A human model of Batten disease shows role of CLN3 in phagocytosis at the photoreceptor-RPE interface.

Mutations in CLN3 lead to photoreceptor cell loss in CLN3 disease, a lysosomal storage disorder characterized by childhood-onset vision loss, neurological impairment, and premature death. However, how CLN3 mutations cause photoreceptor cell death is not known. Here, we show that CLN3 is required for phagocytosis of photoreceptor outer segment (POS) by retinal pigment epithelium (RPE) cells, a cellular process essential for photoreceptor survival. Specifically, a proportion of CLN3 in human, mouse, and iPSC-RPE cells localized to RPE microvilli, the site of POS phagocytosis. Furthermore, patient-derived CLN3 disease iPSC-RPE cells showed decreased RPE microvilli density and reduced POS binding and ingestion. Notably, POS phagocytosis defect in CLN3 disease iPSC-RPE cells could be rescued by wild-type CLN3 gene supplementation. Altogether, these results illustrate a novel role of CLN3 in regulating POS phagocytosis and suggest a contribution of primary RPE dysfunction for photoreceptor cell loss in CLN3 disease that can be targeted by gene therapy.

Microtubule motor transport in the delivery of melanosomes to the actin-rich apical domain of the retinal pigment epithelium.

Melanosomes are motile, light-absorbing organelles that are present in pigment cells of the skin and eye. It has been proposed that melanosome localization, in both skin melanocytes and the retinal pigment epithelium (RPE), involves melanosome capture from microtubule motors by an unconventional myosin, which dynamically tethers the melanosomes to actin filaments. Recent studies with melanocytes have questioned this cooperative capture model. Here, we test the model in RPE cells by imaging melanosomes associated with labeled actin filaments and microtubules, and by investigating the roles of different motor proteins. We found that a deficiency in cytoplasmic dynein phenocopies the lack of myosin-7a, in that melanosomes undergo fewer of the slow myosin-7a-dependent movements and are absent from the RPE apical domain. These results indicate that microtubule-based motility is required for the delivery of melanosomes to the actin-rich apical domain and support a capture mechanism that involves both microtubule and actin motors.

Multistep peripherin-2/rds self-assembly drives membrane curvature for outer segment disk architecture and photoreceptor viability.

Rod and cone photoreceptor outer segment (OS) structural integrity is essential for normal vision; disruptions contribute to a broad variety of retinal ciliopathies. OSs possess many hundreds of stacked membranous disks, which capture photons and scaffold the phototransduction cascade. Although the molecular basis of OS structure remains unresolved, recent studies suggest that the photoreceptor-specific tetraspanin, peripherin-2/rds (P/rds), may contribute to the highly curved rim domains at disk edges. Here, we demonstrate that tetrameric P/rds self-assembly is required for generating high-curvature membranes in cellulo, implicating the noncovalent tetramer as a minimal unit of function. P/rds activity was promoted by disulfide-mediated tetramer polymerization, which transformed localized regions of curvature into high-curvature tubules of extended lengths. Transmission electron microscopy visualization of P/rds purified from OS membranes revealed disulfide-linked tetramer chains up to 100 nm long, suggesting that chains maintain membrane curvature continuity over extended distances. We tested this idea in Xenopus laevis photoreceptors, and found that transgenic expression of nonchain-forming P/rds generated abundant high-curvature OS membranes, which were improperly but specifically organized as ectopic incisures and disk rims. These striking phenotypes demonstrate the importance of P/rds tetramer chain formation for the continuity of rim formation during disk morphogenesis. Overall, this study advances understanding of the normal structure and function of P/rds for OS architecture and biogenesis, and clarifies how pathogenic loss-of-function mutations in P/rds cause photoreceptor structural defects to trigger progressive retinal degenerations. It also introduces the possibility that other tetraspanins may generate or sense membrane curvature in support of diverse biological functions.

Peroxisome turnover and diurnal modulation of antioxidant activity in retinal pigment epithelia utilizes microtubule-associated protein 1 light chain 3B (LC3B).

The retinal pigment epithelium (RPE) supports the outer retina through essential roles in the retinoid cycle, nutrient supply, ion exchange, and waste removal. Each day the RPE removes the oldest ~10% of photoreceptor outer segment (OS) disk membranes through phagocytic uptake, which peaks following light onset. Impaired degradation of phagocytosed OS material by the RPE can lead to toxic accumulation of lipids, oxidative tissue damage, inflammation, and cell death. OSs are rich in very long chain fatty acids, which are preferentially catabolized in peroxisomes. Despite the importance of lipid degradation in RPE function, the regulation of peroxisome number and activity relative to diurnal OS ingestion is relatively unexplored. Using immunohistochemistry, immunoblot analysis, and catalase activity assays, we investigated peroxisome abundance and activity at 6 AM, 7 AM (light onset), 8 AM, and 3 PM, in wild-type (WT) mice and mice lacking microtubule-associated protein 1 light chain 3B (Lc3b), which have impaired phagosome degradation. We found that catalase activity, but not the amount of catalase protein, is 50% higher in the morning compared with 3 PM, in RPE of WT, but not Lc3b-/-, mice. Surprisingly, we found that peroxisome abundance was stable during the day in RPE of WT mice; however, numbers were elevated overall in Lc3b-/- mice, implicating LC3B in autophagic organelle turnover in RPE. Our data suggest that RPE peroxisome function is regulated in coordination with phagocytosis, possibly through direct enzyme regulation, and may serve to prepare RPE peroxisomes for daily surges in ingested lipid-rich OS.

The route of the visual receptor rhodopsin along the cilium.

The photoreceptor outer segment is the most elaborate primary cilium, containing large amounts of rhodopsin (RHO) in disk membranes that grow from a connecting cilium. The movement of RHO along the connecting cilium precedes formation of the disk membranes. However, the route that RHO takes has not been clearly determined; some reports suggest that it follows an intracellular, vesicular route along the axoneme, possibly as an adaptation for the high load of delivery or the morphogenesis of the disk endomembranes. We addressed this question by studying RHO in cilia of IMCD3 cells and mouse rod photoreceptors. In IMCD3 cilia, fluorescence recovery after photobleaching (FRAP) experiments with fluorescently tagged RHO supported the idea of RHO motility in the ciliary plasma membrane and was inconsistent with the hypothesis of RHO motility within the lumen of the cilium. In rod photoreceptors, FRAP of RHO-EGFP was altered by externally applied lectin, supporting the idea of plasmalemmal RHO dynamics. Quantitative immunoelectron microscopy corroborated our live-cell conclusions, as RHO was found to be distributed along the plasma membrane of the connecting cilium, with negligible labeling within the axoneme. Taken together, the present findings demonstrate RHO trafficking entirely via the ciliary plasma membrane.This article has an associated First Person interview with the first author of the paper.

Rapid differentiation of the human RPE cell line, ARPE-19, induced by nicotinamide.

Human RPE cell lines, especially the ARPE-19 cell line, are widely-used in eye research, as well as general epithelial cell studies. In comparison with primary RPE cells, they offer relative convenience and consistency, but cultures derived from these lines are typically not well differentiated. We describe a simple, rapid method to establish cultures from ARPE-19 cells, with significantly improved epithelial cell morphology and cytoskeletal organization, and RPE-related functions. We identify the presence of nicotinamide, a member of the vitamin B family, as an essential factor in promoting the observed differentiation, indicating the importance of metabolism in RPE cell differentiation.

Defective phagosome motility and degradation in cell nonautonomous RPE pathogenesis of a dominant macular degeneration.

Stargardt macular dystrophy 3 (STGD3) is caused by dominant mutations in the ELOVL4 gene. Like other macular degenerations, pathogenesis within the retinal pigment epithelium (RPE) appears to contribute to the loss of photoreceptors from the central retina. However, the RPE does not express ELOVL4, suggesting photoreceptor cell loss in STGD3 occurs through two cell nonautonomous events: mutant photoreceptors first affect RPE cell pathogenesis, and then, second, RPE dysfunction leads to photoreceptor cell death. Here, we have investigated how the RPE pathology occurs, using a STGD3 mouse model in which mutant human ELOVL4 is expressed in the photoreceptors. We found that the mutant protein was aberrantly localized to the photoreceptor outer segment (POS), and that resulting POS phagosomes were degraded more slowly in the RPE. In cell culture, the mutant POSs are ingested by primary RPE cells normally, but the phagosomes are processed inefficiently, even by wild-type RPE. The mutant phagosomes excessively sequester RAB7A and dynein, and have impaired motility. We propose that the abnormal presence of ELOVL4 protein in POSs results in phagosomes that are defective in recruiting appropriate motor protein linkers, thus contributing to slower degradation because their altered motility results in slower basal migration and fewer productive encounters with endolysosomes. In the transgenic mouse retinas, the RPE accumulated abnormal-looking phagosomes and oxidative stress adducts; these pathological changes were followed by pathology in the neural retina. Our results indicate inefficient phagosome degradation as a key component of the first cell nonautonomous event underlying retinal degeneration due to mutant ELOVL4.

Preservation of Photoreceptor Nanostructure for Electron Tomography Using Transcardiac Perfusion Followed by High-Pressure Freezing and Freeze-Substitution.

The phototransductive membrane disks of a vertebrate photoreceptor outer segment (OS) are highly susceptible to perturbations during preservation for electron microscopy. To optimize their preservation for nanostructural studies, such as with electron tomography (ET), we developed a protocol, using a combination of chemical and physical fixation approaches, including transcardiac perfusion, high-pressure freezing, and freeze-substitution.

Three-dimensional organization of nascent rod outer segment disk membranes.

The vertebrate photoreceptor cell contains an elaborate cilium that includes a stack of phototransductive membrane disks. The disk membranes are continually renewed, but how new disks are formed remains poorly understood. Here we used electron microscope tomography to obtain 3D visualization of the nascent disks of rod photoreceptors in three mammalian species, to gain insight into the process of disk morphogenesis. We observed that nascent disks are invariably continuous with the ciliary plasma membrane, although, owing to partial enclosure, they can appear to be internal in 2D profiles. Tomographic analyses of the basal-most region of the outer segment show changes in shape of the ciliary plasma membrane indicating an invagination, which is likely a first step in disk formation. The invagination flattens to create the proximal surface of an evaginating lamella, as well as membrane protrusions that extend between adjacent lamellae, thereby initiating a disk rim. Immediately distal to this initiation site, lamellae of increasing diameter are evident, indicating growth outward from the cilium. In agreement with a previous model, our data indicate that mature disks are formed once lamellae reach full diameter, and the growth of a rim encloses the space between adjacent surfaces of two lamellae. This study provides 3D data of nascent and mature rod photoreceptor disk membranes at unprecedented z-axis depth and resolution, and provides a basis for addressing fundamental questions, ranging from protein sorting in the photoreceptor cilium to photoreceptor electrophysiology.

A comparison of some organizational characteristics of the mouse central retina and the human macula.

Mouse models have greatly assisted our understanding of retinal degenerations. However, the mouse retina does not have a macula, leading to the question of whether the mouse is a relevant model for macular degeneration. In the present study, a quantitative comparison between the organization of the central mouse retina and the human macula was made, focusing on some structural characteristics that have been suggested to be important in predisposing the macula to stresses leading to degeneration: photoreceptor density, phagocytic load on the RPE, and the relative thinness of Bruch's membrane. Light and electron microscopy measurements from retinas of two strains of mice, together with published data on human retinas, were used for calculations and subsequent comparisons. As in the human retina, the central region of the mouse retina possesses a higher photoreceptor cell density and a thinner Bruch's membrane than in the periphery; however, the magnitudes of these periphery to center gradients are larger in the human. Of potentially greater relevance is the actual photoreceptor cell density, which is much greater in the mouse central retina than in the human macula, underlying a higher phagocytic load for the mouse RPE. Moreover, at eccentricities that correspond to the peripheral half of the human macula, the rod to cone ratio is similar between mouse and human. Hence, with respect to photoreceptor density and phagocytic load of the RPE, the central mouse retina models at least the more peripheral part of the macula, where macular degeneration is often first evident.

Rescue of heavy metal effects on cell physiology of the algal model system Micrasterias by divalent ions.

Recent studies have shown that metals such as copper, zinc, aluminum, cadmium, chromium, iron and lead cause severe dose-dependent disturbances in growth, morphogenesis, photosynthetic and respiratory activity as well as on ultrastructure and function of organelles in the algal model system Micrasterias denticulata (Volland et al., 2011, 2012; Andosch et al., 2012). In the present investigation we focus on amelioration of these adverse effects of cadmium, chromium and lead by supplying the cells with different antioxidants and essential micronutrients to obtain insight into metal uptake mechanisms and subcellular metal targets. This seems particularly interesting as Micrasterias is adapted to extremely low-concentrated, oligotrophic conditions in its natural bog environment. The divalent ions of iron, zinc and calcium were able to diminish the effects of the metals cadmium, chromium and lead on Micrasterias. Iron showed most ameliorating effects on cadmium and chromium in short- and long-term treatments and improved cell morphogenesis, ultrastructure, cell division rates and photosynthesis. Analytical transmission electron microscopic (TEM) methods (electron energy loss spectroscopy (EELS) and electron spectroscopic imaging (ESI)) revealed that chromium uptake was decreased when Micrasterias cells were pre-treated with iron, which resulted in no longer detectable intracellular chromium accumulations. Zinc rescued the detrimental effects of chromium on net-photosynthesis, respiration rates and electron transport in PS II. Calcium and gadolinium were able to almost completely compensate the inhibiting effects of lead and cadmium on cell morphogenesis after mitosis, respectively. These results indicate that cadmium is taken up by calcium and iron transporters, whereas chromium appears to enter the algae cells via iron and zinc carriers. It was shown that lead is not taken up into Micrasterias at all but exerts its adverse effects on cell growth by substituting cell wall bound calcium. The antioxidants salicylic acid, ascorbic acid and glutathione were not able to ameliorate any of the investigated metal effects on the green alga Micrasterias when added to the culture medium.

Identification of phytochelatins in the cadmium-stressed conjugating green alga Micrasterias denticulata.

Aquatic environments like peat bogs are affected by anthropogenic metal input into the environment. These ecosystems are inhabited by unicellular green algae of the class Zygnematophyceae. In this study the desmid Micrasterias denticulata was stressed with 600 nM Cd, 10 µM Cr and 300 nM Cu for 3 weeks. GSH levels were measured with HPLC and did not differ between the different treatments or the control. According to the metallo-thiolomics concept, mass spectrometry was used as a method for unambiguous thiol peptide identification. PC2, PC3 and PC4 were clearly identified in the Cd stressed sample with UPLC-MS by their MS spectrum and molecular masses. PC2 and PC3 were determined to be the main thiol compounds, while PC4 was only abundant in traces in Micrasterias. In addition, the identity of PC2 and PC3 was confirmed by MS/MS. No PCs were detected in the Cu stressed algae sample. However, in the Cr stressed sample traces of PC2 were indicated by a peak in UPLC-MS at the retention time of the PC2 standard, but the intensity was too low to acquire reliable MS and MS/MS spectra. In this study PCs have been detected for the first time in a green alga of the division Streptophyta, a close relative to higher plants.

Intracellular chromium localization and cell physiological response in the unicellular alga Micrasterias.

Various contaminants like metals and heavy metals are constantly released into the environment by anthropogenic activities. The heavy metal chromium has a wide industrial use and exists in two stable oxidation states: trivalent and hexavalent. Chromium can cause harm to cell metabolism and development, when it is taken up by plants instead of necessary micronutrients such as for example iron. The uptake of Cr VI into plant cells has been reported to be an active process via carriers of essential anions, while the cation Cr III seems to be taken up inactively. Micrasterias denticulata, an unicellular green alga of the family Desmidiaceae is a well-studied cell biological model organism. Cr III and VI had inhibiting effects on its cell development, while cell division rates were only impaired by Cr VI. Transmission electron microscopy (TEM) revealed ultrastructural changes such as increased vacuolization, condensed cytoplasm and dark precipitations in the cell wall after 3 weeks of Cr VI treatment. Electron energy loss spectroscopy (EELS) and electron spectroscopic imaging (ESI) were applied to measure intracellular chromium distribution. Chromium was only detected after 3 weeks of 10 µM Cr VI treatment in electron dense precipitations found in bag-like structures along the inner side of the cell walls together with iron and elevated levels of oxygen, pointing toward an accumulation respectively extrusion of chromium in form of an iron-oxygen compound. Atomic emission spectroscopy (EMS) revealed that Micrasterias cells are able to accumulate considerable amounts of chromium and iron. During chromium treatment the Cr:Fe ratio shifted in favor of chromium, which implied that chromium may be taken up instead of iron. Significant and rapid increase of ROS production within the first 5 min of treatment confirms an active Cr VI uptake. SOD and CAT activity after Cr VI treatment did not show a response, while the glutathione pool determined by immuno-TEM decreased significantly in chromium treated cells, showing that glutathione is playing a major role in intracellular ROS and chromium detoxification.

INTRACELLULAR METAL COMPARTMENTALIZATION IN THE GREEN ALGAL MODEL SYSTEM MICRASTERIAS DENTICULATA (STREPTOPHYTA) MEASURED BY TRANSMISSION ELECTRON MICROSCOPY-COUPLED ELECTRON ENERGY LOSS SPECTROSCOPY1.

Entry of metals in form of aerosols into areas of high air humidity such as peat bogs represents a serious danger for inhabiting organisms such as the unicellular desmid Micrasterias denticulata Bréb. ex Ralfs (Desmidiaceae, Zynematophyceae, Streptophyta). To understand cellular detoxification and tolerance mechanisms, detailed intracellular localization of metal pollutants is required. This study localizes the metals aluminum (Al), zinc (Zn), copper (Cu), and cadmium (Cd) in the green algal model system Micrasterias after experimental exposure to sulfate solutions by highly sensitive TEM-coupled electron energy loss spectroscopy (EELS). Concentrations of the metals shown to induce inhibiting effects on cell development and cytomorphogenesis were chosen for these experiments. Long-term exposure to these metal concentrations led to a pronounced impact on cell physiology expressed by a general decrease in apparent photosynthesis. After long-term treatment, Zn, Al, and Cu were detected in the cell walls by EELS. Zn was additionally found in vacuoles and mucilage vesicles, and Cu in starch grains and also in mucilage vesicles. Elevated amounts of oxygen in areas where Zn, Al, and Cu were localized suggest sequestration of these metals as oxides. The study demonstrated that Micrasterias can cope differently with metal pollutants. In low doses and during a limited time period, the cells were able to compartmentalize Cu the best, followed by Zn and Al. Cu and Zn were taken up into intracellular compartments, whereas Al was only bound to the cell wall. Cd was not compartmentalized at all, which explains its strongest impact on growth, cell division rate, and photosynthesis in Micrasterias.