Phagosome maturation during endosome interaction revealed by partial rhodopsin processing in retinal pigment epithelium.

Defects in phagocytosis and degradation of photoreceptor outer segments (POS) by the retinal pigment epithelium (RPE) are associated with aging and retinal disease. The daily burst of rod outer segment (ROS) phagocytosis by the RPE provides a unique opportunity to analyse phagosome processing in vivo. In mouse retinae phagosomes containing stacked rhodopsin-rich discs were identified by immuno-electron microscopy. Early apical phagosomes stained with both cytoplasmic and intradiscal domain rhodopsin antibodies. During phagosome maturation a remarkably synchronised loss of the cytoplasmic epitope coincided with movement to the cell body and preceded phagosome:lysosome fusion and disc degradation. Loss of the intradiscal rhodopsin epitope and disc digestion occurred upon fusion with cathepsin D-positive lysosomes. The same sequential stages of phagosome maturation were identified in cultured RPE and macrophages challenged with isolated POS. Loss of the cytoplasmic rhodopsin epitope was insensitive to pH but sensitive to protease inhibition and co-incided with interaction of phagosomes with endosomes. Thus, during prelysosomal maturation of ROS-containing phagosomes limited rhodopsin processing occurs upon interaction with endosomes. This potentially provides a sensitive readout of phagosome:endosome interactions applicable to multiple phagocytes

Photoreceptor phagosome processing defects and disturbed autophagy in retinal pigment epithelium of Cln3Δex1-6 mice modelling juvenile neuronal ceroid lipofuscinosis (Batten disease)

Retinal degeneration and visual impairment are the first signs of juvenile neuronal ceroid lipofuscinosis caused by CLN3 mutations, followed by inevitable progression to blindness. We investigated retinal degeneration in Cln3Δex1-6 null mice, revealing classic ‘fingerprint’ lysosomal storage in the retinal pigment epithelium (RPE), replicating the human disease. The lysosomes contain mitochondrial F0-ATP synthase subunit c along with undigested membranes, indicating a reduced degradative capacity. Mature autophagosomes and basal phagolysosomes, the terminal degradative compartments of autophagy and phagocytosis, are also increased in Cln3Δex1-6 RPE, reflecting disruption to these key pathways that underpin the daily phagocytic turnover of photoreceptor outer segments (POS) required for maintenance of vision. The accumulated autophagosomes have post-lysosome fusion morphology, with undigested internal contents visible, while accumulated phagosomes are frequently docked to cathepsin D-positive lysosomes, without mixing of phagosomal and lysosomal contents. This suggests lysosome-processing defects affect both autophagy and phagocytosis, supported by evidence that phagosomes induced in Cln3Δex1-6-derived mouse embryonic fibroblasts have visibly disorganized membranes, unprocessed internal vesicles and membrane contents, in addition to reduced LAMP1 membrane recruitment. We propose that defective lysosomes in Cln3Δex1-6 RPE have a reduced degradative capacity that impairs the final steps of the intimately connected autophagic and phagocytic pathways that are responsible for degradation of POS. A build-up of degradative organellar by-products and decreased recycling of cellular materials is likely to disrupt processes vital to maintenance of vision by the RPE

Conditional ablation of the choroideremia gene causes age-related changes in mouse retinal pigment epithelium.

The retinal pigment epithelium (RPE) is a pigmented monolayer of cells lying between the photoreceptors and a layer of fenestrated capillaries, the choriocapillaris. Choroideremia (CHM) is an X-linked progressive degeneration of these three layers caused by the loss of function of Rab Escort protein-1 (REP1). REP1 is involved in the prenylation of Rab proteins, key regulators of membrane trafficking. To study the pathological consequences of chronic disruption of membrane traffic in the RPE we used a cell type-specific knock-out mouse model of the disease, where the Chm/Rep1 gene is deleted only in pigmented cells (Chm(Flox), Tyr-Cre+). Transmission electron microscopy (TEM) was used to quantitate the melanosome distribution in the RPE and immunofluorescent staining of rhodopsin was used to quantitate phagocytosed rod outer segments in retinal sections. The ultrastructure of the RPE and Bruch's membrane at different ages was characterised by TEM to analyse age-related changes occurring as a result of defects in membrane traffic pathways. Chm/Rep1 gene knockout in RPE cells resulted in reduced numbers of melanosomes in the apical processes and delayed phagosome degradation. In addition, the RPE accumulated pathological changes at 5-6 months of age similar to those observed in 2-year old controls. These included the intracellular accumulation of lipofuscin-containing deposits, disorganised basal infoldings and the extracellular accumulation of basal laminar and basal linear deposits. The phenotype of the Chm(Flox), Tyr-Cre+ mice suggests that loss of the Chm/Rep1 gene causes premature accumulation of features of aging in the RPE. Furthermore, the striking similarities between the present observations and some of the phenotypes reported in age-related macular degeneration (AMD) suggest that membrane traffic defects may contribute to the pathogenesis of AMD

Remodeling of the Basal Labyrinth of Retinal Pigment Epithelial Cells With Osmotic Challenge, Age, and Disease

PURPOSE: The basal surface of the retinal pigment epithelium (RPE) is folded into a complex basal labyrinth thought to facilitate solute and water transport. We aimed to analyze and define the structural organization of the basal labyrinth of the RPE to enable quantitative analysis of structural changes in age and disease and to better understand the relationship between basal labyrinth structure and efficiency of transepithelial transport. METHODS: Conventional transmission and serial block-face scanning electron microscopy and electron tomography were used to examine the structure of the basal labyrinth in mouse eyes of different ages and genotypes and with and without osmotic shock before fixation. RESULTS: We identified structurally distinct zones (stacked and ribbon-like) within the RPE basal labyrinth that are largely organelle free and cisternal elements that make contact with the endoplasmic reticulum (ER) and mitochondria. These zones are lost in a hierarchic fashion with age and prematurely in a model of the progressive retinal degenerative disease, choroideremia. Junctional complexes crosslink closely opposed infoldings. Spacing between the basal infoldings was affected by subtle osmotic changes while osmotic shock induced dramatic remodeling of the infoldings. CONCLUSION: The basal labyrinth has complex but ordered structural elements that break down with age and in choroideremia. The geometry of these elements and site of contact with ER and mitochondria likely facilitate the ion transport that drives water transport across the basal RPE surface. Changes in structure in response to local osmotic variation may allow transport to be modulated in order to maintain RPE volume

Flat Clathrin Lattices: stable features of the plasma membrane

Clathrin-mediated endocytosis (CME) is a fundamental property of eukaryotic cells. Classical CME proceeds via the formation of clathrin-coated pits (CCP) at the plasma membrane that invaginate to form clathrin-coated vesicles; a process that is well understood. However, clathrin also assembles into flat clathrin lattices (FCL); these structures remain poorly described and their contribution to cell biology is unclear. We have used quantitative imaging to provide the first comprehensive description of FCL and explore their influence on plasma membrane organization. Ultrastructural analysis by electron and super-resolution microscopy revealed two discrete populations of clathrin structures. CCP were typified by their sphericity, small size and homogeneity. FCL were planar, large and heterogeneous, and present on both the dorsal and ventral surfaces of cells. Live microscopy demonstrated that CCP are short-lived and culminate in a peak of dynamin recruitment, consistent with classical CME. In contrast, FCL were long-lived with sustained association with dynamin. We investigated the biological relevance of FCL using the chemokine receptor CCR5 as a model system. Agonist activation leads to sustained recruitment of CCR5 to FCL. Quantitative molecular imaging indicated that FCL partitioned receptors at the cell surface. Our observations suggest that FCL provide stable platforms for the recruitment of endocytic cargo