
695 retinal degenerations: cell biology