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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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GzmB and PFN localizes in gigantosomes in target cells during NK cell lysisYT-Indy NK cells incubated with 721.221 target cells were stained at indicated times for GzmB (a) or PFN (b). Arrows indicate GzmB or PFN signal (pseudocolor) in target cells. After 10 min, GzmB-containing gigantosomes are visible, but at 20 min, GzmB staining is more dispersed. After 10 min, PFN staining (Pf80) in gigantosomes is visible, but disappears at 20 min. Images were acquired by spinning disk confocal microscopy. Representative z stack series projections from two independent experiments are shown. Color bars and associated numbers indicate fluorescence intensity. Scale bars, 10 μm. Dashed lines, plasma membrane. (c) YT-Indy NK cells expressing EGFP-GzmB were incubated with 721.221 target cells and imaged by widefield live imaging every minute. GzmB-containing gigantosomes are visible 2 min after conjugate formation, but after 15 min, GzmB staining is more dispersed. A representative time-lapse series from two independent experiments is shown. Numbers represent min after conjugate formation. Phase contrast is displayed in red. To visualize the low GzmB signal in the target cell, the EGFP channel was over-exposed. A control YT-Indy cell (bottom row) imaged with regular exposure time confirms the granular expression of EGFP-GzmB. Scale bars, 10 μm.
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Figure 6: GzmB and PFN localizes in gigantosomes in target cells during NK cell lysisYT-Indy NK cells incubated with 721.221 target cells were stained at indicated times for GzmB (a) or PFN (b). Arrows indicate GzmB or PFN signal (pseudocolor) in target cells. After 10 min, GzmB-containing gigantosomes are visible, but at 20 min, GzmB staining is more dispersed. After 10 min, PFN staining (Pf80) in gigantosomes is visible, but disappears at 20 min. Images were acquired by spinning disk confocal microscopy. Representative z stack series projections from two independent experiments are shown. Color bars and associated numbers indicate fluorescence intensity. Scale bars, 10 μm. Dashed lines, plasma membrane. (c) YT-Indy NK cells expressing EGFP-GzmB were incubated with 721.221 target cells and imaged by widefield live imaging every minute. GzmB-containing gigantosomes are visible 2 min after conjugate formation, but after 15 min, GzmB staining is more dispersed. A representative time-lapse series from two independent experiments is shown. Numbers represent min after conjugate formation. Phase contrast is displayed in red. To visualize the low GzmB signal in the target cell, the EGFP channel was over-exposed. A control YT-Indy cell (bottom row) imaged with regular exposure time confirms the granular expression of EGFP-GzmB. Scale bars, 10 μm.

Mentions: The most physiologically relevant system to study PFN’s actions is CTL- or NK-mediated lysis of target cells. However, no published studies have visualized PFN or Gzm trafficking in cells subjected to killer cell-mediated destruction, presumably because the amount of native enzyme that enters a target cell is limited. The data presented above were obtained by incubating target cells with sublytic amounts of PFN (with and without GzmB), which is considered a good surrogate for killer cell-mediated cell death, since it reproduces the apoptotic features of the target cell. We previously showed that the two components of the cellular membrane repair response (fusion of internal vesicles with the plasma membrane and rapid endocytosis of the damaged membrane) occur in cells targeted by CD8 T cells and NK cells23, 24. Moreover, EEA-1+ gigantosomes form in target cells during killer cell attack23, 24. To test further whether the two-step model of Gzm delivery by PFN via endosomes applies to the physiologically most relevant model of killer cell attack, we incubated YT-Indy NK cells with 721.221 target cells and examined NK:target cell conjugates at various times over 20 min on slides stained for GzmB or PFN. Within NK cells, GzmB and PFN stained in granules that concentrated at the interface with the target cell, as expected (Fig. 6a,b). Although GzmB or PFN staining was not apparent in most target cells, in a few cells we were able to visualize GzmB and PFN within enlarged cytosolic vesicles sized like gigantosomes. Target cells that stained with GzmB or PFN typically had one or a few gigantosomes visible near the killer cell:target interface. After 20 min incubation, in a few cells GzmB was detected dispersed in the target cell cytosol. At the same time we could not detect PFN staining using the conformation-sensitive Pf80 anti-PFN Ab in any target cell (Fig. 6b). To follow GzmB trafficking in target cells, we also imaged YT-Indy (F6) cells, expressing EGFP-GzmB, as they targeted 721.221 cells (Fig. 6c). EGFP-GzmB first concentrated in a gigantosome-like structure before dispersing in the cytosol about 10–17 min later. Therefore, GzmB and PFN endocytosis into gigantosomes and PFN-induced release of GzmB from gigantosomes to the cytosol of target cell also occurs during killer cell attack.


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)

GzmB and PFN localizes in gigantosomes in target cells during NK cell lysisYT-Indy NK cells incubated with 721.221 target cells were stained at indicated times for GzmB (a) or PFN (b). Arrows indicate GzmB or PFN signal (pseudocolor) in target cells. After 10 min, GzmB-containing gigantosomes are visible, but at 20 min, GzmB staining is more dispersed. After 10 min, PFN staining (Pf80) in gigantosomes is visible, but disappears at 20 min. Images were acquired by spinning disk confocal microscopy. Representative z stack series projections from two independent experiments are shown. Color bars and associated numbers indicate fluorescence intensity. Scale bars, 10 μm. Dashed lines, plasma membrane. (c) YT-Indy NK cells expressing EGFP-GzmB were incubated with 721.221 target cells and imaged by widefield live imaging every minute. GzmB-containing gigantosomes are visible 2 min after conjugate formation, but after 15 min, GzmB staining is more dispersed. A representative time-lapse series from two independent experiments is shown. Numbers represent min after conjugate formation. Phase contrast is displayed in red. To visualize the low GzmB signal in the target cell, the EGFP channel was over-exposed. A control YT-Indy cell (bottom row) imaged with regular exposure time confirms the granular expression of EGFP-GzmB. Scale bars, 10 μm.
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Figure 6: GzmB and PFN localizes in gigantosomes in target cells during NK cell lysisYT-Indy NK cells incubated with 721.221 target cells were stained at indicated times for GzmB (a) or PFN (b). Arrows indicate GzmB or PFN signal (pseudocolor) in target cells. After 10 min, GzmB-containing gigantosomes are visible, but at 20 min, GzmB staining is more dispersed. After 10 min, PFN staining (Pf80) in gigantosomes is visible, but disappears at 20 min. Images were acquired by spinning disk confocal microscopy. Representative z stack series projections from two independent experiments are shown. Color bars and associated numbers indicate fluorescence intensity. Scale bars, 10 μm. Dashed lines, plasma membrane. (c) YT-Indy NK cells expressing EGFP-GzmB were incubated with 721.221 target cells and imaged by widefield live imaging every minute. GzmB-containing gigantosomes are visible 2 min after conjugate formation, but after 15 min, GzmB staining is more dispersed. A representative time-lapse series from two independent experiments is shown. Numbers represent min after conjugate formation. Phase contrast is displayed in red. To visualize the low GzmB signal in the target cell, the EGFP channel was over-exposed. A control YT-Indy cell (bottom row) imaged with regular exposure time confirms the granular expression of EGFP-GzmB. Scale bars, 10 μm.
Mentions: The most physiologically relevant system to study PFN’s actions is CTL- or NK-mediated lysis of target cells. However, no published studies have visualized PFN or Gzm trafficking in cells subjected to killer cell-mediated destruction, presumably because the amount of native enzyme that enters a target cell is limited. The data presented above were obtained by incubating target cells with sublytic amounts of PFN (with and without GzmB), which is considered a good surrogate for killer cell-mediated cell death, since it reproduces the apoptotic features of the target cell. We previously showed that the two components of the cellular membrane repair response (fusion of internal vesicles with the plasma membrane and rapid endocytosis of the damaged membrane) occur in cells targeted by CD8 T cells and NK cells23, 24. Moreover, EEA-1+ gigantosomes form in target cells during killer cell attack23, 24. To test further whether the two-step model of Gzm delivery by PFN via endosomes applies to the physiologically most relevant model of killer cell attack, we incubated YT-Indy NK cells with 721.221 target cells and examined NK:target cell conjugates at various times over 20 min on slides stained for GzmB or PFN. Within NK cells, GzmB and PFN stained in granules that concentrated at the interface with the target cell, as expected (Fig. 6a,b). Although GzmB or PFN staining was not apparent in most target cells, in a few cells we were able to visualize GzmB and PFN within enlarged cytosolic vesicles sized like gigantosomes. Target cells that stained with GzmB or PFN typically had one or a few gigantosomes visible near the killer cell:target interface. After 20 min incubation, in a few cells GzmB was detected dispersed in the target cell cytosol. At the same time we could not detect PFN staining using the conformation-sensitive Pf80 anti-PFN Ab in any target cell (Fig. 6b). To follow GzmB trafficking in target cells, we also imaged YT-Indy (F6) cells, expressing EGFP-GzmB, as they targeted 721.221 cells (Fig. 6c). EGFP-GzmB first concentrated in a gigantosome-like structure before dispersing in the cytosol about 10–17 min later. Therefore, GzmB and PFN endocytosis into gigantosomes and PFN-induced release of GzmB from gigantosomes to the cytosol of target cell also occurs during killer cell attack.

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