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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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Early phagosomes interact with a pre-lysosomal compartment of the endoytic pathway in primary porcine RPE cells. Monolayers of primary porcine RPE cells on Transwell® membrane inserts were incubated in the lower chamber with 5-nm BSA–gold for 3 h prior to the addition of POS, and BSA–gold was maintained throughout the duration of the experiment. Cells were then challenged with POS from the apical chamber for 1 h, washed to remove unbound POS, chased for 2 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) A double-labelled early phagosome (earlyP) that contains no 5-nm gold particles. (B) A double-labelled early phagosome containing monodisperse BSA–gold (black arrowheads) and ILVs (white arrowheads) at one pole, suggesting recent fusion of the early phagosome with a multivesicular endocytic compartment. (C) Maturing phagosomes (matP) containing 5-nm BSA–gold. Note that the density of PAG 10 nm is greatly reduced compared with that of the early phagosome shown in A and B. A lysosome (L) containing aggregated 5-nm gold particles can be readily identified. (D) Phagolysosome (PL) containing PAG 15 nm only and aggregated 5-nm gold particles. Scale bars: 100 nm (A,B,D), 200 nm (C). Black and white arrows indicate rhodopsin labelling with RET-P1 and 1D4, respectively.
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f06: Early phagosomes interact with a pre-lysosomal compartment of the endoytic pathway in primary porcine RPE cells. Monolayers of primary porcine RPE cells on Transwell® membrane inserts were incubated in the lower chamber with 5-nm BSA–gold for 3 h prior to the addition of POS, and BSA–gold was maintained throughout the duration of the experiment. Cells were then challenged with POS from the apical chamber for 1 h, washed to remove unbound POS, chased for 2 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) A double-labelled early phagosome (earlyP) that contains no 5-nm gold particles. (B) A double-labelled early phagosome containing monodisperse BSA–gold (black arrowheads) and ILVs (white arrowheads) at one pole, suggesting recent fusion of the early phagosome with a multivesicular endocytic compartment. (C) Maturing phagosomes (matP) containing 5-nm BSA–gold. Note that the density of PAG 10 nm is greatly reduced compared with that of the early phagosome shown in A and B. A lysosome (L) containing aggregated 5-nm gold particles can be readily identified. (D) Phagolysosome (PL) containing PAG 15 nm only and aggregated 5-nm gold particles. Scale bars: 100 nm (A,B,D), 200 nm (C). Black and white arrows indicate rhodopsin labelling with RET-P1 and 1D4, respectively.

Mentions: As loss of the cytoplasmic rhodopsin epitope occurred before lysosomal fusion, the endosome was a likely source of the protease that cleaves the cytoplasmic rhodopsin epitope during phagosome maturation. Although phagosome–lysosome interactions are relatively easy to measure through the acquisition of lysosomal enzymes like cathepsin D, most studies of interaction with the endocytic pathway have relied on measuring the acquisition of endosomal markers or the acquisition of overexpressed Rab proteins. We sought a more direct measure of phagosome–endosome interactions, and took advantage of the polarised nature of RPE cells cultured on Transwell® membrane inserts that allow the phagocytic and endocytic pathways to be loaded from opposite sides of the monolayer. The presence of particles endocytosed from the basal surface in phagosomes containing ROS taken up from the apical surface would indicate phagosome–endosome interaction. This approach relies on probes endocytosed from the basal surface having access to those parts of the endocytic pathway that can potentially interact with phagosomes. In polarised MDCK cells, probes endocytosed from basal and apical surfaces have been shown to meet in a common apical recycling endosome (Apodaca et al., 1994; Futter et al., 1998). To determine whether this would also be the case in the RPE, cells were incubated with 10-nm bovine serum albumin (BSA)–gold and fluid-phase horseradish peroxidase (HRP) for 2 h from opposite chambers. The HRP reaction product and BSA–gold particles colocalised in vacuoles with the morphological characteristics of endosomes – these vacuoles were electron luscent and contained discrete intraluminal vesicles (ILVs) (supplementary material Fig. S2A,B insets). These pre-lysosomal compartments could be clearly distinguished from lysosomes where the two probes also colocalised because the lysosomes were electron dense, had multiple membranous content and the gold particles within the lumen were aggregated, indicating that they were contained within a degradative compartment (supplementary material Fig. S2A, inset). Having established that RPE cells, in common with MDCK cells, have a common apical recycling endosome that can be accessed from the basal surface of polarized RPE, cells cultured on Transwell® membrane inserts were incubated with 5-nm BSA–gold in the basal chamber for 3 h prior to challenging the cells with POS in the apical chamber, as described above. Cells were then processed for cryo-immuno-electron microscopy and subsequently labelled for rhodopsin with antibodies against both C-terminal cytoplasmic (1D4) and N-terminal intradiscal (RET-P1) epitopes. Gold-loaded elements of the endocytic pathway that did not have phagocytic content could be readily identified. Monodisperse BSA–gold particles could be found in endocytic vacuoles, most of which were multivesicular endosomes/bodies (MVBs), whereas aggregated BSA–gold could be found in electron dense lysosomes (supplementary material Fig. S3A). POS added to the upper chamber contained no gold particles (supplementary material Fig. S3D,E), confirming that the monolayer remained intact during the course of the experiment. Within the cells, the maturing phagosomes could be divided into roughly four categories: (i) double-positive early phagosomes that contained no BSA–gold, indicating that they had not yet fused with the endocytic pathway (Fig. 6A); (ii) BSA–gold-containing double-positive phagosomes, where, in some cases (e.g. Fig. 6B), one pole of the phagosome contained BSA–gold and ILVs largely separate from the double-positive ROS discs, suggesting recent fusion between the phagosome and MVB; (iii) phagosomes that had lost most of the C-terminal 1D4 epitope where the endocytosed gold particles and phagocytosed ROS are mixed (maturing phagosomes in Fig. 6C) – importantly, in this category, the BSA–gold particles remained monodisperse, confirming that they were derived from a pre-lyosomal endocytic compartment; and (iv) phagolysosomes containing only the N-terminal (RET-P1) epitope with aggregated 5-nm gold particles, indicating that the phagosome had fused with the lysosome (Fig. 6D). These data show that loss of the 1D4 epitope only occurs after interaction of the phagosome with the endocytic pathway.


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)

Early phagosomes interact with a pre-lysosomal compartment of the endoytic pathway in primary porcine RPE cells. Monolayers of primary porcine RPE cells on Transwell® membrane inserts were incubated in the lower chamber with 5-nm BSA–gold for 3 h prior to the addition of POS, and BSA–gold was maintained throughout the duration of the experiment. Cells were then challenged with POS from the apical chamber for 1 h, washed to remove unbound POS, chased for 2 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) A double-labelled early phagosome (earlyP) that contains no 5-nm gold particles. (B) A double-labelled early phagosome containing monodisperse BSA–gold (black arrowheads) and ILVs (white arrowheads) at one pole, suggesting recent fusion of the early phagosome with a multivesicular endocytic compartment. (C) Maturing phagosomes (matP) containing 5-nm BSA–gold. Note that the density of PAG 10 nm is greatly reduced compared with that of the early phagosome shown in A and B. A lysosome (L) containing aggregated 5-nm gold particles can be readily identified. (D) Phagolysosome (PL) containing PAG 15 nm only and aggregated 5-nm gold particles. Scale bars: 100 nm (A,B,D), 200 nm (C). Black and white arrows indicate rhodopsin labelling with RET-P1 and 1D4, respectively.
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Related In: Results  -  Collection

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f06: Early phagosomes interact with a pre-lysosomal compartment of the endoytic pathway in primary porcine RPE cells. Monolayers of primary porcine RPE cells on Transwell® membrane inserts were incubated in the lower chamber with 5-nm BSA–gold for 3 h prior to the addition of POS, and BSA–gold was maintained throughout the duration of the experiment. Cells were then challenged with POS from the apical chamber for 1 h, washed to remove unbound POS, chased for 2 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) A double-labelled early phagosome (earlyP) that contains no 5-nm gold particles. (B) A double-labelled early phagosome containing monodisperse BSA–gold (black arrowheads) and ILVs (white arrowheads) at one pole, suggesting recent fusion of the early phagosome with a multivesicular endocytic compartment. (C) Maturing phagosomes (matP) containing 5-nm BSA–gold. Note that the density of PAG 10 nm is greatly reduced compared with that of the early phagosome shown in A and B. A lysosome (L) containing aggregated 5-nm gold particles can be readily identified. (D) Phagolysosome (PL) containing PAG 15 nm only and aggregated 5-nm gold particles. Scale bars: 100 nm (A,B,D), 200 nm (C). Black and white arrows indicate rhodopsin labelling with RET-P1 and 1D4, respectively.
Mentions: As loss of the cytoplasmic rhodopsin epitope occurred before lysosomal fusion, the endosome was a likely source of the protease that cleaves the cytoplasmic rhodopsin epitope during phagosome maturation. Although phagosome–lysosome interactions are relatively easy to measure through the acquisition of lysosomal enzymes like cathepsin D, most studies of interaction with the endocytic pathway have relied on measuring the acquisition of endosomal markers or the acquisition of overexpressed Rab proteins. We sought a more direct measure of phagosome–endosome interactions, and took advantage of the polarised nature of RPE cells cultured on Transwell® membrane inserts that allow the phagocytic and endocytic pathways to be loaded from opposite sides of the monolayer. The presence of particles endocytosed from the basal surface in phagosomes containing ROS taken up from the apical surface would indicate phagosome–endosome interaction. This approach relies on probes endocytosed from the basal surface having access to those parts of the endocytic pathway that can potentially interact with phagosomes. In polarised MDCK cells, probes endocytosed from basal and apical surfaces have been shown to meet in a common apical recycling endosome (Apodaca et al., 1994; Futter et al., 1998). To determine whether this would also be the case in the RPE, cells were incubated with 10-nm bovine serum albumin (BSA)–gold and fluid-phase horseradish peroxidase (HRP) for 2 h from opposite chambers. The HRP reaction product and BSA–gold particles colocalised in vacuoles with the morphological characteristics of endosomes – these vacuoles were electron luscent and contained discrete intraluminal vesicles (ILVs) (supplementary material Fig. S2A,B insets). These pre-lysosomal compartments could be clearly distinguished from lysosomes where the two probes also colocalised because the lysosomes were electron dense, had multiple membranous content and the gold particles within the lumen were aggregated, indicating that they were contained within a degradative compartment (supplementary material Fig. S2A, inset). Having established that RPE cells, in common with MDCK cells, have a common apical recycling endosome that can be accessed from the basal surface of polarized RPE, cells cultured on Transwell® membrane inserts were incubated with 5-nm BSA–gold in the basal chamber for 3 h prior to challenging the cells with POS in the apical chamber, as described above. Cells were then processed for cryo-immuno-electron microscopy and subsequently labelled for rhodopsin with antibodies against both C-terminal cytoplasmic (1D4) and N-terminal intradiscal (RET-P1) epitopes. Gold-loaded elements of the endocytic pathway that did not have phagocytic content could be readily identified. Monodisperse BSA–gold particles could be found in endocytic vacuoles, most of which were multivesicular endosomes/bodies (MVBs), whereas aggregated BSA–gold could be found in electron dense lysosomes (supplementary material Fig. S3A). POS added to the upper chamber contained no gold particles (supplementary material Fig. S3D,E), confirming that the monolayer remained intact during the course of the experiment. Within the cells, the maturing phagosomes could be divided into roughly four categories: (i) double-positive early phagosomes that contained no BSA–gold, indicating that they had not yet fused with the endocytic pathway (Fig. 6A); (ii) BSA–gold-containing double-positive phagosomes, where, in some cases (e.g. Fig. 6B), one pole of the phagosome contained BSA–gold and ILVs largely separate from the double-positive ROS discs, suggesting recent fusion between the phagosome and MVB; (iii) phagosomes that had lost most of the C-terminal 1D4 epitope where the endocytosed gold particles and phagocytosed ROS are mixed (maturing phagosomes in Fig. 6C) – importantly, in this category, the BSA–gold particles remained monodisperse, confirming that they were derived from a pre-lyosomal endocytic compartment; and (iv) phagolysosomes containing only the N-terminal (RET-P1) epitope with aggregated 5-nm gold particles, indicating that the phagosome had fused with the lysosome (Fig. 6D). These data show that loss of the 1D4 epitope only occurs after interaction of the phagosome with the endocytic pathway.

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