The biosynthesis of A2E, a fluorophore of aging retina, involves the formation of the precursor, A2-PE, in the photoreceptor outer segment membrane

The autofluorescent lipofuscin that accumulates in retinal pigment epithelial cells with age may contribute to an age-related decline in cell function. The major lipofuscin fluorophore, A2E, is a pyridinium bisretinoid. We previously proposed that the biogenesis of A2E involves the following: (i) formation of the Schiff base, N-retinylidene phosphatidylethanolamine from all-trans-retinal and phosphatidylethanolamine in the photoreceptor outer segment membrane; (ii) further reaction of N-retinylidene phosphatidylethanolamine with retinal to yield phosphatidylethanolamine-bisretinoid, A2-PE; (iii) hydrolysis of A2-PE to generate A2E. To provide evidence for this biogenic scheme, all-trans-retinal was reacted with dipalmitoyl-l-alpha-phosphatidylethanolamine to yield DP-A2-PE (A2-PE), as confirmed by UV, with mass spectrometry revealing the molecular ion at m/z 1222.9 (C(77)H(124)O(8)PN) accompanied by product ion at m/z 672.8, representing the phosphoryl-A2E fragment of A2-PE. In reaction mixtures of retinal and outer segments and in samples of Royal College of Surgeons rat retina containing outer segment membranous debris, A2-PE was detected as a series of high performance liquid chromatography peaks, each with UV similar to reference A2-PE. By mass spectrometry, A2-PE consisted of multiple peaks, representing fatty acids with different chain lengths, and the phosphoryl-A2E moiety, m/z 673. Incubation of the retinal/outer segment reaction mixture with phospholipase D generated A2E, as detected by high performance liquid chromatography, thus confirming A2-PE as the A2E precursor.     

Biogenesis of myeloid bodies in regenerating newt (Notophthalmus viridescens) retinal pigment epithelium

Summary Myeloid bodies are believed to be differentiated areas of smooth endoplasmic reticulum membranes, and they are found within the retinal pigment epithelium in a number of lower vertebrates. Previous studies demonstrated a correlation between phagocytosis of outer segment disc membranes and myeloid body numbers in the retinal pigment epithelium of the newt. To test the hypothesis that myeloid bodies are directly involved in outer segment lipid metabolism and to further characterize the origin and functional significance of these organelles, we examined the effects on myeloid bodies of eliminating the source of outer segment membrane lipids (neural retina removal) and of the subsequent return of outer segments (retinal regeneration) in the newt Notophthalmus viridescens. Light- and electron-microscopic analysis demonstrated that myeloid bodies disappeared from the pigment epithelium within six days of neural retina removal. By week 6 of regeneration, rudimentary photoreceptor outer segments were present but myeloid bodies were still absent. However, at this time, the smooth endoplasmic reticulum in some areas of the retinal pigment epithelial cells had become flattened, giving rise to small (0.5 m long), two-to-four layer-thick lamellar units, which are myeloid body precursors. Small myeloid bodies were first observed one week later at week 7 of retinal regeneration. This study revealed that newt myeloid bodies are specialized areas of smooth endoplasmic reticulum. It also showed that a contact between functional photoreceptors and the retinal pigment epithelium is essential to the presence of myeloid bodies in the epithelial cells.     

The fine structure of the pigment epithelium and photoreceptor cells of the newt,Triturus viridescens dorsalis (Rafinesque)

The morphology of the retinal pigment epithelium and photoreceptor cells has been studied in the common newt Triturus viridescens dorsalis by light, conventional transmission and scanning electron microscopy. The pigment epithelium is formed by a single layer of low rectangular cells, separated by a multilayered membrane (Bruch's membrane) from the vessels of the choriocapillaris. The scleral border of the pigment epithelium is highly infolded and each epithelial cell contains smooth endoplasmic reticulum, myeloid bodies, mitochondria, lysosomes, phagosomes and an oval nucleus. Inner, pigment laden, epithelial processes surround the photoreceptor outer and inner segments. The three retinal photoreceptor types, rods, single cones and double cones, differ in both external and internal appearance. The newt, rod, outer segments appear denser than the cones in both light and electron micrographs, due to a greater number of rod lamellae per unit distance of outer segment and to the presence of electron dense intralamellar bands. The rod outer segments possess deep incisures in the lamellae while the cone lamellae lack incisures. Both rod and cone outer segments are supported by a peripheral array of dendritic processes containing longitudinal filaments which originate in the inner segment. The inner segment mitochondria, forming the rod ellipsoid, arelong and narrow while those in the cone are spherical to oval in shape. The inner segments of all three receptor cell types also contain a glycogen-filled paraboloid and a myoid region, just outside the nucleus, rich in both rough and smooth endoplasmic reticulum. The elongate, cylindrical nuclei differ in density. The rod nuclei are denser than those of the cones, contain clumped chromatin and usually extend further vitreally. Similarly, the cytoplasm of the rod synaptic terminal is denser than its cone counterpart and contains synaptic vesicles almost twice as large as those of the cones. Photoreceptor synapses in rods and cones are established by both superficial and invaginated contacts with bipolar or horizontal cells.     

The effect of inhibitors of glycoprotein synthesis and processing on the phagocytosis of rod outer segments by cultured retinal pigment epithelial cells

Retinal pigment epithelial cells selectively phagocytize rod outer segments by a process that may be mediated by specific cell surface receptors. Since many receptors are glycoproteins, we have studied the effect of tunicamycin, an inhibitor of N-linked oligosaccharide synthesis, and of castanospermine and swainsonine, which are inhibitors of oligosaccharide processing, on the ability of cultured retinal pigment epithelial cells to phagocytize rod outer segment. Tunicamycin inhibits the glycosylation of newly synthesized glycoproteins by 85-90%; concomitantly, the phagocytosis of rod outer segments is inhibited by 70-80%. The effect of tunicamycin is to initially reduce rod outer segments binding, and therefore the subsequent ingestion of rod outer segments. SDS-PAGE analysis and autoradiography of [35S]methionine labelled extracts of tunicamycin-treated cells, demonstrates the disappearance of a number of glycoprotein bands, and the appearance of a number of protein bands of lower Mr. Kinetic analysis of the disappearance and reappearance of specific glycoproteins suggests that the lower Mr bands are the non-glycosylated forms of the higher Mr bands. By contrast, castanospermine and swainsonine have no effect on the ability of retinal pigment epithelial cells to phagocytize rod outer segments, or on the SDS-PAGE pattern of treated cells, although they were shown to inhibit oligosaccharide processing as expected. These results support the hypothesis that rod outer segment phagocytosis by retinal pigment epithelial cells is mediated by specific glycoprotein receptors. N-Glycosylation of these receptors is required for their function, or for their insertion into the plasma membrane, whereas processing of the N-linked oligosaccharide chains of these receptors is not crucial for rod outer segment phagocytosis by retinal pigment epithelial cells.     

PHOTORECEPTOR-PIGMENT EPITHELIAL CELL RELATIONSHIPS IN RATS WITH INHERITED RETINAL DEGENERATION : Radioautographic and Electron Microscope Evidence for a Dual Source of Extra Lamellar Material

Protein synthesis and displacement in photoreceptor and pigment epithelial cells of inbred normal (Fisher) and mutant (RCS) rats with inherited retinal degeneration has been studied by light and electron microscope radioautography. Groups of animals 14, 15, 17, 19, 27, 35, and 50 days of age were injected with amino acids-H³ and killed at subsequent time intervals. In normal rats, radioactive protein synthesized in the rod inner segments was incorporated into outer segment saccules and displaced outward; the total renewal time of outer segments at all ages was approximately 9 days. In RCS photoreceptors, outer segment displacement was slowed from the normal rate before day 17 and at all subsequent stages. Most of the newly synthesized protein appeared to migrate only into the basal third of the outer segments. Labeling of pigment epithelial cells in RCS rats was always heavier than in controls. Labeled protein was displaced as early as 1 hr postinjection from pigment epithelial cell somas into the apical processes, and by 2 hr postinjection was located in the adjacent lamellar whorls characteristic of the mutant rat retina. After 1 day, radioactivity was present in the 14, 15, 17, and 19 day series of RCS rats in the apical third of the outer segment layer (occupied mainly by extra lamellar material) while there were few silver grains in the middle third of the layer (occupied mainly by distal parts of outer segments). The RCS pigment epithelial cells thus have an unusual synthetic role and appear to be a source of the extra lamellar material. Electron microscope examination revealed that many intact pigment epithelial cell processes were incorporated into the large whorls of extra lamellae. In addition, many disorganized outer segment saccules were observed in continuity with longer membranous lamellae and large lamellar whorls. The extra lamellar material therefore appears to be derived from both rod outer segments and pigment epithelial cells.     

ATP-binding cassette transporter ABCA4: Molecular properties and role in vision and macular degeneration

ABCA4, also known as ABCR or the rim protein, is a member of the ABCA subfamily of ATP binding cassette (ABC) transporters expressed in vertebrate rod and cone photoreceptor cells and localized to outer segment disk membranes. ABCA4 is organized in two tandem halves, each consisting of a transmembrane segment followed successively by a large exocytoplasmic domain, a multispanning membrane domain, and a nucleotide-binding domain. Over 400 mutations in ABCA4 have been linked to Stargardt macular degeneration and related retinal degenerative diseases that cause severe vision loss in affected individuals. Direct binding studies and ATPase activation measurements have identified N-retinylidene-phosphatidylethanolamine, a product generated from the photobleaching of rhodopsin, as the substrate for ABCA4. Mice deficient in ABCA4 accumulate phosphatidylethanolamine, all-trans retinal, and N-retinylidene-phosphatidylethanolamine in photoreceptors and the diretinal pyridinium compound A2E in retinal pigment epithelial cells. On the basis of these studies, ABCA4 is proposed to actively transport or flip N-retinylidene-phosphatidylethanolamine from the lumen to the cytoplasmic side of disc membranes following the photobleaching of rhodopsin. This transport activity insures that retinoids do not accumulate in disc membranes. Disease-linked mutations in ABCA4 that result in diminished transport activity lead to an accumulation of all-trans retinal and N-retinylidene-PE in disc membranes which react to produce A2E precursors. A2E progressively accumulates as lipofuscin deposits in retinal pigment epithelial cells as a result of phagocytosis of outer segment discs. A2E and photo-oxidation products cause RPE cell death and consequently photoreceptor degeneration resulting in a loss in vision in individuals with Stargardt macular degeneration and other retinal degenerative diseases associated with mutations in ABCA4.     

Phospholipid meets all-trans-retinal: the making of RPE bisretinoids

The lipid phase of the photoreceptor outer segment membrane is essential to the photon capturing and signaling functions of rhodopsin. Rearrangement of phospholipids in the bilayer accompanies the formation of the active intermediates of rhodopsin following photon absorption. Furthermore, evidence for the formation of a condensation product between the photolyzed chromophore all-trans-retinal and phosphatidylethanolamine indicates that phospholipid may also participate in the movement of the retinoid in the membrane. The downside of these interactions is the formation of bisretinoid-phosphatidylethanolamine compounds that accumulate in retinal pigment epithelial cells with age and that are particularly abundant in some retinal disorders. The propensity of these compounds to negatively impact on the cells has been linked to the pathogenesis of some retinal disorders including juvenile onset recessive Stargardt disease and age-related macular degeneration.     

Immunocytochemical localization of vitamin A in the retina and pineal organ of the frog, Rana esculenta

Vitamin A immunoreactive sites were studied in the retina and pineal organ of the frog, Rana esculenta, by the peroxidase antiperoxidase, avidin-biotinperoxidase and immunogold methods. In dark-adapted material, strong immunoreaction was found in the outer and inner segments of the photoreceptor cells of both retina and pineal organ, as well as in the pigment epithelium, retinal Müller cells and pineal ependymal cells. In light-adapted retina, cones and green (blue-sensitive) rods were immunopositive. At the electron microscopic level, immunogold particles were found on the membranes of the photoreceptor outer segments as well as on the membranes of the endoplasmic reticulum and mitochondria. Individual retinal photorecptor cells exhibited strong immunoreaction in the distal portion of the inner segment, the ciliary connecting piece and the electron-dense material covering the outer segment. In the pigment epithelium, the immunolabeling varied in intensity in the basal and apical cytoplasm and phagocytosed outer segments. The immunocytochemical results indicate that retinoids (retinal, retinol and possibly retinoic acid) are present not only in the photoreceptor cells of the retina but also in those of the pineal organ. The light-dependent differences in the immunoreactivity of vitamin A underlines its essential role in the visual cycle of the photopigments. Our results suggest that the pineal ependyma plays a role comparable to that of the Müller cells and pigment epithelium of the retina with regard to the transport and storage of vitamin A. The presence of a retinoid in nuclei, mitochondria and cytoplasmic membranes suggests an additional role of vitamin A in other metabolic processes.     

Photoreceptors of cubozoan jellyfish

The anatomically sophisticated visual system of the cubozoan jellyfish Carybdea marsupialis is described. Individual cubomedusae have eight complex eyes, each with a cornea, lens, and retina of ciliated photoreceptor cells, eight slit ocelli, and eight dimple ocelli. The photoreceptor cells of the complex eyes are bipolar and resemble vertebrate rod cells. Each photoreceptor has an outer cylindrical light-receptive segment that projects into a vitreous space that separates the lens and the retina, an inner segment rich in pigment granules, and a basal region housing the nucleus. The outer segment is a modified cilium with a 9 + 2 arrangement of microtubules plus stacks of membrane. These stacks of membrane form numerous discs that are oriented transversely to the long axis of the cell. The outer segment is connected to the inner segment by a slender stalk. The basal end of each photoreceptor forms an axon that projects into an underlying layer of interneurons. Each ocellus is composed of ciliated photoreceptor cells containing pigment granules. Rhodopsin-like and opsin-like proteins are found in the membrane stacks of the outer segments of the photoreceptors of the complex eyes. An ultraviolet-sensing opsin-like protein is present in the inner segments and basal regions of some of the photoreceptors of the complex eyes. Rhodopsin-like proteins are also detected in the photoreceptors of the slit ocelli. The cellular lens, composed of crystallin proteins, shows a paucity of organelles and a high concentration of homogeneous cytoplasm. Neurons expressing RFamide (Arg-Phe-amide) comprise a subset of interneurons found beneath the retinas of the complex eyes. RFamide-positive fibers extend from these neurons into the stalks of the rhopalia, eventually entering into the subumbrellar nerve ring. Vision may play a role in the navigation, feeding, and reproduction of the cubomedusae.     

VITAMIN A RECEPTORS: MULTIPLE SPECIES IN RETINA AND BRAIN AND POSSIBLE COMPARTMENTALIZATION IN RETINAL PHOTORECEPTORS

Abstract— As assessed by sucrose density gradient ultracentrifugation, bovine retinal cytosol exhibits 2S and 7S vitamin A binding species ('receptors'). Upon fractionation of the retina, outer segment photoreceptor units are enriched in 7S receptor whereas the outer segment poor layers of the retina have a decreased amount of 7S receptor. The 2S vitamin A receptor is found both in the photoreceptor fraction and in the rod-poor layers of the retina. The supernatant fraction of fetal retina demonstrates 2S binding but no 7S binding; a small 7S peak observed in adult pigment epithelial supernatant preparations is also not seen in the supernatant fraction of fetal pigment epithelial cells. The binding pattern in the newborn retina is similar to that in adult retina, i.e. extensive 7S as well as 2S binding. Adult bovine brain exhibits a large 7S receptor peak which is missing in fetal brain supernatant and virtually absent in newborn brain. The 7S receptor may thus be compartmentalized in retinal photoreceptors but is not unique to the retina since it is also observed in brain. The ontogenic patterns in the two tissues are different however.     

The influence of substrate elastic modulus on retinal pigment epithelial cell phagocytosis

To better understand if a complex process such as phagocytosis is influenced by substrate stiffness, we investigated the influence of substrate elastic modulus on phagocytosis in the retinal pigment epithelial (RPE) cell line ARPE-19. RPE cells lie on Bruch?s membrane, directly under the retina, and phagocytose the shed photoreceptor outer segments. Bruch?s membrane is known to increase in stiffness by an order of magnitude with age and thus, this study has potential relevance in explaining retinal changes in age-related macular degeneration.     

The molecular regulation of organelle transport in mammalian retinal pigment epithelial cells

Retinal pigment epithelial cells contain large numbers of melanosomes that can enter the apical processes extending between the outer segments of the overlying photoreceptors. Every day the distal portion of the photoreceptor outer segment is shed and phagocytosed by the retinal pigment epithelial cell. The phagosome is then transported into the cell body and the contents degraded by lysosomal enzymes. This review focuses on recent progress made in the identification of molecules that regulate the transport of melanosomes into the apical processes and the transport of phagosomes into the cell body. Myosin VIIa is a key player in both processes and, at least in the case of melanosome movement, myosin VIIa is recruited to the melanosome via the GTPase, Rab27a. The possible role played by defects in the transport of melanosomes and phagosomes in the development of retinal degenerative diseases is discussed.     

Immunocytochemical localization of Vitamin A in the retina and pineal organ of the frog,Rana esculenta

Summary Vitamin A immunoreactive sites were studied in the retina and pincal organ of the frog,Rana esculenta, by the peroxidase antiperoxidase, avidin-biotinperoxidase and immunogold methods. Indark-adapted material, strong immunoreaction was found in the outer and inner segments of the photoreceptor cells of both retina and pineal organ, as well as in the pigment epithelium, retinal Müller cells and pineal ependymal cells. Inlight-adapted retina, cones and green (blue-sensitive) rods were immunopositive.At the electron microscopic level, immunogold particles were found on the membranes of the photoreceptor outer segments as well as on the membranes of the endoplasmic reticulum and mitochondria. Individual retinal photoreceptor cells exhibited strong immunoreaction in the distal portion of the inner segment, the ciliary connecting piece and the electron-dense material covering the outer segment. In the pigment epithelium, the immunolabeling varied in intensity in the basal and apical cytoplasm and phagocytosed outer segments.The immunocytochemical results indicate that retinoids (retinal, retinol and possibly retinoic acid) are present not only in the photoreceptor cells of the retina but also in those of the pineal organ. The light-dependent differences in the immunoreactivity of vitamin A underlines its essential role in the visual cycle of the photopigments. Our results suggest that the pineal ependyma plays a role comparable to that of the Müller cells and pigment epithelium of the retina with regard to the transport and storage of vitamin A. The presence of a retinoid in nuclei, mitochondria and cytoplasmic membranes suggests an additional role of vitamin A in other metabolic processes.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthdaySupported by the Hungarian OTKA grant Nr. 1619 to B.V., and a grant from the Pardee Foundation to G.H.W.     

Annexin A2 Regulates Phagocytosis of Photoreceptor Outer Segments in the Mouse Retina

The daily phagocytosis of shed photoreceptor outer segments by pigment epithelial cells is critical for the maintenance of the retina. In a subtractive polymerase chain reaction analysis, we found that functional differentiation of human ARPE19 retinal pigment epithelial (RPE) cells is accompanied by up-regulation of annexin (anx) A2, a major Src substrate and regulator of membrane–cytoskeleton dynamics. Here, we show that anx A2 is recruited to the nascent phagocytic cup in vitro and in vivo and that it fully dissociates once the phagosome is internalized. In ARPE19 cells depleted of anx A2 by using small interfering RNA and in ANX A2^−/− mice the phagocytosis of outer segments was impaired, and in ANX A2^−/− mice there was an accumulation of phagocytosed outer segments in the RPE apical processes, indicative of retarded phagosome transport. We show that anx A2 is tyrosine phosphorylated at the onset of phagocytosis and that the synchronized activation of focal adhesion kinase and c-Src is abnormal in ANX A2^−/− mice. These findings reveal that anx A2 is involved in the circadian regulation of outer segment phagocytosis, and they provide new insight into the protein machinery that regulates phagocytic function in RPE cells.     

Retinal Pigment Epithelium–Calycal Processes Association in the Catfish Clarias batrachus(Linnaeus, 1758)

Abstract In the duplex retina of the catfish Clarias batrachus(Linnaeus, 1758), the apical processes of the pigment epithelial cells have been found by transmission electron microscopy to be in intimate contact with the calycal processes around the basal portion of the photoreceptor outer segments. It is hypothesized that the retinal pigment epithelium effectively transports synthesized products and metabolites to the photoreceptor inner segments via the anatomical zone of the apical–calycal processes interface in this species.     

Characterization of dihydro-A2PE: an intermediate in the A2E biosynthetic pathway

Bisretinoid lipofuscin pigments that accumulate in retinal pigment epithelial cells are implicated in the etiology of several forms of macular degeneration, including juvenile onset Stargardt disease, Best vitelliform macular degeneration, and age-related macular degeneration. One of these compounds, A2E, is generated by phosphate hydrolysis of a phosphatidyl-pyridinium bisretinoid (A2PE) that forms within photoreceptor outer segments. Here, we demonstrate that the formation of the aromatic pyridinium ring of A2PE follows from the oxidation of a dihydropyridinium intermediate. Time-dependent density functional theory calculation, based on the structure of dihydro-A2E, produced a simulated UV-visible absorbance spectrum characterized by maxima of 494 and 344 nm. Subsequently, a compound exhibiting similar UV-visible absorbance maxima (lambdamax 490 and 330 nm) was identified in the A2E biomimetic reaction mixture. By liquid chromatography-mass spectrometry (LC-MS) this bischromophore had the expected mass of the dihydro-pyridinium bisretinoid. The compound also exhibited the behavior of a biosynthetic intermediate since it formed in advance of the final product A2E and was consumed as A2E accumulated. Moreover, under deoxygenated conditions, conversion to the aromatic pyridinium bisretinoid was inhibited. Taken together, these findings indicate that A2E biosynthesis involves the oxidation of a dihydropyridinium intermediate dihydro-A2PE. An understanding of the biosynthetic pathways of retinal pigment epithelial lipofuscin pigments is critical to the development of therapies for macular degeneration that are based on limiting the formation of these damaging compounds.     

Embryonic fissure and photoreceptor differentiation in the eye of adult Garra rufa Heckel 1843 (Cyprinidae, Teleostei)

The retina of the adult teleost Garra rufa retains a curved, open embryonic fissure indicating an asymmetrical postembryonic retinal growth. Undifferentiated, oval photoreceptors are observed on both sides of the middle of the fissure with their larger diameter running parallel to the fissure to which they may attach by desmosomes. They detach from the fissure, rotate to become perpendicular to it and begin an active process of differentiation as they slide along the temporal side of the outer half of the fissure. This process is divided into stages for simplicity. The photoreceptors develop stumpy inner segments extending into a ventricular space that appears between the retinal pigment epithelium and the photoreceptors. Calycal processes arise from the inner segments and the distal centriole of each photoreceptor forms a connecting cilium. The proximal centriole is retained for some time after the outer segment develops. The formation of rod spherules and cone pedicles takes place almost concomitantly with the outer segments. Double cones appear first as single cones before pairing. One or more of the principal cone mitochondria accumulate electron-dense material and merge to form the ellipsosome. The retinal pigment epithelium undergoes a parallel differentiation. The developmental events described in the present work conform those recorded in embryonic teleostean retinas.     

猫外层视网膜和脉络膜的年龄相关变化

取老年猫(12龄,2.5~3 kg)和青年猫(1~3龄,2~2.5 kg)各4只的视网膜,经4%多聚甲醛处理后用H.E染色以显示视网膜和脉络膜的结构。光学显微镜下观察感光细胞层、玻璃膜(Bruch’s membrane)结构的变化,计数色素上皮层(retinal pigment epithelium,RPE)细胞数、脉络膜毛细血管数,测量玻璃膜、脉络膜厚度,脉络膜毛细血管之间的距离。结果显示,与青年猫比较,老年猫视网膜感光细胞层结构杂乱;色素上皮细胞数显著下降;玻璃膜厚度无显著变化,出现较多碎片;脉络膜厚度明显变薄,脉络膜毛细血管数显著减小,脉络膜毛细血管之间的距离显著增大。推测老年猫脉络膜的退化可能是导致玻璃膜、色素上皮层的退化,进而导致感光细胞的功能衰退的重要原因。     

Localization of peripherin/rds in the disk membranes of cone and rod photoreceptors: relationship to disk membrane morphogenesis and retinal degeneration

The outer segments of vertebrate rod photoreceptor cells consist of an ordered stack of membrane disks, which, except for a few nascent disks at the base of the outer segment, is surrounded by a separate plasma membrane. Previous studies indicate that the protein, peripherin or peripherin/rds, is localized along the rim of mature disks of rod outer segments. A mutation in the gene for this protein has been reported to be responsible for retinal degeneration in the rds mouse. In the present study, we have shown by immunogold labeling of rat and ground squirrel retinas that peripherin/rds is present in the disk rims of cone outer segments as well as rod outer segments. Additionally, in the basal regions of rod and cone outer segments, where disk morphogenesis occurs, we have found that the distribution of peripherin/rds is restricted to a region that is adjacent to the cilium. Extension of its distribution from the cilium coincides with the formation of the disk rim. These results support the model of disk membrane morphogenesis that predicts rim formation to be a second stage of growth, after the first stage in which the ciliary plasma membrane evaginates to form open nascent disks. The results also indicate how the proteins of the outer segment plasma membrane and the disk membranes are sorted into their separate domains: different sets of proteins may be incorporated into membrane outgrowths during different growth stages of disk morphogenesis. Finally, the presence of peripherin/rds protein in both cone and rod outer segment disks, together with the phenotype of the rds mouse, which is characterized by the failure of both rod and cone outer segment formation, suggest that the same rds gene is expressed in both types of photoreceptor cells.     

Contribution of Müller cells toward the regulation of photoreceptor outer segment assembly

The assembly of photoreceptor outer segments into stacked discs is a complicated process, the precise regulation of which remains a mystery. It is known that the integrity of the outer segment is heavily dependent upon surrounding cell types including the retinal pigment epithelium and Müller cells; however the role played by Müller cells within this photoreceptor-specific process has not been fully explored. Using an RPE-deprived but otherwise intact Xenopus laevis eye rudiment preparation, we reveal that Müller cell involvement in outer segment assembly is dependent upon the stimulus provided to the retina. Pigment epithelium-derived factor is able to support proper membrane folding after inhibition of Müller cell metabolism by alpha-aminoadipic acid, while isopropyl beta-D-thiogalactoside, a permissive glycan, requires intact Müller cell function. These results demonstrate that both intrinsic and extrinsic redundant mechanisms exist to support the ability of photoreceptors to properly assemble their outer segments. Our study further suggests that the receptor for pigment epithelium-derived factor resides in photoreceptors themselves while that for permissive glycans is likely localized to Müller cells, which in turn communicate with photoreceptors to promote proper membrane assembly.