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Clare Futter, Ingrid P. Meschede, Silene T. Wavre, Mafalda Lopes Da Silva, Tanya Tolmachova, Miguel C. Seabra; Phagosome Maturation And Interactions With The Endocytic Pathway In Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3701. doi: https://doi.org/.
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Phagosome maturation in macrophages involves sequential interactions with the endocytic pathway and the acquisition of Rab GTPases before fusion with the lysosome. Despite the huge phagocytic load of retinal pigment epithelial (RPE) cells, which phagocytose shed photoreceptor outer segments everyday, the regulation of phagosome maturation and degradation in the RPE is not well characterised. We aimed to develop assays to monitor phagosome maturation in RPE cells and determine whether the maturing phagosome interacts with the endocytic pathway before lysosomal fusion and degradation.
To identify sequential phagocytic compartments in mouse retinal sections rhodopsin processing was monitored, in conjunction with cathepsin D staining, by cryo-immunoEM. To identify endocytic compartments, the endocytosis of fluid phase probes and transferrin from apical and basal surfaces of cultured primary porcine RPE was followed. To monitor interactions between phagocytic and endocytic pathways RPE cells were incubated with gold-labelled endocytic probes from the basal surface, and photoreceptor outer segments from the apical surface, and the appearance of gold probes in the maturing phagosome was identified by electron microscopy.
Antibodies to different rhodopsin epitopes allowed the identification of sequential stages of phagosome maturation. Cultured RPE cells engulf and process photoreceptor outer segments and endocytose probes from both apical and basolateral surfaces. Probes endocytosed from the basolateral surface meet apically endocytosed probes in a pre-lysosomal compartment that contains transferrin receptor. When the endocytic pathway is loaded with gold particles from the basal surface gold particles appear in the maturing phagosome before lysosomal delivery.
Endosomes in RPE cells can be accessed from both apical and basolateral surfaces via clathrin coated pits. Sequential stages of phagosome maturation can be identified by monitoring rhodopsin processing. As phagosomes mature they interact with the endocytic compartment before fusing with the lysosome. The assays that we have developed will allow the determination of the effects of retinal disease-causing mutations on phagosome maturation and interactions with the endocytic pathway.
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