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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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Loss of the N-terminal intradiscal rhodopsin epitope occurs on fusion with the lysosome. Immunogold labelling of cryosections of mouse retina collected 2.5 h after light onset (9.30am). (A,B) Double labelling of rhodopsin C-terminal epitope (with 1D4; PAG, 15 nm) and cathepsin D (PAG, 10 nm). (C,D) Double labelling of rhodopsin N-terminal epitope (with RET-P1; PAG, 15 nm) and cathepsin D (PAG, 10 nm). Outer segment discs are visible in all phagosomes and, to some extent, in phagolysosomes. They are less visible in B (asterisk) and they are completely absent from the smaller phagolysosome in D. Scale bars: 500 nm. EarlyP, early phagosome; P, phagosome; M, melanosome; PL, phagolysosome; L, lysosome; black arrows indicate examples of PAG15; white arrows indicate examples of PAG10.
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f03: Loss of the N-terminal intradiscal rhodopsin epitope occurs on fusion with the lysosome. Immunogold labelling of cryosections of mouse retina collected 2.5 h after light onset (9.30am). (A,B) Double labelling of rhodopsin C-terminal epitope (with 1D4; PAG, 15 nm) and cathepsin D (PAG, 10 nm). (C,D) Double labelling of rhodopsin N-terminal epitope (with RET-P1; PAG, 15 nm) and cathepsin D (PAG, 10 nm). Outer segment discs are visible in all phagosomes and, to some extent, in phagolysosomes. They are less visible in B (asterisk) and they are completely absent from the smaller phagolysosome in D. Scale bars: 500 nm. EarlyP, early phagosome; P, phagosome; M, melanosome; PL, phagolysosome; L, lysosome; black arrows indicate examples of PAG15; white arrows indicate examples of PAG10.

Mentions: In all phagocytes, phagosomes are eventually degraded upon fusion with lysosomes. In RPE cells, the lysosomal enzyme cathepsin D is very highly expressed (Rakoczy et al., 1999; Yamada et al., 1990; Zhang et al., 2002; Zimmerman et al., 1983), and RPE cells expressing a mutant inactive form of cathepsin D accumulate undigested POS products (Rakoczy et al., 2002), suggesting an important role for this enzyme in POS degradation. We therefore co-stained ultrathin cryosections for cathepsin D and rhodopsin, using 1D4 or RET-P1 (Fig. 3). Interestingly, we found that early phagosomes positive for 1D4 never stained for cathepsin D (Fig. 3A). Cathepsin-D-positive phagosomes or phagolysosomes were negative for 1D4 but could be identified as phagolysosomes (rather than lysosomes) by the presence of ROS discs. However, the discs in phagolysosomes were often morphologically less well defined (Fig. 3B, black asterisk), consistent with some degradation taking place.


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)

Loss of the N-terminal intradiscal rhodopsin epitope occurs on fusion with the lysosome. Immunogold labelling of cryosections of mouse retina collected 2.5 h after light onset (9.30am). (A,B) Double labelling of rhodopsin C-terminal epitope (with 1D4; PAG, 15 nm) and cathepsin D (PAG, 10 nm). (C,D) Double labelling of rhodopsin N-terminal epitope (with RET-P1; PAG, 15 nm) and cathepsin D (PAG, 10 nm). Outer segment discs are visible in all phagosomes and, to some extent, in phagolysosomes. They are less visible in B (asterisk) and they are completely absent from the smaller phagolysosome in D. Scale bars: 500 nm. EarlyP, early phagosome; P, phagosome; M, melanosome; PL, phagolysosome; L, lysosome; black arrows indicate examples of PAG15; white arrows indicate examples of PAG10.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f03: Loss of the N-terminal intradiscal rhodopsin epitope occurs on fusion with the lysosome. Immunogold labelling of cryosections of mouse retina collected 2.5 h after light onset (9.30am). (A,B) Double labelling of rhodopsin C-terminal epitope (with 1D4; PAG, 15 nm) and cathepsin D (PAG, 10 nm). (C,D) Double labelling of rhodopsin N-terminal epitope (with RET-P1; PAG, 15 nm) and cathepsin D (PAG, 10 nm). Outer segment discs are visible in all phagosomes and, to some extent, in phagolysosomes. They are less visible in B (asterisk) and they are completely absent from the smaller phagolysosome in D. Scale bars: 500 nm. EarlyP, early phagosome; P, phagosome; M, melanosome; PL, phagolysosome; L, lysosome; black arrows indicate examples of PAG15; white arrows indicate examples of PAG10.
Mentions: In all phagocytes, phagosomes are eventually degraded upon fusion with lysosomes. In RPE cells, the lysosomal enzyme cathepsin D is very highly expressed (Rakoczy et al., 1999; Yamada et al., 1990; Zhang et al., 2002; Zimmerman et al., 1983), and RPE cells expressing a mutant inactive form of cathepsin D accumulate undigested POS products (Rakoczy et al., 2002), suggesting an important role for this enzyme in POS degradation. We therefore co-stained ultrathin cryosections for cathepsin D and rhodopsin, using 1D4 or RET-P1 (Fig. 3). Interestingly, we found that early phagosomes positive for 1D4 never stained for cathepsin D (Fig. 3A). Cathepsin-D-positive phagosomes or phagolysosomes were negative for 1D4 but could be identified as phagolysosomes (rather than lysosomes) by the presence of ROS discs. However, the discs in phagolysosomes were often morphologically less well defined (Fig. 3B, black asterisk), consistent with some degradation taking place.

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