Phagosome maturation during endosome interaction revealed by partial rhodopsin processing in retinal pigment epithelium.
Bottom Line: Loss of the cytoplasmic rhodopsin epitope was insensitive to pH but sensitive to protease inhibition and coincided with the interaction of phagosomes with endosomes.Thus, during pre-lysosomal maturation of ROS-containing phagosomes, limited rhodopsin processing occurs upon interaction with endosomes.This potentially provides a sensitive readout of phagosome-endosome interactions that is applicable to multiple phagocytes.
Affiliation: Molecular Medicine Section, National Heart and Lung Institute, Imperial College London, London SW7 2AZ, UK UCL Institute of Ophthalmology, University College London, London EC1V 9EL, UK.Show MeSH
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Mentions: In this study, we use an approach that does not rely on the acquisition of endocytic markers by the maturing phagosome. Instead, we focused on the phagosome content and used cryo-immuno-electron microscopy to simultaneously analyse rhodopsin processing and phagosome content. Immuno-electron microscopy is unaffected by melanin quenching, which limits immunofluorescent analysis of the RPE, and the resolution of immune-electron microscopy allows the ready distinction between surface and internalised ROS and between apical and basal phagosomes. Using this approach, we have shown that sequential stages in phagosome maturation can be identified (Fig. 8). Early phagosomes stain for both intradiscal and cytoplasmically exposed rhodopsin epitopes. During phagosome maturation in RPE cells in situ, a cytoplasmically exposed C-terminal rhodopsin epitope is lost before fusion with the lysosome, allowing the identification of a second ‘mature’ stage in phagosome maturation that retains the intradiscal N-terminal rhodopsin epitope. Loss of the intradiscal epitope occurs only upon fusion with the lysosome and subsequent degradation of the ROS discs, allowing mature phagosomes to be distinguished from phagolysosomes.
Affiliation: Molecular Medicine Section, National Heart and Lung Institute, Imperial College London, London SW7 2AZ, UK UCL Institute of Ophthalmology, University College London, London EC1V 9EL, UK.