Outer Segment Disk Research Articles

Photoreceptor sensory cilia and ciliopathies: focus on CEP290, RPGR and their interacting proteins

Ciliopathies encompass a broad array of clinical findings associated with genetic defects in biogenesis and/or function of the primary cilium, a ubiquitous organelle involved in the transduction of diverse biological signals. Degeneration or dysfunction of retinal photoreceptors is frequently observed in diverse ciliopathies. The sensory cilium in a photoreceptor elaborates into unique outer segment discs that provide extensive surface area for maximal photon capture and efficient visual transduction. The daily renewal of approximately 10% of outer segments requires a precise control of ciliary transport. Here, we review the ciliopathies with associated retinal degeneration, describe the distinctive structure of the photoreceptor cilium, and discuss mouse models that allow investigations into molecular mechanisms of cilia biogenesis and defects. We have specifically focused on two ciliary proteins – CEP290 and RPGR – that underlie photoreceptor degeneration and syndromic ciliopathies. Mouse models of CEP290 and RPGR disease, and of their multiple interacting partners, have helped unravel new functional insights into cell type-specific phenotypic defects in distinct ciliary proteins. Elucidation of multifaceted ciliary functions and associated protein complexes will require concerted efforts to assimilate diverse datasets from in vivo and in vitro studies. We therefore discuss a possible framework for investigating genetic networks associated with photoreceptor cilia biogenesis and pathology.

Detecting single photons: A supramolecular matter?

Rod photoreceptors detect single photons through a tradeoff of light collecting ability, amplification and speed. Key roles are played by rhodopsin (Rh) and transducin (Gt), whose complex supramolecular organization in outer segment disks begs for a functional interpretation. Here we review past and recent evidence of a temperature-dependence of photon detection by mammalian rods, and link this phenomenon with the putative oligomeric organization of Rh and new ideas on the dynamics of Rh–Gt interaction. Identifying an electrophysiological correlate of the supramolecular organization of Rh and Gt may shed light on the evolutionary advantage it confers to night vision.

Three-Dimensional Architecture of the Rod Sensory Cilium and Its Disruption in Retinal Neurodegeneration

Defects in primary cilia lead to devastating disease because of their roles in sensation and developmental signaling but much is unknown about ciliary structure and mechanisms of their formation and maintenance. We used cryo-electron tomography to obtain 3D maps of the connecting cilium and adjacent cellular structures of a modified primary cilium, the rod outer segment, from wild-type and genetically defective mice. The results reveal the molecular architecture of the cilium and provide insights into protein functions. They suggest that the ciliary rootlet is involved in cellular transport and stabilizes the axoneme. A defect in the BBSome membrane coat caused defects in vesicle targeting near the base of the cilium. Loss of the proteins encoded by the Cngb1 gene disrupted links between the disk and plasma membranes. The structures of the outer segment membranes support a model for disk morphogenesis in which basal disks are enveloped by the plasma membrane.

Overexpression of Guanylate Cyclase Activating Protein 2 in Rod Photoreceptors In Vivo Leads to Morphological Changes at the Synaptic Ribbon

Guanylate cyclase activating proteins are EF-hand containing proteins that confer calcium sensitivity to retinal guanylate cyclase at the outer segment discs of photoreceptor cells. By making the rate of cGMP synthesis dependent on the free intracellular calcium levels set by illumination, GCAPs play a fundamental role in the recovery of the light response and light adaptation. The main isoforms GCAP1 and GCAP2 also localize to the synaptic terminal, where their function is not known. Based on the reported interaction of GCAP2 with Ribeye, the major component of synaptic ribbons, it was proposed that GCAP2 could mediate the synaptic ribbon dynamic changes that happen in response to light. We here present a thorough ultrastructural analysis of rod synaptic terminals in loss-of-function (GCAP1/GCAP2 double knockout) and gain-of-function (transgenic overexpression) mouse models of GCAP2. Rod synaptic ribbons in GCAPs−/− mice did not differ from wildtype ribbons when mice were raised in constant darkness, indicating that GCAPs are not required for ribbon early assembly or maturation. Transgenic overexpression of GCAP2 in rods led to a shortening of synaptic ribbons, and to a higher than normal percentage of club-shaped and spherical ribbon morphologies. Restoration of GCAP2 expression in the GCAPs−/− background (GCAP2 expression in the absence of endogenous GCAP1) had the striking result of shortening ribbon length to a much higher degree than overexpression of GCAP2 in the wildtype background, as well as reducing the thickness of the outer plexiform layer without affecting the number of rod photoreceptor cells. These results indicate that preservation of the GCAP1 to GCAP2 relative levels is relevant for maintaining the integrity of the synaptic terminal. Our demonstration of GCAP2 immunolocalization at synaptic ribbons at the ultrastructural level would support a role of GCAPs at mediating the effect of light on morphological remodeling changes of synaptic ribbons.

The kinesin superfamily protein KIF17: one protein with many functions

Kinesins are ATP-dependent molecular motors that carry cargos along microtubules, generally in an anterograde direction. They are classified into 14 distinct families with varying structural and functional characteristics. KIF17 is a member of the kinesin-2 family that is plus end-directed. It is a homodimer with a pair of head motor domains that bind microtubules, a coiled-coil stalk, and a tail domain that binds cargos. In neurons, KIF17 transports N-methyl-D-aspartate receptor NR2B subunit, kainate receptor GluR5, and potassium Kv4.2 channels from cell bodies exclusively to dendrites. These cargos are necessary for synaptic transmission, learning, memory, and other functions. KIF17's interaction with NXF2 enables the transport of mRNA bidirectionally in dendrites. KIF17 or its homolog OSM-3 also mediates intraflagellar transport of cargos to the distal tips of flagella or cilia, thereby aiding in ciliogenesis. In many invertebrate and vertebrate sensory cells, KIF17 delivers cargos that contribute to chemosensory perception and signal transduction. In vertebrate photoreceptors, KIF17 is necessary for outer segment development and disc morphogenesis. In the testis, KIF17 (KIF17b) mediates microtubule-independent delivery of ACT from the nucleus to the cytoplasm and microtubule-dependent transport of Spatial-ε, both are presumably involved in spermatogenesis. KIF17 is also implicated in epithelial polarity and morphogenesis, placental transport and development, and the development of specific brain regions. The transcriptional regulation of KIF17 has recently been found to be mediated by nuclear respiratory factor 1 (NRF-1), which also regulates NR2B as well as energy metabolism in neurons. Dysfunctions of KIF17 are linked to a number of pathologies.

Cholesterol Enhances or Reduces Metarhodopsin II Formation Depending on Bilayer Thickness

Cholesterol is one of the most efficient modulators of G Protein-Coupled Receptor (GPCR) function. We monitored the effect of cholesterol on rhodopsin function for a set of bilayers with different hydrophobic thickness. Surprisingly, cholesterol shifts the Metarhodopsin-I (MI)/Metarhodopsin-II (MII) equilibrium toward MII for bilayers thinner than the average length of hydrophobic transmembrane helices (2.7 nm), and to MI for thicker bilayers. In previous work conducted on rod outer segment disks and model membranes, increasing cholesterol concentration always shifted the equilibrium towards MI. It was proposed that the cholesterol effect is primarily related to a tighter packing of lipid hydrocarbon chains which generates a less permissive environment for the formation of MII. To gain deeper insights into mechanisms, we followed changes in lipid-rhodopsin interaction by 2H NMR using deuterated lipids. It was reported by us and the Brown laboratory that an increase of bilayer hydrophobic thickness in the absence of cholesterol favors MII with a turnover to MI for bilayers that are very thick. Indeed, the cholesterol-induced shifts towards MII for thinner membranes correlated nicely with the cholesterol-induced increase of bilayer hydrophobic thickness measured by NMR suggesting that the increase in bilayer thickness by cholesterol plays a major role in controlling the energetics of the MI-MII equilibrium. Furthermore, changes in average lipid order parameters due to the presence of rhodopsin were much larger in cholesterol-containing membranes than in cholesterol-free membranes suggesting strongly that the perturbation in the lipid matrix from protein insertion reaches much further away from the protein. This is expected for membranes that are stiffer due to the presence of cholesterol. The consequences of these findings for lipid mediated shifts in rhodopsin function and rhodopsin-rhodopsin interactions will be discussed.

Lipid and Detergent Effects on the Kinetic Stability of the GPCR, Rhodopsin

The photoreceptor, rhodopsin is a kinetically stable G-Protein Coupled Receptor that is located in rod outer segment disk membranes. Differential scanning calorimetry (DSC) studies have shown that rhodopsin exhibits an irreversible scan rate dependent endothermic transition (Tm) at approximately 72 oC. The activation energy for thermal denaturation (Eact) calculated from the scan rate dependence of the Tm is sensitive to the integrity of the lipid bilayer. It is also sensitive to proteolytic cleavage of the extramembraneous loops of rhodopsin. Here we investigate the influence of palmitoylation, membrane cholesterol and digitonin solubilization on rhodopsin kinetic stability. DSC experiments were performed using a MicroCal VP-DSC microcalorimeter. Samples were scanned at 15, 30, 60 and 90o/hr. Because the protein transitions are irreversible, a second scan was used to determine the baseline. Rhodopsin palmitoylation at cys 322 and cys 323 anchors the C-terminus to the membrane. The Eact for rhodopsin in disk membranes treated with hydroxylamine to remove these palmitate groups was approximately 35 Kcal/mole less than that of native rhodopsin. The Eact for rhodopsin was also determined at different membrane cholesterol levels. These data indicated a decrease in stability both above and below native membrane cholesterol levels. Finally, unlike solubilization in other detergents, solubilization in digitonin had little effect on rhodopsin kinetic stability.

Biochemical Crosslinking and Liquid Chromatography-Mass Spectrometry Demonstrate a Rhodopsin Dimerization Interface Mediated by Helices 1 and 8

Heptahelical G protein-coupled receptors (GPCRs) can exist as monomers and a ternary complex of ligand, GPCR and heterotrimeric G protein is the basic signaling unit. GPCRs also tend to form dimers and higher-order oligomers in membranes, although the functional consequences of these interactions are in most cases unknown. Furthermore, the precise protein-protein interface(s) in receptor dimers and the mechanisms mediating dimerization remain controversial. Two-dimensional and three-dimensional densities obtained from electron microscopy, as well as X-ray data on packing of rhodopsin crystals, suggest that the primary dimer contacts involve transmembrane helix (H) 1 and cytoplasmic H8. Biochemical crosslinking studies with dopamine D2 receptors hint that this interface may be broadly significant. We crosslinked rhodopsin dimers in native rod outer segment disk membranes to demonstrate the proximity of H8 between adjacent receptors. Four homobifunctional cysteine-reactive crosslinkers were used, two with maleimide groups and two with methanethiosulfonate groups. The formation of dimers and oligomers was verified by SDS-PAGE, size-exclusion chromatography, and immunoblot analysis. We used partial proteolysis and high-resolution liquid chromatography-mass spectrometry (LC-MS) to identify the site of a crosslink between Cys316-Cys316. Cys316 is one of two reactive cysteines in rhodopsin and is located in H8. The spacer length of the crosslinkers that formed the intermolecular Cys316-Cys316 crosslink is consistent with the distance predicted in a H1/H8 dimer model. This result corroborates findings from coarse-grained molecular dynamics (CGMD) potential of mean force (PMF) calculations, which show that the H1/H8 orientation is by far the most stable among the possible dimer orientations tested. Together these results strongly suggest the existence of this interface in native membranes. Given the high degree of homology across class A GPCRs, these results may be relevant for other receptors.

Steric volume exclusion sets soluble protein concentrations in photoreceptor sensory cilia

Proteins segregate into discrete subcellular compartments via a variety of mechanisms, including motor protein transport, local binding, and diffusion barriers. This physical separation of cell functions serves, in part, as a mechanism for controlling compartment activity by allowing regulation of local protein concentrations. In this study we explored how soluble protein size impacts access to the confined space within the retinal photoreceptor outer segment signaling compartment and discovered a strikingly steep relationship. We find that GFP monomers, dimers, and trimers expressed transgenically in frog rods are present in the outer segment at 1.8-, 2.9-, and 6.8-fold lower abundances, relative to the cell body, than the small soluble fluorescent marker, calcein. Theoretical analysis, based on statistical-mechanical models of molecular access to polymer meshes, shows that these observations can be explained by the steric hindrance of molecules occupying the highly constrained spaces between outer segment disc membranes. This mechanism may answer a long-standing question of how the soluble regulatory protein, arrestin, is effectively excluded from the outer segments of dark-adapted rods and cones. Generally, our results suggest an alternate mode for the control of protein access to cell domains based on dynamic, size-dependent compartmental partitioning that does not require diffusion barriers, active transport, or large numbers of immobile binding sites.

Phase-sensitive imaging of the outer retina using optical coherence tomography and adaptive optics.

The cone photoreceptor’s outer segment (OS) experiences changes in optical path length, both in response to visible stimuli and as a matter of its daily course of renewal and shedding. These changes are of interest, to quantify function in healthy cells and assess dysfunction in diseased ones. While optical coherence tomography (OCT), combined with adaptive optics (AO), has permitted unprecedented three-dimensional resolution in the living retina, it has not generally been able to measure these OS dynamics, whose scale is smaller than OCT’s axial resolution of a few microns. A possible solution is to take advantage of the phase information encoded in the OCT signal. Phase-sensitive implementations of spectral-domain optical coherence tomography (SD-OCT) have been demonstrated, capable of resolving sample axial displacements much smaller than the imaging wavelength, but these have been limited to ex vivo samples. In this paper we present a novel technique for retrieving phase information from OCT volumes of the outer retina. The key component of our technique is quantification of phase differences within the retina. We provide a quantitative analysis of such phase information and show that–when combined with appropriate methods for filtering and unwrapping–it can improve the sensitivity to OS length change by more than an order of magnitude, down to 45 nm, slightly thicker than a single OS disc. We further show that phase sensitivity drops off with retinal eccentricity, and that the best location for phase imaging is close to the fovea. We apply the technique to the measurement of sub-resolution changes in the OS over matters of hours. Using custom software for registration and tracking, these microscopic changes are monitored in hundreds of cones over time. In two subjects, the OS was found to have average elongation rates of 150 nm/hr, values which agree with our previous findings.

Biallelic Mutations in PLA2G5, Encoding Group V Phospholipase A2, Cause Benign Fleck Retina

Flecked-retina syndromes, including fundus flavimaculatus, fundus albipunctatus, and benign fleck retina, comprise a group of disorders with widespread or limited distribution of yellow-white retinal lesions of various sizes and configurations. Three siblings who have benign fleck retina and were born to consanguineous parents are the basis of this report. A combination of homozygosity mapping and exome sequencing helped to identify a homozygous missense mutation, c.133G>T (p.Gly45Cys), in PLA2G5, a gene encoding a secreted phospholipase (group V phospholipase A(2)). A screen of a further four unrelated individuals with benign fleck retina detected biallelic variants in the same gene in three patients. In contrast, no loss of function or common (minor-allele frequency>0.05%) nonsynonymous PLA2G5 variants have been previously reported (EVS, dbSNP, 1000 Genomes Project) or were detected in an internal database of 224 exomes (from subjects with adult onset neurodegenerative disease and without a diagnosis of ophthalmic disease). All seven affected individuals had fundoscopic features compatible with those previously described in benign fleck retina and no visual or electrophysiological deficits. No medical history of major illness was reported. Levels of low-density lipoprotein were mildly elevated in two patients. Optical coherence tomography and fundus autofluorescence findings suggest that group V phospholipase A(2) plays a role in the phagocytosis of photoreceptor outer-segment discs by the retinal pigment epithelium. Surprisingly, immunohistochemical staining of human retinal tissue revealed localization of the protein predominantly in the inner and outer plexiform layers.

A Novel Bisretinoid of Retina Is an Adduct on Glycerophosphoethanolamine

Fluorescent bisretinoid compounds accumulate in retinal pigment epithelial (RPE) cells as a consequence of two processes: random reactions of vitamin A aldehyde in photoreceptor cell outer segments, and phagocytosis of discarded photoreceptor outer segment discs by RPE. The formation of bisretinoid is accentuated in some forms of retinal degeneration. The detection of a novel bisretinoid fluorophore that is a conjugate of all-trans-retinal and glycerophosphoethanolamine is reported. Human RPE/choroid, eyes harvested from Abca4 (ATP-binding cassette transporter 4) null mutant mice, and biosynthetic reaction mixtures were analyzed by ultra performance liquid chromatography coupled to mass spectrometry and by nuclear magnetic resonance spectra and spectrofluorometry. A fluorescent compound in mouse eyes and in human RPE/choroid corresponded to the product of the reaction between all-trans-retinal and glycerophosphoethanolamine (A2-GPE), as determined on the basis of molecular weight (m/z 746), absorbance (approximately 338,443 nm), and retention time. Nuclear magnetic resonance spectra were consistent with a pyridinium molecule with a glycerophosphate moiety. The emission maximum of A2-GPE was approximately 610 nm. A2-GPE accumulated with age in mouse eyes and was more abundant in Abca4(-/-) mice, a model of recessive Stargardt disease. To date, several bisretinoids of RPE lipofuscin have been isolated and characterized, and for all of these, formation involves the membrane phospholipid phosphatidylethanolamine. Conversely, the bisretinoid A2-GPE is detected as sn-glycero-3-phosphoethanolamine (GPE) derivatized by two all-trans-retinal. The pathways by which A2-GPE may form under conditions of increased availability of all-trans-retinal, for instance in the Abca4(-/-) mouse, are discussed.

Intraflagellar transport proteins in ciliogenesis of photoreceptor cells

The assembly and maintenance of cilia depend on IFT (intraflagellar transport) mediated by molecular motors and their interplay with IFT proteins. Here, we have analysed the involvement of IFT proteins in the ciliogenesis of mammalian photoreceptor cilia. Electron microscopy revealed that ciliogenesis in mouse photoreceptor cells follows an intracellular ciliogenesis pathway, divided into six distinct stages. The first stages are characterized by electron-dense centriolar satellites and a ciliary vesicle, whereas the formations of the ciliary shaft and the light-sensitive outer segment discs are features of the later stages. IFT proteins were associated with ciliary apparatus during all stages of photoreceptor cell development. Our data conclusively provide evidence for the participation of IFT proteins in photoreceptor cell ciliogenesis, including the formation of the ciliary vesicle and the elongation of the primary cilium. In advanced stages of ciliogenesis the ciliary localization of IFT proteins indicates a role in IFT as is seen in mature cilia. A prominent accumulation of IFT proteins in the periciliary cytoplasm at the base of the cilia in these stages most probably resembles a reserve pool of IFT molecules for further delivery into the growing ciliary shaft and their subsequent function in IFT. Nevertheless, the cytoplasmic localization of IFT proteins in the absence of a ciliary shaft in early stages of ciliogenesis may indicate roles of IFT proteins beyond their well-established function for IFT in mature cilia and flagella.

Targeting of mouse guanylate cyclase 1 ( Gucy2e) to Xenopus laevis rod outer segments

Photoreceptor guanylate cyclase (GC1) is a transmembrane protein and responsible for synthesis of cGMP, the secondary messenger of phototransduction. It consists of an extracellular domain, a single transmembrane domain, and an intracellular domain. It is unknown how GC1 targets to the outer segments where it resides. To identify a putative GC1 targeting signal, we generated a series of peripheral membrane and transmembrane constructs encoding extracellular and intracellular mouse GC1 fragments fused to EGFP. The constructs were expressed in Xenopus laevis rod photoreceptors under the control of the rhodopsin promoter. We examined the localization of GFP-GC1 fusion proteins containing the complete GC1 sequence, or partial GC1 sequences, which were membrane-associated via either the GC1 transmembrane domain or the rhodopsin C-terminal palmitoyl chains. Full-length GFP-GC1 targeted to the rod outer segment disk rims. As a group, fusion proteins containing the entire cytoplasmic domain of GC1 targeted to the OS, whereas other fusion proteins containing portions of the cytoplasmic or the extracellular domains did not. We conclude that GC1 likely has no single linear peptide-based OS targeting signal. Our results suggest targeting is due to either multiple weak signals in the cytoplasmic domain of GC1, or co-transport to the OS with an accessory protein.

The Bilayer Enhances Rhodopsin Kinetic Stability in Bovine Rod Outer Segment Disk Membranes

Rhodopsin is a kinetically stable protein constituting >90% of rod outer segment disk membrane protein. To investigate the bilayer contribution to rhodopsin kinetic stability, disk membranes were systematically disrupted by octyl- β-D-glucopyranoside. Rhodopsin kinetic stability was examined under subsolubilizing (rhodopsin in a bilayer environment perturbed by octyl- β-D-glucopyranoside) and under fully solubilizing conditions (rhodopsin in a micelle with cosolubilized phospholipids). As determined by DSC, rhodopsin exhibited a scan-rate-dependent irreversible endothermic transition at all stages of solubilization. The transition temperature ( T m) decreased in the subsolubilizing stage. However, once the rhodopsin was in a micelle environment there was little change of the T m as the phospholipid/rhodopsin ratio in the mixed micelles decreased during the fully solubilized stage. Rhodopsin thermal denaturation is consistent with the two-state irreversible model at all stages of solubilization. The activation energy of denaturation ( E act) was calculated from the scan rate dependence of the T m and from the rate of rhodopsin thermal bleaching at all stages of solubilization. The E act as determined by both techniques decreased in the subsolubilizing stage, but remained constant once fully solubilized. These results indicate the bilayer structure increases the E act to rhodopsin denaturation.

Rhodopsin is spatially heterogeneously distributed in rod outer segment disk membranes

The visual photoreception takes place in the retina, where specialized rod and cone photoreceptor cells are located. The rod outer segments contain a stack of 500-2,000 sealed membrane disks. Rhodopsin is the visual pigment located in rod outer segment disks, it is a member of the G-protein-coupled receptor (GPCR) superfamily, an important group of membrane proteins responsible for the majority of physiological responses to stimuli such as light, hormones, peptides, etc. Alongside rhodopsin, peripherin/Rom proteins located in the disk rims are thought to be responsible for disk morphology. Here we describe the supramolecular structure of rod outer segment disk membranes and the spatial organization of rhodopsin and peripherin/Rom molecules. Using atomic force microscopy operated in physiological buffer solution, we found that rhodopsin is loosely packed in the central region of the disks, in average about 26 000 molecules covering approximately one third of the disk surface. Peripherin/Rom proteins form dense assemblies in the rim region. A protein-free lipid bilayer girdle separates the rhodopsin and peripherin/Rom domains. The described supramolecular assembly of rhodospin, peripherin/Rom and lipids in native rod outer segment disks is consistent with the functional requirements of photoreception.

Peripherin/rds co-distributes with putative binding partners in basal rod outer segment disks

The retinal degeneration slow protein peripherin/rds, (a.k.a. RDS), encoded by the rds gene, is essential for the formation, organization and maintenance of outer segments (Arikawa et al., 1992; Connell et al., 1991; Connell and Molday, 1990; Kedzierski et al., 2001; Molday, 1998). In humans, any one of over 160 mutations within this gene results in a broad variety of late onset progressive retinal dystrophies characterized by abnormal photoreceptor structure (Dryja et al., 1997; Keen and Inglehearn, 1996; Kohl et al., 1998). The pathogenic mechanism(s) underlying peripherin/rds mediated retinal dystrophies are unknown, however new insight has been gained in our understanding of peripherin/rds function based upon recent structural studies identifying the C-terminal domain of this protein as homologous to structures called intrinsically disordered domains (IDD) (Dyson, 2005; Edrington, 2007b; Ritter, 2005). IDDs are unstructured domains that become structurally stabilized by interacting with one or with several binding partners (Uversky et al., 2008). Structural flexibility imparts multi-functionality to the protein and binding partners act as regulators of its function (Lakoucheva et al., 2002; Vucetic et al., 2007). In humans, numerous mutations in the rds C-terminus result in rod/cone or cone/rod dystrophies. In a murine model of retinitis pigmentosa, deletion of codon 307 in the rds gene results in severely dysmorphic outer segments (OSs) in mice (McNally et al., 2002). The rds 307 del mouse provides the first in vivo evidence for a correlation between intrinsic disorder and retinopathy. In these mice disruption of the peripherin/rds C-terminus results in a gene product with a more intrinsically ordered C-terminal domain characterized by an increase in α-helical content.

Rhodopsin - Rhodopsin Oligomerization in Model Lipid Bilayers - Functional Implications

We studied rhodopsin oligomerization as a function of rhodopsin concentration and lipid composition and related oligomerization to shifts in rhodopsin function. In the rod outer segment disks of the retina, rhodopsin is densely packed in phospholipid bilayers with a high content of polyunsaturated acyl chains. In model membranes, increasing rhodopsin packing density was linked to a shift in the metarhodopsin-I (MI)/metarhodopsin-II (MII) equilibrium towards MI as well as to lower rates of MII formation. We reconstituted rhodopsin into various phosphatidylcholine bilayers at rhodopsin/lipid ratios ranging from 1:1,000 to 1:70 and followed rhodopsin oligomerization by cross linking of rhodopsin and changes in lipid-rhodopsin interactions by 2H NMR. The amount of MII formed after photoactivation was determined by UV/vis spectroscopy and the rate of transducin activation studied with a GTPγS-assay. At low rhodopsin concentrations (1/300 and lower) rhodopsin appears to be predominantly monomeric. At rhodopsin/lipid ratios higher than 1/300, the level of oligomerization increases in a highly cooperative fashion with concentration such that at physiological concentrations rhodopsin is mostly oligomeric. Protein function correlated tightly with rhodopsin oligomerization. Data on the influence of bilayer properties on the monomer - oligomer transition of rhodopsin and the rate of transducin activation will be presented.

The Disk Membrane Bilayer Enhances Bovine Rhodopsin Kinetic Stability

The photoreceptor, rhodopsin is a kinetically stable membrane protein. This G-protein coupled receptor consitiutes greater than 90 percent of the membrane proteins of rod outer segment disk membranes. Therefore biophysical studies of rhodopsin can be carried out in its native membrane. The role of the native bilayer in maintaining the kinetic stability rhodopsin was investigated. The disks were systematically disrupted by the detergent, octyl-β-D-glucopyranoside (OG). Rhodopsin kinetic stability was examined under sub-solubilizing conditions in which the bilayer was partially disrupted as well as fully solubilizing conditions in which rhodopsin was largely delipidated. In differential scanning calorimetry (DSC) studies rhodopsin exhibited an irreversible scan rate dependent endothermic transition and a scan rate dependent exothermic transition at all stages of solubilization. The endothermic Tm decreased and the exothermic Tm increased as the OG partitioned into the bilayer. There was little change once the fully solubilized stage was achieved even though phospholipids were present in the mixed micelles. The activation energy of denaturation (Eact) was calculated at each stage of membrane disruption from the scan rate dependence of the Tm. The endothermic Eact decreased rapidly in the sub-solubilizing phase, but not once the membrane was fully solubilized. The Eact determined from the rate of thermal bleaching was in agreement with the DSC data. The degree of solubilization had no effect on the exothermic transition Eact. The calorimetric enthalpy (ΔHcal) was independent of the extent of solubilization. The thermal transition broadened during the sub-solubilizing phase suggesting increased protein motional freedom. There was no further broadening in the fully solubilized stage. These results indicate that the phospholipids present in detergent micelles do not kinetically stabilize the protein. The membrane bilayer enhances the kinetic stability rhodopsin by increasing the energy barrier to denaturation.

Mutation in the βA3/A1-crystallin gene impairs phagosome degradation in the retinal pigmented epithelium of the rat

Phagocytosis of the shed outer segment discs of photoreceptors is a major function of the retinal pigmented epithelium (RPE). We demonstrate for the first time that βA3/A1-crystallin, a major structural protein of the ocular lens, is expressed in RPE cells. Further, by utilizing the Nuc1 rat, in which the βA3/A1-crystallin gene is mutated, we show that this protein is required by RPE cells for proper degradation of outer segment discs that have been internalized in phagosomes. We also demonstrate that in wild-type RPE, βA3/A1-crystallin is localized to the lysosomes. However, in the Nuc1 RPE, βA3/A1-crystallin fails to translocate to the lysosomes, perhaps because misfolding of the mutant protein masks sorting signals required for proper trafficking. The digestion of phagocytized outer segments requires a high level of lysosomal enzyme activity, and cathepsin D, the major enzyme responsible for proteolysis of the outer segments, is decreased in mutant RPE cells. Interestingly, our results also indicate a defect in the autophagy process in the Nuc1 RPE, which is probably also linked to impaired lysosomal function, because phagocytosis and autophagy might share common mechanisms in degradation of their targets. βA3/A1-crystallin is a novel lysosomal protein in RPE, essential for degradation of phagocytosed material.