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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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Reduced pH does not affect binding to the C-terminal cytoplasmic rhodopsin epitope. POS were incubated in neutral (pH 7.0) or low (pH 5.0) pH solution and were prepared for cryo-immuno-electron microscopy. Ultrathin sections were labelled with 1D4 (PAG, 10 nm) and RET-P1 (PAG, 15 nm). The pH did not affect labelling with the antibody against the cytoplasmic 1D4 epitope. Areas outlined in white are shown at higher magnification in the lower-right corner. Scale bars: 200 nm.
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f04: Reduced pH does not affect binding to the C-terminal cytoplasmic rhodopsin epitope. POS were incubated in neutral (pH 7.0) or low (pH 5.0) pH solution and were prepared for cryo-immuno-electron microscopy. Ultrathin sections were labelled with 1D4 (PAG, 10 nm) and RET-P1 (PAG, 15 nm). The pH did not affect labelling with the antibody against the cytoplasmic 1D4 epitope. Areas outlined in white are shown at higher magnification in the lower-right corner. Scale bars: 200 nm.

Mentions: The synchronised loss of the C-terminal rhodopsin epitope raised the possibility that an irreversible conformational change or post-translational modification of rhodopsin in the maturing phagosome might lead to loss of this epitope. Alternatively, a protease present on the ROS themselves might become activated upon phagosome maturation. The most likely activator of any of these changes would be the progressive lowering of the luminal pH of the phagosome that has been shown to occur in other systems (Deguchi et al., 1994; Horwitz and Maxfield, 1984; Kielian and Cohn, 1980). We therefore isolated porcine POS, incubated them in low pH solution (pH 5.0) and determined the effects on C-terminal rhodopsin epitope antibody binding by cryo-immuno-electron microscopy. As shown in Fig. 4, the binding of C-terminal 1D4 and N-terminal RET-P1 antibodies was unaffected by incubation in low pH solution, suggesting that the loss of cytoplasmic 1D4 epitope is not caused by a lowering of pH within the phagosome.


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)

Reduced pH does not affect binding to the C-terminal cytoplasmic rhodopsin epitope. POS were incubated in neutral (pH 7.0) or low (pH 5.0) pH solution and were prepared for cryo-immuno-electron microscopy. Ultrathin sections were labelled with 1D4 (PAG, 10 nm) and RET-P1 (PAG, 15 nm). The pH did not affect labelling with the antibody against the cytoplasmic 1D4 epitope. Areas outlined in white are shown at higher magnification in the lower-right corner. Scale bars: 200 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f04: Reduced pH does not affect binding to the C-terminal cytoplasmic rhodopsin epitope. POS were incubated in neutral (pH 7.0) or low (pH 5.0) pH solution and were prepared for cryo-immuno-electron microscopy. Ultrathin sections were labelled with 1D4 (PAG, 10 nm) and RET-P1 (PAG, 15 nm). The pH did not affect labelling with the antibody against the cytoplasmic 1D4 epitope. Areas outlined in white are shown at higher magnification in the lower-right corner. Scale bars: 200 nm.
Mentions: The synchronised loss of the C-terminal rhodopsin epitope raised the possibility that an irreversible conformational change or post-translational modification of rhodopsin in the maturing phagosome might lead to loss of this epitope. Alternatively, a protease present on the ROS themselves might become activated upon phagosome maturation. The most likely activator of any of these changes would be the progressive lowering of the luminal pH of the phagosome that has been shown to occur in other systems (Deguchi et al., 1994; Horwitz and Maxfield, 1984; Kielian and Cohn, 1980). We therefore isolated porcine POS, incubated them in low pH solution (pH 5.0) and determined the effects on C-terminal rhodopsin epitope antibody binding by cryo-immuno-electron microscopy. As shown in Fig. 4, the binding of C-terminal 1D4 and N-terminal RET-P1 antibodies was unaffected by incubation in low pH solution, suggesting that the loss of cytoplasmic 1D4 epitope is not caused by a lowering of pH within the phagosome.

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