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Perforin pores in the endosomal membrane trigger the release of endocytosed granzyme B into the cytosol of target cells.

Thiery J, Keefe D, Boulant S, Boucrot E, Walch M, Martinvalet D, Goping IS, Bleackley RC, Kirchhausen T, Lieberman J - Nat. Immunol. (2011)

Bottom Line: As a consequence, both perforin and granzymes are endocytosed into enlarged endosomes called 'gigantosomes'.Here we show that perforin formed pores in the gigantosome membrane, allowing endosomal cargo, including granzymes, to be gradually released.Thus, perforin delivers granzymes by a two-step process that involves first transient pores in the cell membrane that trigger the endocytosis of granzyme and perforin and then pore formation in endosomes to trigger cytosolic release.

View Article: PubMed Central - PubMed

Affiliation: Immune Disease Institute and Program in Cellular and Molecular Medicine, Children's Hospital, Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT
How the pore-forming protein perforin delivers apoptosis-inducing granzymes to the cytosol of target cells is uncertain. Perforin induces a transient Ca2+ flux in the target cell, which triggers a process to repair the damaged cell membrane. As a consequence, both perforin and granzymes are endocytosed into enlarged endosomes called 'gigantosomes'. Here we show that perforin formed pores in the gigantosome membrane, allowing endosomal cargo, including granzymes, to be gradually released. After about 15 min, gigantosomes ruptured, releasing their remaining content. Thus, perforin delivers granzymes by a two-step process that involves first transient pores in the cell membrane that trigger the endocytosis of granzyme and perforin and then pore formation in endosomes to trigger cytosolic release.

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Endocytosed cargo is released from gigantosomes into the cytosol(a) Sublytic rat PFN induces rapid enhanced uptake of Texas Red (TR)-dextran in EGFP-EEA-1-transfected HeLa cells. Data are representative of six independent experiments. (b) Representative gigantosomes 10–17 min after EGFP-EEA-1-transfected HeLa cells were incubated with TR-dextran and sublytic PFN. Images obtained at 10–12 min suggest focal release of dextran, while at later times (15–17 min) dextran is released as gigantosomes rupture. (c) Time lapse confocal microscopy images acquired every 10 sec of EGFP-EEA-1+ HeLa cells beginning 10 min after treatment with sublytic PFN and TR-dextran. Data are representative of three different experiments. Supplementary Movie 1 shows the movie from which these images were extracted. Discrete TR-dextran release is observed initially (white arrowhead), but after ~15 min of PFN treatment, gigantosomes lose EEA-1 staining, form tubulations and rupture, leading to dextran dispersal (empty arrowhead). (d) Dextran intensity within a PFN-induced gigantosome or normal endosome (-PFN) and in the local surrounding area. Background dextran intensity was also measured in a region devoid of gigantosomes/endosomes. Corresponding images are shown below. Color bars indicate fluorescence intensity. Scale bars, 5 μm (a), 2μm (b–d).
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Figure 5: Endocytosed cargo is released from gigantosomes into the cytosol(a) Sublytic rat PFN induces rapid enhanced uptake of Texas Red (TR)-dextran in EGFP-EEA-1-transfected HeLa cells. Data are representative of six independent experiments. (b) Representative gigantosomes 10–17 min after EGFP-EEA-1-transfected HeLa cells were incubated with TR-dextran and sublytic PFN. Images obtained at 10–12 min suggest focal release of dextran, while at later times (15–17 min) dextran is released as gigantosomes rupture. (c) Time lapse confocal microscopy images acquired every 10 sec of EGFP-EEA-1+ HeLa cells beginning 10 min after treatment with sublytic PFN and TR-dextran. Data are representative of three different experiments. Supplementary Movie 1 shows the movie from which these images were extracted. Discrete TR-dextran release is observed initially (white arrowhead), but after ~15 min of PFN treatment, gigantosomes lose EEA-1 staining, form tubulations and rupture, leading to dextran dispersal (empty arrowhead). (d) Dextran intensity within a PFN-induced gigantosome or normal endosome (-PFN) and in the local surrounding area. Background dextran intensity was also measured in a region devoid of gigantosomes/endosomes. Corresponding images are shown below. Color bars indicate fluorescence intensity. Scale bars, 5 μm (a), 2μm (b–d).

Mentions: We next used live cell imaging to visualize the release of gigantosome cargo from PFN-treated cells. Time-lapse spinning disk confocal microscopy was used to image the trafficking of TR-Dextran in PFN-treated HeLa cells transfected to express EGFP-EEA-1. As previously described24, PFN enhanced 10 kDa TR-Dextran endocytosis, and TR-Dextran remained localized to gigantosomes after 10 min (Fig. 5a). Similar results were obtained when mRFP-EEA-1-transfected cells were treated with 10 kDa cationic rhodamine green-dextran and PFN (data not shown). After 10 min, we began to observe discrete and localized release of TR-Dextran from gigantosomes into the cytosol, while the gigantosome membrane appeared to remain intact (Fig. 5b and Supplementary Fig. 6a). A little later (~15–17 min after PFN–TR-Dextran loading), the gigantosome membrane became unstable. EEA-1 staining of gigantosomes disappeared and endosomal tubulations formed, which was followed by rupture of the gigantosome membrane, leading to complete release and diffusion of dextran into the cytosol (Fig. 5b,c, Supplementary Fig. 6b, Movies S1–S3). As dextran diffuses, it becomes difficult to detect. To confirm our impression that TR-Dextran was released from gigantosomes to the cytosol before they ruptured, we imaged PFN and dextran-treated cells by live cell 4D spinning disk confocal imaging beginning 7 min after adding PFN and dextran. TR-Dextran staining intensity was measured in the gigantosome or endosomes and in the surrounding cytoplasm (Fig. 5d). In the absence of PFN, the TR-Dextran signal in endosomes gradually increased as more dextran was incorporated, but the signal in the surrounding cytosol remained low and was stable with some fluctuation. However, in cells treated with PFN, TR-dextran signal intensity in the gigantosome gradually decreased as TR staining in the surrounding cytoplasm increased. As a control, we measured TR-Dextran background intensity in a region of the cytosol that did not contain gigantosomes or endosomes, and found that it did not change. Taken together, these data suggest that PFN pores in the gigantosome membrane allow slow release of endosomal cargo before completely destabilizing the endosomal membrane, which leads to endosomolysis and rapid release of the remaining cargo to the cytosol. A model for PFN delivery of Gzms is shown in Supplementary Fig. 7.


Perforin pores in the endosomal membrane trigger the release of endocytosed granzyme B into the cytosol of target cells.

Thiery J, Keefe D, Boulant S, Boucrot E, Walch M, Martinvalet D, Goping IS, Bleackley RC, Kirchhausen T, Lieberman J - Nat. Immunol. (2011)

Endocytosed cargo is released from gigantosomes into the cytosol(a) Sublytic rat PFN induces rapid enhanced uptake of Texas Red (TR)-dextran in EGFP-EEA-1-transfected HeLa cells. Data are representative of six independent experiments. (b) Representative gigantosomes 10–17 min after EGFP-EEA-1-transfected HeLa cells were incubated with TR-dextran and sublytic PFN. Images obtained at 10–12 min suggest focal release of dextran, while at later times (15–17 min) dextran is released as gigantosomes rupture. (c) Time lapse confocal microscopy images acquired every 10 sec of EGFP-EEA-1+ HeLa cells beginning 10 min after treatment with sublytic PFN and TR-dextran. Data are representative of three different experiments. Supplementary Movie 1 shows the movie from which these images were extracted. Discrete TR-dextran release is observed initially (white arrowhead), but after ~15 min of PFN treatment, gigantosomes lose EEA-1 staining, form tubulations and rupture, leading to dextran dispersal (empty arrowhead). (d) Dextran intensity within a PFN-induced gigantosome or normal endosome (-PFN) and in the local surrounding area. Background dextran intensity was also measured in a region devoid of gigantosomes/endosomes. Corresponding images are shown below. Color bars indicate fluorescence intensity. Scale bars, 5 μm (a), 2μm (b–d).
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Figure 5: Endocytosed cargo is released from gigantosomes into the cytosol(a) Sublytic rat PFN induces rapid enhanced uptake of Texas Red (TR)-dextran in EGFP-EEA-1-transfected HeLa cells. Data are representative of six independent experiments. (b) Representative gigantosomes 10–17 min after EGFP-EEA-1-transfected HeLa cells were incubated with TR-dextran and sublytic PFN. Images obtained at 10–12 min suggest focal release of dextran, while at later times (15–17 min) dextran is released as gigantosomes rupture. (c) Time lapse confocal microscopy images acquired every 10 sec of EGFP-EEA-1+ HeLa cells beginning 10 min after treatment with sublytic PFN and TR-dextran. Data are representative of three different experiments. Supplementary Movie 1 shows the movie from which these images were extracted. Discrete TR-dextran release is observed initially (white arrowhead), but after ~15 min of PFN treatment, gigantosomes lose EEA-1 staining, form tubulations and rupture, leading to dextran dispersal (empty arrowhead). (d) Dextran intensity within a PFN-induced gigantosome or normal endosome (-PFN) and in the local surrounding area. Background dextran intensity was also measured in a region devoid of gigantosomes/endosomes. Corresponding images are shown below. Color bars indicate fluorescence intensity. Scale bars, 5 μm (a), 2μm (b–d).
Mentions: We next used live cell imaging to visualize the release of gigantosome cargo from PFN-treated cells. Time-lapse spinning disk confocal microscopy was used to image the trafficking of TR-Dextran in PFN-treated HeLa cells transfected to express EGFP-EEA-1. As previously described24, PFN enhanced 10 kDa TR-Dextran endocytosis, and TR-Dextran remained localized to gigantosomes after 10 min (Fig. 5a). Similar results were obtained when mRFP-EEA-1-transfected cells were treated with 10 kDa cationic rhodamine green-dextran and PFN (data not shown). After 10 min, we began to observe discrete and localized release of TR-Dextran from gigantosomes into the cytosol, while the gigantosome membrane appeared to remain intact (Fig. 5b and Supplementary Fig. 6a). A little later (~15–17 min after PFN–TR-Dextran loading), the gigantosome membrane became unstable. EEA-1 staining of gigantosomes disappeared and endosomal tubulations formed, which was followed by rupture of the gigantosome membrane, leading to complete release and diffusion of dextran into the cytosol (Fig. 5b,c, Supplementary Fig. 6b, Movies S1–S3). As dextran diffuses, it becomes difficult to detect. To confirm our impression that TR-Dextran was released from gigantosomes to the cytosol before they ruptured, we imaged PFN and dextran-treated cells by live cell 4D spinning disk confocal imaging beginning 7 min after adding PFN and dextran. TR-Dextran staining intensity was measured in the gigantosome or endosomes and in the surrounding cytoplasm (Fig. 5d). In the absence of PFN, the TR-Dextran signal in endosomes gradually increased as more dextran was incorporated, but the signal in the surrounding cytosol remained low and was stable with some fluctuation. However, in cells treated with PFN, TR-dextran signal intensity in the gigantosome gradually decreased as TR staining in the surrounding cytoplasm increased. As a control, we measured TR-Dextran background intensity in a region of the cytosol that did not contain gigantosomes or endosomes, and found that it did not change. Taken together, these data suggest that PFN pores in the gigantosome membrane allow slow release of endosomal cargo before completely destabilizing the endosomal membrane, which leads to endosomolysis and rapid release of the remaining cargo to the cytosol. A model for PFN delivery of Gzms is shown in Supplementary Fig. 7.

Bottom Line: As a consequence, both perforin and granzymes are endocytosed into enlarged endosomes called 'gigantosomes'.Here we show that perforin formed pores in the gigantosome membrane, allowing endosomal cargo, including granzymes, to be gradually released.Thus, perforin delivers granzymes by a two-step process that involves first transient pores in the cell membrane that trigger the endocytosis of granzyme and perforin and then pore formation in endosomes to trigger cytosolic release.

View Article: PubMed Central - PubMed

Affiliation: Immune Disease Institute and Program in Cellular and Molecular Medicine, Children's Hospital, Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT
How the pore-forming protein perforin delivers apoptosis-inducing granzymes to the cytosol of target cells is uncertain. Perforin induces a transient Ca2+ flux in the target cell, which triggers a process to repair the damaged cell membrane. As a consequence, both perforin and granzymes are endocytosed into enlarged endosomes called 'gigantosomes'. Here we show that perforin formed pores in the gigantosome membrane, allowing endosomal cargo, including granzymes, to be gradually released. After about 15 min, gigantosomes ruptured, releasing their remaining content. Thus, perforin delivers granzymes by a two-step process that involves first transient pores in the cell membrane that trigger the endocytosis of granzyme and perforin and then pore formation in endosomes to trigger cytosolic release.

Show MeSH
Related in: MedlinePlus