Mertk triggers uptake of photoreceptor outer segments during phagocytosis by cultured retinal pigment epithelial cells

The RCS rat is a widely studied model of recessively inherited retinal degeneration. The genetic defect, known as rdy (retinal dystrophy), results in failure of the retinal pigment epithelium (RPE) to phagocytize shed photoreceptor outer segment membranes. We previously used positional cloning and in vivo genetic complementation to demonstrate that Mertk is the gene for rdy. We have now used a rat primary RPE cell culture system to demonstrate that the RPE is the site of action of Mertk and to obtain functional evidence for a key role of Mertk in RPE phagocytosis. We found that Mertk protein is absent from RCS, but not wild-type, tissues and cultured RPE cells. Delivery of rat Mertk to cultured RCS RPE cells by means of a recombinant adenovirus restored the cells to complete phagocytic competency. Infected RCS RPE cells ingested exogenous outer segments to the same extent as wild-type RPE cells, but outer segment binding was unaffected. Mertk protein progressively co-localized with outer segment material during phagocytosis by primary RPE cells, and activated Mertk accumulated during the early stages of phagocytosis by RPE-J cells. We conclude that Mertk likely functions directly in the RPE phagocytic process as a signaling molecule triggering outer segment ingestion.     

Ezrin, a membrane-organizing protein, as a polarization marker of the retinal pigment epithelium in vertebrates

Immunoreactivity for ezrin, a membrane-organizing phosphoprotein that tethers actin microfilaments to cell membrane proteins, was evaluated as a polarization marker in the intraocular neuroepithelial cells of vertebrates, especially in the retinal pigment epithelium (RPE). Six fetal human eyes representing the 14th-28th gestational weeks, 9 normal adult eyes, 12 eyes with intraocular tumors, and 26 eyes from 15 other vertebrate species were analyzed by immunohistochemistry using the avidin-biotinylated peroxidase complex (ABC) method and monoclonal antibody (mAb) 3C12 to ezrin. The apical cytoplasm and microvilli of the human RPE always reacted with mAb 3C12, but the basal cytoplasm was labeled in reactive RPE only. In autopsy eyes and if fixation was delayed, ezrin immunoreactivity in RPE was more diffuse. Developing RPE became gradually immunoreactive from the 14th week of gestation onward. The microvilli of the baboon, pig, raccoon dog, cow, and rat RPE cells were likewise labeled, and their basal cytoplasm was variably immunoreactive as well, but the microvilli of the avian RPE did not react with the antibody used. In all six mammals mentioned, both layers of the ciliary epithelium and the anterior iris epithelium reacted for ezrin, and the posterior epithelium was weakly labeled in pig, cow, and rat eyes. Normal peripheral and reactive human retina, and normal baboon, pig, raccoon dog, cow, rat, black grouse, and jay eyes, showed immunoreaction for ezrin in Müller cells, usually in their microvilli. Ezrin is widely found in RPE and anterior segment neuroepithelia of the mammalian eye, in which it may segregate membrane proteins to specific membrane surfaces, especially to the apical microvilli of the RPE, which intimately interact with outer segments of photoreceptor cells. The ezrin gene on human chromosome 6q25-26 is consequently a candidate gene for causing retinal degenerations.     

Membranes of retinal pigment epithelial cells in vitro are damaged in the phagocytotic process of the photoreceptor outer segment discs peroxidized by ferrous ions

The ferrous ions released from haemoglobin and storage-transferrin ions cause oxidative stress in the eyes. We observed the phagocytotic process of the photoreceptor outer segment discs peroxidized by ferrous ions in the retinal pigment epithelial (RPE) cells in vitro, and investigated how the ferrous ions influenced RPE in vitro and the photoreceptor outer segment discs. We obtained isolated photoreceptor outer segment discs using sucrose gradient of specific gravity after homogenizing porcine retinas. After bovine RPE cells were cultured with isolated photoreceptor outer segment discs containing FeCl2 for 5 and 24 h, we incubated the specimens with rhodamine phalloidin, antimouse alpha-tubulin antibody and antimouse Ig G (FITC and rhodamine labelled). We observed the specimens by a laser scanning microscopy, and made the ultrathin sections with or without 2% uranyl acetate and 2% lead acetate for examination by transmission electron microscopy. Actin filaments and microtubules of specialized cells such as RPE cells were actively involved in phagocytosis of the photoreceptor outer segment discs. Microtubules were damaged during the phagocytotic process of the photoreceptor outer segment discs peroxidized by ferrous ions. The peroxidation increased the granular and aggregated autofluorescence of the photoreceptor outer segment discs. The membranes of the disc and the phagosomes, and lysosomes in RPE cells were damaged by ferrous ions and had fine particles with high electron density staining without uranium acetate and lead citrate. The cytoskeletons such as actin filaments and microtubules, and the membranes of the phagosomes and the lysosomes in RPE cells in vitro were damaged during the phagocytotic process of the photoreceptor outer segment discs peroxidized by ferrous ions.     

Products of lipid peroxidation induce missorting of the principal lysosomal protease in retinal pigment epithelium

Phagocytosis of photoreceptor outer segments (OS) by retinal pigment epithelium (RPE) is essential for OS renewal and survival of photoreceptors. Internalized, oxidatively modified macromolecules perturb the lysosomal function of the RPE and can lead to impaired processing of photoreceptor outer segments. In this study, we sought to investigate the impact of intracellular accumulation of oxidatively damaged lipid-protein complexes on maturation and distribution of cathepsin D, the major lysosomal protease in the RPE. Primary cultures of human RPE cells were treated with copper-oxidized low density lipoprotein (LDL) and then challenged with serum-coated latex beads to stimulate phagocytosis. Three observations were noted to occur in this experimental system. First, immature forms of cathepsin D (52 and 46 kDa) were exclusively associated with latex-containing phagosomes. Second, maturation of cathepsin D was severely impaired in RPE cells loaded with oxidized LDL (oxLDL) prior to the phagocytic challenge. Third, pre-treatment with oxLDL caused sustained secretion of pro-cathepsin D and the latent form of gelatinase A into the extracellular space in a dose-dependent manner. These data stimulate the hypothesis that intracellular accumulation of poorly degradable, oxidized lipid-protein cross-links, may alter the turnover of cathepsin D, causing its mistargeting into the extracellular space together with the enhanced secretion of a gelatinase.     

Mesd extrinsically promotes phagocytosis by retinal pigment epithelial cells

Phagocytosis is a critical process to maintain tissue homeostasis. In the retina, photoreceptor cells renew their photoexcitability by shedding photoreceptor outer segments (POSs) in a diurnal rhythm. Shed POSs are phagocytosed by retinal pigment epithelial (RPE) cells to prevent debris accumulation, retinal degeneration, and blindness. Phagocytosis ligands are the key to understanding how RPE recognizes shed POSs. Here, we characterized mesoderm development candidate 2 (Mesd or Mesdc2), an endoplasmic reticulum (ER) chaperon for low-density lipoprotein receptor-related proteins (LRPs), to extrinsically promote RPE phagocytosis. The results showed that Mesd stimulated phagocytosis of fluorescence-labeled POS vesicles by D407 RPE cells. Ingested POSs were partially degraded within 3 h in some RPE cells to dispense undegradable fluorophore throughout the cytoplasm. Internalized POSs were colocalized with phagosome biomarker Rab7, suggesting that Mesd-mediated engulfment is involved in a phagocytosis pathway. Mesd also facilitated phagocytosis of POSs by primary RPE cells. Mesd bound to unknown phagocytic receptor(s) on RPE cells. Mesd was detected in the cytoplasm, but not nuclei, of different retinal layers and is predominantly expressed in the ER-free cellular compartment of POSs. Mesd was not secreted into medium from healthy cells but passively released from apoptotic cells with increased membrane permeability. Released Mesd selectively bound to the surface of POS vesicles and apoptotic cells, but not healthy cells. These results suggest that Mesd may be released from and bind to shed POSs to facilitate their phagocytic clearance.     

Increased phagocytosis of outer segments in the presence of serum by cultured normal, but not dystrophic, rat retinal pigment epithelium

Cultured normal rat retinal pigment epithelium (PE) ingested six times more rod outer segments in the presence of 20% fetal bovine serum than in serum-free medium. PE cultured from Royal College of Surgeons (RCS) rats with hereditary retinal dystrophy, known to have a defect in vivo in the phagocytosis of shed outer segment tips, ingested amounts of outer segments comparable to normal PE in serum-free medium but did not show an increase in the presence of serum. In both strains of rat PE phagocytosis of latex spheres was similar in the absence of serum and was six-fold higher in the presence of serum, showing that the RCS phagocytic deficiency for outer segments in vitro is not due to a general defect in the phagocytic capacity of the cell. Increased phagocytosis of outer segments by normal PE was observed in the presence of the high molecular weight fraction of ultrafiltered serum but was not seen with serum that was heated at 93 degrees C or precipitated with 5% trichloroacetic acid. Bovine serum albumin had no effect on phagocytosis. These results are consistent with the idea that the phagocytosis of outer segments by cultured normal rat PE, but not by cultured RCS rat PE, is increased in the presence of a specific protein or other macromolecular component of fetal bovine serum.     

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.     

Rat retinal pigment epithelial cells show specificity of phagocytosis in vitro

The retinal pigment epithelial (RPE) cell of the eye normally phagocytozes only retinal rod outer segments (ROS). The specificity of this phagocytic process was examined by incubating RPE cells with a variety of particle types. Confluent RPE cell cultures were incubated for 3 h at 37 degrees C in the presence of rat ROS, rat red blood cells (RBC), algae, bacteria, or yeast. Other cell cultures were incubated with equal numbers of ROS and one other particle type. Quantitative scanning electron microscopy was used to determine the numbers and morphology of particles bound to RPE cells, while double immunofluorescence labeling (Chaitin, M. H., and M. O. Hall, 1983, Invest. Ophthalmol. Vis. Sci., 24:812-820) was used to quantitate particle binding and ingestion. Both assays demonstrated phagocytosis to be a highly specific process. RPE cells bound 40-250 X more ROS than RBC, 30 X more ROS than algae, and 5 X more ROS than bacteria or yeast. Ingestion was more specific than binding; RPE cells ingested 970 X more ROS than RBC, 140 X more ROS than bacteria, and 35 X more ROS than yeast. The phagocytic preference for ROS was maintained in competition experiments with other particle types. Serum was found to be essential for phagocytosis. This study demonstrates that both the binding and ingestion phases of phagocytosis are highly specific processes.     

A study of photoreceptor-retinal pigment epithelium complex: age-related changes in monkeys

Retinas of 4-, 10-, and 20-year-old monkeys were studied by light microscopy, electron microscopy, and scanning electron microscopy. Sections from the midperipheral region of every retina were selected for comparison. Although no significant differences were found between 4- and 10-year-old retinas, four major changes were found in 20-year-old monkey retinas: (i) increased number of displaced photoreceptor cells (DPC), (ii) increased number of macrophages of different morphology in subretinal space, (iii) increase in pigment granules in retinal pigment epithelium (RPE) cells, and (iv) altered morphology of Muller cells. DPC included both rods and cones. Their location and morphology depended on the stage of their displacement. These cells were usually oval or rounded in shape and were found either among the outer segments of other photoreceptor cells, having stalks extending into the outer nuclear layer, or were located in the subretinal space and had no stalk. A narrow space around the DPC stalks, indicating a change in the intercellular connection between photoreceptor cells and Muller cells, was observed. Furthermore, the Muller cells related to DPC had shortened and markedly reduced microvilli. Two types of macrophages were found in the subretinal space of aged monkey retinas. One type was similar in morphology to RPE cells. Some of these cells were noticed detaching from RPE. Other types of macrophages were nonpigmented. The modifications in RPE were closely related to the changes in the associated neuroretina. The RPE cells in aged retina were devoid of microvilli or had a few thin microvilli. The pleomorphic pigment granules were dispersed throughout the cytoplasm. These cells varied in their size, shape, and surface features. These changes could significantly alter the retinal metabolic equilibrium and may be indicative of age related degenerative processes.     

X-Ray Microanalysis and Phagocytotic Activity of Cultured Retinal Pigment Epithelial Cells in Hypoxia

Purpose: To investigate the influence of the functional and morphological changes induced in retinal pigment epithelial (RPE) cells by retinal ischemia, we evaluated the phagocytotic activity, the concentration of various elements, and ultrastructure in cultured RPE cells in hypoxia. Methods: The concentrations of oxygen in incubators were adjusted to 20, 10, and 1% by the addition of nitrogen for 72 hr. To observe phagocytotic activity and its relationship to actin filaments, the filaments of RPE cells incubated with fluoresbrite carboxylate YG microspheres were stained with rhodamine phalloidin. Some of the specimens were subjected to X-ray microanalysis by scanning electron microscope after being fixed, freeze-dried, and coated with carbon to investigate the cytoplasmic concentration of elements. A part of the latter specimens was also observed by transmission electron microscope after being embedded in epon and cut into ultrathin sections to see the ultra-structural changes inside cell. Results: Lowering oxygen concentrations from 20% to 1% swelled RPE cells and decreased the number of fluoresbrite carboxylate YG microspheres phagocytized by RPE cells. Phagocytosis of a large amount of latex beads (30 μl) for 24 hr in 1% oxygen caused a disruption of RPE cells. Na, S, and P were detected in RPE cells cultured in 20% oxygen. Reducing the oxygen concentration from 20 to 10 or 1% significantly decreased Na and increased S. Mitochondria were observed in RPE cells in 20 and 10% oxygen, but many vacuoles were observed in the cytoplasm in 1% oxygen. Conclusion: Hypoxia as low as 1% oxygen induced malfunction of phagocytosis and the fragility of RPE cells. We could speculate the imbalance of the electrolytes such as Na or a decrease of antioxidants such as glutathione containing S as a reason of disturbance of cell viability.     

Electron microscopic studies of the endocytotic process of cationized ferritin in cultured human retinal pigment epithelial cells

Summary The endocytotic process in cultured human RPE cells was observed after 1 min, 20 min, and 2 h incubation with cationized ferritin. Within 1 min the ferritin particles were seen to attach to the cell membrane, especially between microvilli. Uncoated and coated pits could be recognized on the cell membranes, and uncoated and coated endocytotic vesicles were found in the cytoplasm after 20 min of incubation. These vesicles were surrounded by abundant microfilaments and had no visible membranes. Loss of membrane may be an initial step in the process of developing into the irregular clumps of ferritin particles found inside the plasma membrane. With time, more irregular clumps of ferritin, smaller than the particles introduced during incubation, appeared just beneath the cell membrane. Lysosomes were adjacent to the clumps of ferritin particles associated with microtobules and finally degraded these particles. The phagolysosomes containing many particles were surrounded by many microtubules. Small ferritin particles surrounded but had not entered the rough endoplasmic reticulums, and no particles were seen either around or in the Golgi apparatus. Presented at the 7th International Congress of Eye Research, Nagoya, Japan, 27 September 1986.     

Experimental autoimmune uveoretinitis initiated by non‐phagocytic destruction of inner segments of photoreceptor cells by Mac‐1+ mononuclear cells

EAU in mice is a model of human posterior uveitis. EAU is a Th1‐dependent disease that has been assumed to target the neural retina and related tissues; however, in situ effector cells and the target have not yet been clearly demonstrated. In the present study, we induced EAU in B10R mice by immunizing them with human interphotoreceptor retinoid‐binding protein peptide 161–180. Histological examinations revealed that EAU occurred approximately 11 days after the immunization and reached a peak on day 14. Retinae from normal or EAU mice were treated with proteases to obtain mono‐dispersed cells. The mono‐dispersed cells thus obtained were separated into three to four fractions by discontinuous Percoll density‐gradient (e.g. PBS/40/60) centrifugation. In normal mice, 94% of the total cells were recovered in two fractions (i.e. PBS/40 and pellet); and these fractions mainly contained inner and outer segments and cell bodies of photoreceptor cells and RPE cells, respectively. In EAU mice, additional cells (i.e. inflammatory cells) were obtained at the 40/60 interface. Electron microscopic examination showed that tissue damage during EAU was initiated by non‐phagocytic destruction of inner segments by Mac‐1⁺ mononuclear cells on day 11, followed by phagocytic activity of macrophages against outer segments and RPE cells on day 14. In vitro culturing of normal retinal cells with EAU infiltrates suggested the involvement of TNF‐α and NO in the tissue damage. These results indicate that EAU was initiated by non‐phagocytic destruction of inner segments of photoreceptor cells by Mac‐1⁺ mononuclear cells.     

Immunocytochemical localization of two retinoid-binding proteins in vertebrate retina

The recent discovery and characterization of several proteins that purify with endogenous, bound retinoid have given rise to the suggestion that these proteins, which are abundant in retina, perform a role in transport and function of vitamin A. Immunocytochemical techniques were used to localize two retinoid-binding proteins in the retina of four species. Antisera to cellular retinal-binding protein (CRALBP) and an interphotoreceptor retinoid-binding protein (IRBP) were obtained from rabbits immunized with antigens purified from bovine retina. Antibodies from each antiserum reacted with a single component in retinal homogenates and supernatants which corresponded to the molecular weight and charge of the respective antigen (non-SDS and SDS PAGE, electrophoretic transfer to nitrocellulose, immunochemical staining). Immunocytochemistry controls were antibodies from nonimmune serum and antibodies absorbed with purified antigen. Antigens were localized on frozen-sectioned bovine, rat, monkey, and human retina using immunofluorescence and the peroxidase-antiperoxidase technique. Specific staining with anti-IRBP was found in the space that surrounds photoreceptor outer segments, with heaviest labeling in a line corresponding to the retinal pigment epithelium (RPE) apical surface. Cone outer segments were positive. Staining with anti-CRALBP was found in two cell types in all species: the RPE and the Muller glial cell. Within the RPE, labeling filled the cytoplasm and was heaviest apically, with negative nuclei. Labeling of Muller cells produced Golgi- like silhouettes with intense staining of all cytoplasmic compartments. Staining of the external limiting membrane was heavy, with labeled microvilli projecting into the interphotoreceptor space. Localization of IRBP to this space bordered by three cell types (RPE, photoreceptor, and Muller) is consistent with its proposed role in transport of retinoids among cells. Localization of CRALBP in RPE corroborates previous biochemical studies; its presence in the Muller cell suggests that this glial cell may play a hitherto unsuspected role in vitamin A metabolism in retina.     

Anatomy and fine structure of the alimentary canal of the spittlebug Lepyronia coleopterata (L.) (Hemiptera: Cercopoidea)

The alimentary canal of the spittlebug Lepyronia coleopterata (L.) differentiates into esophagus, filter chamber, midgut (conical segment, tubular midgut), and hindgut (ileum, rectum). The filter chamber is composed of the anterior extremity of the midgut, posterior extremity of the midgut, proximal Malpighian tubules, and proximal ileum; it is externally enveloped by a thin cellular sheath and thick muscle layers. The sac-like anterior extremity of the midgut is coiled around by the posterior extremity of the midgut and proximal Malpighian tubules. The tubular midgut is subdivided into an anterior tubular midgut, mid-midgut, posterior tubular midgut, and distal tubular midgut. Four Malpighian tubules run alongside the ileum, and each terminates in a rod closely attached to the rectum. Ultrastructurally, the esophagus is lined with a cuticle and enveloped by circular muscles; its cytoplasm contains virus-like fine granules of high electron-density. The anterior extremity of the midgut consists of two cellular types: (1) thin epithelia with well-developed and regularly arranged microvilli, and (2) large cuboidal cells with short and sparse microvilli. Cells of the posterior extremity of the midgut have regularly arranged microvilli and shallow basal infoldings devoid of mitochondria. Cells of the proximal Malpighian tubule possess concentric granules of different electron-density. The internal proximal ileum lined with a cuticle facing the lumen and contains secretory vesicles in its cytoplasm. Dense and long microvilli at the apical border of the conical segment cells are coated with abundant electron-dense fine granules. Cells of the anterior tubular midgut contain spherical secretory granules, oval secretory vesicles of different size, and autophagic vacuoles. Ferritin-like granules exist in the mid-midgut cells. The posterior tubular midgut consists of two cellular types: 1) cells with shallow and bulb-shaped basal infoldings containing numerous mitochondria, homocentric secretory granules, and fine electron-dense granules, and 2) cells with well-developed basal infoldings and regularly-arranged apical microvilli containing vesicles filled with fine granular materials. Cells of the distal tubular midgut are similar to those of the conical segment, but lack electron-dense fine granules coating the microvilli apex. Filamentous materials coat the microvilli of the conical segment, anterior and posterior extremities of the midgut, which are possibly the perimicrovillar membrane closely related to the nutrient absorption. The lumen of the hindgut is lined with a cuticle, beneath which are cells with poorly-developed infoldings possessing numerous mitochondria. Single-membraned or double-membraned microorganisms exist in the anterior and posterior extremities of the midgut, proximal Malpighian tubule and ileum; these are probably symbiotic.     

Generation of retinal pigment epithelial cells from human embryonic stem cell-derived spherical neural masses

Dysfunction and loss of retinal pigment epithelium (RPE) are major pathologic changes observed in various retinal degenerative diseases such as aged-related macular degeneration. RPE generated from human pluripotent stem cells can be a good candidate for RPE replacement therapy. Here, we show the differentiation of human embryonic stem cells (hESCs) toward RPE with the generation of spherical neural masses (SNMs), which are pure masses of hESCs-derived neural precursors. During the early passaging of SNMs, cystic structures arising from opened neural tube-like structures showed pigmented epithelial morphology. These pigmented cells were differentiated into functional RPE by neuroectodermal induction and mechanical purification. Most of the differentiated cells showed typical RPE morphologies, such as a polygonal-shaped epithelial monolayer, and transmission electron microscopy revealed apical microvilli, pigment granules, and tight junctions. These cells also expressed molecular markers of RPE, including Mitf, ZO-1, RPE65, CRALBP, and bestrophin. The generated RPE also showed phagocytosis of isolated bovine photoreceptor outer segment and secreting pigment epithelium-derived factor and vascular endothelial growth factor. Functional RPE could be generated from SNM in our method. Because SNMs have several advantages, including the capability of expansion for long periods without loss of differentiation capability, easy storage and thawing, and no need for feeder cells, our method for RPE differentiation may be used as an efficient strategy for generating functional RPE cells for retinal regeneration therapy.     

Changes in retinal pigment epithelial cell autofluorescence and protein expression associated with phagocytosis of rod outer segments in vitro.

The accumulation of autofluorescent lipofuscin was quantified in cultured human retinal pigment epithelial (RPE) cells phagocytosing bovine rod outer segments (BROS) and the expression of proteins in these cells was investigated. Results showed a steady increase in autofluorescence of RPE cells over a 4-week period as measured by fluorophotometric flow cytometry. A significantly greater increase in autofluorescence was found in the cultured RPE cells from a 7-year-old donor compared with those from a 47-year-old donor. Within both groups the BROS-challenged cells had significantly higher fluorescence readings than the control cells which were not challenged. Autoradiography of 35S-labelled proteins separated by polyacrylamide gel electrophoresis (PAGE) revealed a small distinct band at 102 kDa in BROS-challenged RPE cells of both bovine and human origin that did not appear in control or microsphere-phagocytosing RPE cells. The intensity of the signal was unrelated to the duration of the challenge period.     

Structure of pollen grains of Tradescantia reflexa with special reference to the generative cell and the ER around it

A pollen grain in Tradescantia reflexa consists of two cells, the generative and the vegetative cells, the generative cell being surrounded completely by the vegetative cell. The generative cell has many lobes or surface invaginations. A complicated network of rER extends throughout the entire vegetative cytoplasm, forming a system of channels made up by the cisternae of rER. Lipid granules are surrounded by ER. Branches of the rER enter all the concavities of the invaginations and attach to the plasma membranes at the bottoms of the invaginations. In the generative cell, no reserve substances, such as lipids, are seen. There is little ER, mitochondria are few in number, and Golgi bodies seem to be less active within this type of cell. Bundles of microtubules run parallel to the long axis of the generative cell. No microtubules or microfilaments can be detected at or near the bottoms of concavities, either on the generative or the vegetative side. ER is the sole cell element that bears a positional relationship to the invaginations. It appears, therefore, that rER is intimately involved in the shaping of the invaginations. This is the first report that a cell element other than microtubules and microfilaments can be involved in the formation of the outer shape of a cell. The possibility that materials from decomposed lipid droplets are transported through the rER to the generative cell is also discussed.     

The Retinal Pigment Epithelium Utilizes Fatty Acids for Ketogenesis: IMPLICATIONS FOR METABOLIC COUPLING WITH THE OUTER RETINA*

Every day, shortly after light onset, photoreceptor cells shed approximately a tenth of their outer segment. The adjacent retinal pigment epithelial (RPE) cells phagocytize and digest shed photoreceptor outer segment, which provides a rich source of fatty acids that could be utilized as an energy substrate. From a microarray analysis, we found that RPE cells express particularly high levels of the mitochondrial HMG-CoA synthase 2 (Hmgcs2) compared with all other tissues (except the liver and colon), leading to the hypothesis that RPE cells, like hepatocytes, can produce β-hydroxybutyrate (β-HB) from fatty acids. Using primary human fetal RPE (hfRPE) cells cultured on Transwell filters with separate apical and basal chambers, we demonstrate that hfRPE cells can metabolize palmitate, a saturated fatty acid that constitutes ≈15% of all lipids in the photoreceptor outer segment, to produce β-HB. Importantly, we found that hfRPE cells preferentially release β-HB into the apical chamber and that this process is mediated primarily by monocarboxylate transporter isoform 1 (MCT1). Using a GC-MS analysis of ¹³C-labeled metabolites, we showed that retinal cells can take up and metabolize ¹³C-labeled β-HB into various TCA cycle intermediates and amino acids. Collectively, our data support a novel mechanism of RPE-retina metabolic coupling in which RPE cells metabolize fatty acids to produce β-HB, which is transported to the retina for use as a metabolic substrate.     

Organ of Bellonci of an Antarctic crustacean,the marine isopod Glyptonotus antarcticus

The paired organ of Bellonci protrudes from the optic lobe of the giant Antarctic isopod, Glyptonotus antarcticus. It is linked to the cortex by a broad peduncle. No connection to the cuticle or “sensory pore organ” was found. A cluster of sensory-like cells forms two outer ciliary segments branching into numerous microvilli with microtubules. The putative sensory somata are irregular in shape and contain a very high density of glycogen granules. The two outer segments sprout from two pits of the soma in different directions, forming a right angle. Glial cells wrap around the sensory cells and also delimit lacunae into which bundles of microvilli project. These lacunae contain electron-dense granules of small size and with species-specific patterns. Lacunae and dense granules show features typical of a degeneration process in the sensory cells. This general morphology corresponds to the unilobular type of organ of Bellonci, known in other isopods; it differs from the plurilobular type with onion bodies found in other Crustacea.     

Phagocytosis of lectin-coated beads by dystrophic and normal retinal pigment epithelium

In the dystrophic pigmented Royal College of Surgeons (RCS) rat, the retinal pigment epithelium (RPE) has a diminished capacity to phagocytose shed photoreceptor outer segments (ROS). An alteration in phagocytic recognition or ligand-receptor interactions between the RPE and ROS's could contribute to this defect. To this end, we have examined whether or not RPE lectin receptors are implicated in phagocytosis in the normal and dystrophic rat RPE by comparing differences in phagocytic uptake of lectin-coated beads. To test this, the following lectins were bound either indirectly to sugar-coated latex beads or directly to activated beads: Concanavalin A (conA), specific for mannose; Ulex europeus (ULEX), specific for fucose; Lens culinaris (LcH), specific for mannose; and wheat germ agglutinin (WGA), specific for N-acetyl glucosamine and sialic acid. The distribution of the lectin binding around beads was visualized and confirmed using lectin-Ferritin conjugates. Lectin-coated beads were fed to normal and dystrophic pigmented RPE tissue explants to determine differences in phagocytic uptake. We found that whether beads were directly or indirectly coated, similar results were obtained, but that there were differences in uptake of two types of lectin-coated beads by dystrophic as compared with normal animals. The dystrophic RPE phagocytosed greater numbers of conA-mannose beads (6.9/cell) than the normal RPE (3.6/cell). LcH-mannose beads were also phagocytosed by dystrophic (2.7/cell) but not by the normal (0/cell). A similar number of ULEX-fucose beads were taken up by dystrophic (3.8/cell) and normal (3.4/cell) RPE and neither took up WGA-N-acetyl glucosamine beads (0/cell). These results showing that the dystrophic RPE takes up greater numbers of conA and LcH-coated beads than the normal RPE suggest that a ligand-receptor interaction involving mannose may contribute to this difference in phagocytic uptake.