Limits...
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.

Show MeSH

Related in: MedlinePlus

Sequential stages of phagosome maturation can also be identified by monitoring rhodopsin in macrophages challenged with POS. J774A.1 macrophages seeded onto Transwell® membrane inserts were challenged with POS 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) Double-labelled ROS at the plasma membrane. (B) A double-labelled early phagosome (earlyP). (C) A maturing phagosome (matP) containing only very little 1D4 on some discs. (D) A phagolysosome (PL) containing disorganised discs where RET-P1 has started to be degraded. Scale bars: 200 nm (A,C), 100 nm (B,D). Black and white arrows indicate rhodopsin labelling with RET-P1 and 1D4, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4150067&req=5

f07: Sequential stages of phagosome maturation can also be identified by monitoring rhodopsin in macrophages challenged with POS. J774A.1 macrophages seeded onto Transwell® membrane inserts were challenged with POS 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) Double-labelled ROS at the plasma membrane. (B) A double-labelled early phagosome (earlyP). (C) A maturing phagosome (matP) containing only very little 1D4 on some discs. (D) A phagolysosome (PL) containing disorganised discs where RET-P1 has started to be degraded. Scale bars: 200 nm (A,C), 100 nm (B,D). Black and white arrows indicate rhodopsin labelling with RET-P1 and 1D4, respectively.

Mentions: A range of different surface molecules are used by phagocytes to recognise and engulf particles, although there are also some common components of the core phagocytic machinery. Less is known about the molecular regulation of phagosome maturation, but proteomic studies of maturing phagosomes suggest that a combination of core and cell-type-specific and particle-type-specific components are likely to be used. Our finding that proteolytic processing of rhodopsin occurs following fusion with endosomes suggests that rhodopsin processing might prove to be a very sensitive readout of a phagosome–endosome interaction that is central to phagosome maturation in multiple phagocytes. Therefore, we investigated whether the C-terminal rhodopsin processing also occurred during phagosome maturation in the macrophage cell line J774.1, when challenged with POS. After a 1-h pulse and a 2-h chase, cryo-immuno-electron microscopy using both rhodopsin antibodies, 1D4 and RET-P1, showed that the cytoplasmic 1D4 epitope was lost before the intradiscal RET-P1 epitope (Fig. 7), as observed in RPE cells. Loss of the cytoplasmic epitope was rapid in the macrophage cell line, such that most phagosomes had a lower density of cytoplasmic 1D4 staining than the ROS before engulfment (compare Fig. 7A with Fig. 7B). Even in phagosomes that had largely lost the 1D4 epitope, individual discs could sometimes be observed that retained 1D4 staining (Fig. 7C). As with RPE cells, loss of the intradiscal RET-P1 epitope was only observed in phagosomes that appeared to have fused with electron-dense lysosomes and had lost morphologically identifiable discs (Fig. 7D).


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 can also be identified by monitoring rhodopsin in macrophages challenged with POS. J774A.1 macrophages seeded onto Transwell® membrane inserts were challenged with POS 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) Double-labelled ROS at the plasma membrane. (B) A double-labelled early phagosome (earlyP). (C) A maturing phagosome (matP) containing only very little 1D4 on some discs. (D) A phagolysosome (PL) containing disorganised discs where RET-P1 has started to be degraded. Scale bars: 200 nm (A,C), 100 nm (B,D). Black and white arrows indicate rhodopsin labelling with RET-P1 and 1D4, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f07: Sequential stages of phagosome maturation can also be identified by monitoring rhodopsin in macrophages challenged with POS. J774A.1 macrophages seeded onto Transwell® membrane inserts were challenged with POS 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) Double-labelled ROS at the plasma membrane. (B) A double-labelled early phagosome (earlyP). (C) A maturing phagosome (matP) containing only very little 1D4 on some discs. (D) A phagolysosome (PL) containing disorganised discs where RET-P1 has started to be degraded. Scale bars: 200 nm (A,C), 100 nm (B,D). Black and white arrows indicate rhodopsin labelling with RET-P1 and 1D4, respectively.
Mentions: A range of different surface molecules are used by phagocytes to recognise and engulf particles, although there are also some common components of the core phagocytic machinery. Less is known about the molecular regulation of phagosome maturation, but proteomic studies of maturing phagosomes suggest that a combination of core and cell-type-specific and particle-type-specific components are likely to be used. Our finding that proteolytic processing of rhodopsin occurs following fusion with endosomes suggests that rhodopsin processing might prove to be a very sensitive readout of a phagosome–endosome interaction that is central to phagosome maturation in multiple phagocytes. Therefore, we investigated whether the C-terminal rhodopsin processing also occurred during phagosome maturation in the macrophage cell line J774.1, when challenged with POS. After a 1-h pulse and a 2-h chase, cryo-immuno-electron microscopy using both rhodopsin antibodies, 1D4 and RET-P1, showed that the cytoplasmic 1D4 epitope was lost before the intradiscal RET-P1 epitope (Fig. 7), as observed in RPE cells. Loss of the cytoplasmic epitope was rapid in the macrophage cell line, such that most phagosomes had a lower density of cytoplasmic 1D4 staining than the ROS before engulfment (compare Fig. 7A with Fig. 7B). Even in phagosomes that had largely lost the 1D4 epitope, individual discs could sometimes be observed that retained 1D4 staining (Fig. 7C). As with RPE cells, loss of the intradiscal RET-P1 epitope was only observed in phagosomes that appeared to have fused with electron-dense lysosomes and had lost morphologically identifiable discs (Fig. 7D).

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