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

Wavre-Shapton ST, Meschede IP, Seabra MC, Futter CE - J. Cell. Sci. (2014)

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.

View Article: PubMed Central - PubMed

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.

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Sequential stages of phagosome maturation in the RPE. Early phagosomes are positive for the C-terminal cytoplasmic 1D4 and N-terminal intradiscal RET-P1 epitopes. As the phagosomes mature, 1D4 staining is lost and mature phagosomes are only positive for RET-P1. Maturing phagosomes also show little staining for the lysosomal protein cathepsin D. After fusion of the mature phagosome with the cathepsin D (CatD)-positive lysosome (phagolysosome), RET-P1 staining is lost. Loss of the C-terminal 1D4 epitope occurs only after interaction with endosomes. Profiles indicative of an interaction of melanosomes with both maturing phagosomes and multivesicular endosomes have also been observed.
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f08: Sequential stages of phagosome maturation in the RPE. Early phagosomes are positive for the C-terminal cytoplasmic 1D4 and N-terminal intradiscal RET-P1 epitopes. As the phagosomes mature, 1D4 staining is lost and mature phagosomes are only positive for RET-P1. Maturing phagosomes also show little staining for the lysosomal protein cathepsin D. After fusion of the mature phagosome with the cathepsin D (CatD)-positive lysosome (phagolysosome), RET-P1 staining is lost. Loss of the C-terminal 1D4 epitope occurs only after interaction with endosomes. Profiles indicative of an interaction of melanosomes with both maturing phagosomes and multivesicular endosomes have also been observed.

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.


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

Wavre-Shapton ST, Meschede IP, Seabra MC, Futter CE - J. Cell. Sci. (2014)

Sequential stages of phagosome maturation in the RPE. Early phagosomes are positive for the C-terminal cytoplasmic 1D4 and N-terminal intradiscal RET-P1 epitopes. As the phagosomes mature, 1D4 staining is lost and mature phagosomes are only positive for RET-P1. Maturing phagosomes also show little staining for the lysosomal protein cathepsin D. After fusion of the mature phagosome with the cathepsin D (CatD)-positive lysosome (phagolysosome), RET-P1 staining is lost. Loss of the C-terminal 1D4 epitope occurs only after interaction with endosomes. Profiles indicative of an interaction of melanosomes with both maturing phagosomes and multivesicular endosomes have also been observed.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150067&req=5

f08: Sequential stages of phagosome maturation in the RPE. Early phagosomes are positive for the C-terminal cytoplasmic 1D4 and N-terminal intradiscal RET-P1 epitopes. As the phagosomes mature, 1D4 staining is lost and mature phagosomes are only positive for RET-P1. Maturing phagosomes also show little staining for the lysosomal protein cathepsin D. After fusion of the mature phagosome with the cathepsin D (CatD)-positive lysosome (phagolysosome), RET-P1 staining is lost. Loss of the C-terminal 1D4 epitope occurs only after interaction with endosomes. Profiles indicative of an interaction of melanosomes with both maturing phagosomes and multivesicular endosomes have also been observed.
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.

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.

View Article: PubMed Central - PubMed

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
Related in: MedlinePlus