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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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Partial proteolytic processing of the C-terminal rhodopsin epitope during phagosome maturation in porcine RPE cells. Monolayers of primary porcine RPE cells on Transwell® membrane inserts were challenged with POS apically for 1 h, washed to remove unbound POS, chased for 2 h or 4 h at 37°C and processed for cryo-immuno-electron microscopy. Ultrathin sections were double labelled for rhodopsin with antibodies against the C-terminal epitope (1D4; PAG, 10 nm) and N-terminal epitope (RET-P1; PAG, 15 nm). (A) An overview of RPE cells and ROS. The inset shows double labelling in ROS. (B) A double-labelled early phagosome with same density of both gold particles as seen on ROS. (C) A single-labelled late phagosome lacking the cytoplasmic 1D4 epitope. Scale bars: 500 nm (A), 200 nm (B,C). M, melanosome; EarlyP, early phagosome; LateP, late phagosome. (D) Quantification shows the percentage of total phagosomes that are positive for both 1D4 and RET-P1 or for RET-P1 only after 2 h and 4 h of chase, in the presence or absence of the protease inhibitor leupeptin (L). Data show the mean±s.e.m. (three independent experiments).
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f05: Partial proteolytic processing of the C-terminal rhodopsin epitope during phagosome maturation in porcine RPE cells. Monolayers of primary porcine RPE cells on Transwell® membrane inserts were challenged with POS apically for 1 h, washed to remove unbound POS, chased for 2 h or 4 h at 37°C and processed for cryo-immuno-electron microscopy. Ultrathin sections were double labelled for rhodopsin with antibodies against the C-terminal epitope (1D4; PAG, 10 nm) and N-terminal epitope (RET-P1; PAG, 15 nm). (A) An overview of RPE cells and ROS. The inset shows double labelling in ROS. (B) A double-labelled early phagosome with same density of both gold particles as seen on ROS. (C) A single-labelled late phagosome lacking the cytoplasmic 1D4 epitope. Scale bars: 500 nm (A), 200 nm (B,C). M, melanosome; EarlyP, early phagosome; LateP, late phagosome. (D) Quantification shows the percentage of total phagosomes that are positive for both 1D4 and RET-P1 or for RET-P1 only after 2 h and 4 h of chase, in the presence or absence of the protease inhibitor leupeptin (L). Data show the mean±s.e.m. (three independent experiments).

Mentions: To further investigate the mechanisms underlying the loss of the cytoplasmic rhodopsin epitope, it was necessary to develop a system of cultured RPE cells. As insufficient numbers of cultured mouse RPE cells could be obtained for cryo-immuno-electron microscopy, we developed an in vitro assay using primary porcine RPE cells challenged with porcine isolated POS. In order to mimic the in vivo situation as closely as possible, primary cells were used only after a single passage and were cultured on Transwell® membrane inserts for 5–10 days in the presence of low serum. Under these conditions, the cells developed a transepithelial resistance of 160–300 Ωcm2, and conventional electron microscopy showed that they had the characteristics of polarised RPE cells, with apical processes, melanosomes localised close to the apical membrane and mitochondria around the basolateral border (supplementary material Fig. S1A). To mimic the in vivo phagocytosis process as closely as possible, purified POS (supplementary material Fig. S1C) were sonicated for 10 min before addition to the cells. After sonication, the size of the isolated POS (supplementary material Fig. S1D) resembled that of ROS engulfed by RPE in vivo (supplementary material Fig. S1B, asterisk). Furthermore, >90% of the isolated porcine POS stained strongly with both 1D4 and RET-P1 antibodies (data not shown), indicating that the majority of this preparation is composed of ROS. To determine whether the same stages of phagosome maturation identified in vivo could be identified using our in vitro system, cultured porcine RPE cells were challenged with porcine POS for 1 h and chased for 2 h or 4 h (Fig. 5A–C). After 2 h of chase, ∼50% of RET-P1-positive phagosomes were also positive for 1D4 (Fig. 5B,D). After 4 h of chase, most (>70%) of the RET-P1-positive phagosomes were negative for 1D4 (Fig. 5C,D). Thus, there was a progression over time from double-labelled to single-labelled phagosomes that occurred over a similar timescale to that observed on retinal sections, suggesting that phagosome maturation in cultured cells proceeds through similar stages to that occurring in vivo.


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)

Partial proteolytic processing of the C-terminal rhodopsin epitope during phagosome maturation in porcine RPE cells. Monolayers of primary porcine RPE cells on Transwell® membrane inserts were challenged with POS apically for 1 h, washed to remove unbound POS, chased for 2 h or 4 h at 37°C and processed for cryo-immuno-electron microscopy. Ultrathin sections were double labelled for rhodopsin with antibodies against the C-terminal epitope (1D4; PAG, 10 nm) and N-terminal epitope (RET-P1; PAG, 15 nm). (A) An overview of RPE cells and ROS. The inset shows double labelling in ROS. (B) A double-labelled early phagosome with same density of both gold particles as seen on ROS. (C) A single-labelled late phagosome lacking the cytoplasmic 1D4 epitope. Scale bars: 500 nm (A), 200 nm (B,C). M, melanosome; EarlyP, early phagosome; LateP, late phagosome. (D) Quantification shows the percentage of total phagosomes that are positive for both 1D4 and RET-P1 or for RET-P1 only after 2 h and 4 h of chase, in the presence or absence of the protease inhibitor leupeptin (L). Data show the mean±s.e.m. (three independent experiments).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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f05: Partial proteolytic processing of the C-terminal rhodopsin epitope during phagosome maturation in porcine RPE cells. Monolayers of primary porcine RPE cells on Transwell® membrane inserts were challenged with POS apically for 1 h, washed to remove unbound POS, chased for 2 h or 4 h at 37°C and processed for cryo-immuno-electron microscopy. Ultrathin sections were double labelled for rhodopsin with antibodies against the C-terminal epitope (1D4; PAG, 10 nm) and N-terminal epitope (RET-P1; PAG, 15 nm). (A) An overview of RPE cells and ROS. The inset shows double labelling in ROS. (B) A double-labelled early phagosome with same density of both gold particles as seen on ROS. (C) A single-labelled late phagosome lacking the cytoplasmic 1D4 epitope. Scale bars: 500 nm (A), 200 nm (B,C). M, melanosome; EarlyP, early phagosome; LateP, late phagosome. (D) Quantification shows the percentage of total phagosomes that are positive for both 1D4 and RET-P1 or for RET-P1 only after 2 h and 4 h of chase, in the presence or absence of the protease inhibitor leupeptin (L). Data show the mean±s.e.m. (three independent experiments).
Mentions: To further investigate the mechanisms underlying the loss of the cytoplasmic rhodopsin epitope, it was necessary to develop a system of cultured RPE cells. As insufficient numbers of cultured mouse RPE cells could be obtained for cryo-immuno-electron microscopy, we developed an in vitro assay using primary porcine RPE cells challenged with porcine isolated POS. In order to mimic the in vivo situation as closely as possible, primary cells were used only after a single passage and were cultured on Transwell® membrane inserts for 5–10 days in the presence of low serum. Under these conditions, the cells developed a transepithelial resistance of 160–300 Ωcm2, and conventional electron microscopy showed that they had the characteristics of polarised RPE cells, with apical processes, melanosomes localised close to the apical membrane and mitochondria around the basolateral border (supplementary material Fig. S1A). To mimic the in vivo phagocytosis process as closely as possible, purified POS (supplementary material Fig. S1C) were sonicated for 10 min before addition to the cells. After sonication, the size of the isolated POS (supplementary material Fig. S1D) resembled that of ROS engulfed by RPE in vivo (supplementary material Fig. S1B, asterisk). Furthermore, >90% of the isolated porcine POS stained strongly with both 1D4 and RET-P1 antibodies (data not shown), indicating that the majority of this preparation is composed of ROS. To determine whether the same stages of phagosome maturation identified in vivo could be identified using our in vitro system, cultured porcine RPE cells were challenged with porcine POS for 1 h and chased for 2 h or 4 h (Fig. 5A–C). After 2 h of chase, ∼50% of RET-P1-positive phagosomes were also positive for 1D4 (Fig. 5B,D). After 4 h of chase, most (>70%) of the RET-P1-positive phagosomes were negative for 1D4 (Fig. 5C,D). Thus, there was a progression over time from double-labelled to single-labelled phagosomes that occurred over a similar timescale to that observed on retinal sections, suggesting that phagosome maturation in cultured cells proceeds through similar stages to that occurring in vivo.

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