Membrane curvature generation by a C-terminal amphipathic helix in peripherin-2/rds, a tetraspanin required for photoreceptor sensory cilium morphogenesis.

Vertebrate vision requires photon absorption by photoreceptor outer segments (OSs), structurally elaborate membranous organelles derived from non-motile sensory cilia. The structure and function of OSs depends on a precise stacking of hundreds of membranous disks. Each disk is fully (as in rods) or partially (as in cones) bounded by a rim, at which the membrane is distorted into an energetically unfavorable high-curvature bend; however, the mechanism(s) underlying disk rim structure is (are) not established. Here, we demonstrate that the intrinsically disordered cytoplasmic C-terminus of the photoreceptor tetraspanin peripherin-2/rds (P/rds) can directly generate membrane curvature. A P/rds C-terminal domain and a peptide mimetic of an amphipathic helix contained within it each generated curvature in liposomes with a composition similar to that of OS disks and in liposomes generated from native OS lipids. Association of the C-terminal domain with liposomes required conical phospholipids, and was promoted by membrane curvature and anionic surface charge, results suggesting that the P/rds C-terminal amphipathic helix can partition into the cytosolic membrane leaflet to generate curvature by a hydrophobic insertion (wedging) mechanism. This activity was evidenced in full-length P/rds by its induction of small-diameter tubulovesicular membrane foci in cultured cells. In sum, the findings suggest that curvature generation by the P/rds C-terminus contributes to the distinctive structure of OS disk rims, and provide insight into how inherited defects in P/rds can disrupt organelle structure to cause retinal disease. They also raise the possibility that tethered amphipathic helices can function for shaping cellular membranes more generally.

In situ visualization of protein interactions in sensory neurons: glutamic acid-rich proteins (GARPs) play differential roles for photoreceptor outer segment scaffolding.

Vertebrate photoreceptors initiate vision via a G-protein-mediated signaling cascade organized within a specialized cilium, the outer segment (OS). The membranous "stacked pancake" architecture of this organelle must be partially renewed daily to maintain cell function and viability; however, neither its static structure nor renewal process is well described in molecular terms. Glutamic acid-rich proteins (GARPs), including the cyclic nucleotide-gated cation channel (CNGB1) and GARP2 (a CNGB1 splice-variant), are proposed to contribute to OS organization in concert with peripherin/rds (P/rds), a retinal tetraspanin. We developed and applied an in situ fluorescence complementation approach that offers an unprecedented glimpse at the formation, trafficking, and localization of GARP-P/rds interactions in transgenic Xenopus laevis rod photoreceptors. Interactions for these (and other) proteins could be readily visualized using confocal microscopy. Nearly all associations, including CNGB1-P/rds interaction, were initiated within inner segments (ISs) before trafficking to OSs. In contrast, GARP2-P/rds interactions were only observed downstream, at or near sites of disk morphogenesis. These results suggest that GARP2-P/rds interaction participates directly in structuring disk stacks but CNGB1-P/rds interaction does not and instead serves mainly to localize plasma membrane ion channels. Altogether, the results lead us to propose that differential interaction of GARPs with P/rds may contribute to the broad phenotypic heterogeneity produced by inherited defects in P/rds. Analogous experiments applied to the synaptic protein RIBEYE suggest that monomers can oligomerize at the level of the IS before ribbon assembly and demonstrate the general applicability of this strategy for in situ analysis of protein interactions in sensory neurons.

An intramembrane glutamic acid governs peripherin/rds function for photoreceptor disk morphogenesis.

PURPOSE: Peripherin/rds (P/rds), the product of the retinal degeneration slow (rds) gene, is a tetraspanin protein that plays a pivotal role for photoreceptor outer segment (OS) structure and is involved in a broad spectrum of inherited retinal degenerations. P/rds interacts with the homologous protein rom-1, previously proposed to regulate P/rds function. The authors examined the significance of an intramembrane glutamic acid conserved in all P/rds proteins (and many other tetraspanins) but absent in all rom-1 orthologs. METHODS: The authors performed isosteric glutamine substitution of the conserved glutamate at position 276, in the fourth transmembrane domain of bovine P/rds, and expressed E276Q P/rds in COS-1 cells and in transgenic mouse photoreceptors of rds +/+, -/+, and -/- backgrounds. Western blot, immunoprecipitation, and sedimentation analyses were used to assess protein structure and interactions. Microscopy and electroretinography were used to characterize transgenic protein localization and retinal photoreceptor structure and function. RESULTS: E276Q P/rds was expressed, assembled, and properly localized in photoreceptor OSs of transgenic mice. In contrast to wild-type (WT) P/rds, however, this mutant did not rescue the OS structural defects observed in rds -/- and -/+ mice. Moreover, E276Q expression did not prevent the retinal degeneration that occurred as a consequence of OS disruption. CONCLUSIONS: E276 plays a critical role in P/rds support of photoreceptor OS structure. This finding provides a molecular rationale for asymmetry in P/rds and rom-1 function and for rom-1 regulation of P/rds activity. These findings also suggest that ionizable intramembrane residues may serve regulatory roles for tetraspanin proteins more generally.

Protective gene expression changes elicited by an inherited defect in photoreceptor structure.

Inherited defects in retinal photoreceptor structure impair visual transduction, disrupt relationship with the retinal pigment epithelium (RPE), and compromise cell viability. A variety of progressive retinal degenerative diseases can result, and knowledge of disease etiology remains incomplete. To investigate pathogenic mechanisms in such instances, we have characterized rod photoreceptor and retinal gene expression changes in response to a defined insult to photoreceptor structure, using the retinal degeneration slow (rds) mouse model. Global gene expression profiling was performed on flow-sorted rds and wild-type rod photoreceptors immediately prior and subsequent to times at which OSs are normally elaborated. Dysregulated genes were identified via microarray hybridization, and selected candidates were validated using quantitative PCR analyses. Both the array and qPCR data revealed that gene expression changes were generally modest and dispersed amongst a variety of known functional networks. Although genes showing major (>5-fold) differential expression were identified in a few instances, nearly all displayed transient temporal profiles, returning to WT levels by postnatal day (P) 21. These observations suggest that major defects in photoreceptor cell structure may induce early homeostatic responses, which function in a protective manner to promote cell viability. We identified a single key gene, Egr1, that was dysregulated in a sustained fashion in rds rod photoreceptors and retina. Egr1 upregulation was associated with microglial activation and migration into the outer retina at times subsequent to the major peak of photoreceptor cell death. Interestingly, this response was accompanied by neurotrophic factor upregulation. We hypothesize that activation of Egr1 and neurotrophic factors may represent a protective immune mechanism which contributes to the characteristically slow retinal degeneration of the rds mouse model.

Uncoupling of photoreceptor peripherin/rds fusogenic activity from biosynthesis, subunit assembly, and targeting: a potential mechanism for pathogenic effects.

Inherited defects in the RDS gene cause a multiplicity of progressive retinal diseases in humans. The gene product, peripherin/rds (P/rds), is a member of the tetraspanin protein family required for normal vertebrate photoreceptor outer segment (OS) architecture. Although its molecular function remains uncertain, P/rds has been suggested to catalyze membrane fusion events required for the OS renewal process. This study investigates the importance of two charged residues within a predicted C-terminal helical region for protein biosynthesis, localization, and interaction with model membranes. Targeted mutagenesis was utilized to neutralize charges at Glu(321) and Lys(324) individually and in combination to generate three mutant variants. Studies were conducted on variants expressed as 1) full-length P/rds in COS-1 cells, 2) glutathione S-transferase fusion proteins in Escherichia coli, and 3) membrane-associated green fluorescent protein fusion proteins in transgenic Xenopus laevis. None of the mutations affected biosynthesis of full-length P/rds in COS-1 cells as assessed by Western blotting, sedimentation velocity, and immunofluorescence microscopy. Although all mutations reside within a recently identified localization signal, none altered the ability of this region to direct OS targeting in transgenic X. laevis retinas. In contrast, individual or simultaneous neutralization of the charged amino acids Glu(321) and Lys(324) abolished the ability of the C-terminal domain to promote model membrane fusion as assayed by lipid mixing. These results demonstrate that, although overlapping, C-terminal determinants responsible for OS targeting and fusogenicity are separable and that fusogenic activity has been uncoupled from other protein properties. The observation that subunit assembly and OS targeting can both proceed normally in the absence of fusogenic activity suggests that properly assembled and targeted yet functionally altered proteins could potentially generate pathogenic effects within the vertebrate photoreceptor.