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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 GzmB is released into the cytosol within ~10 min of PFN loading(a) Within 5–10 min of treatment with sublytic native rat PFN and native human GzmB, GzmB begins to be released from gigantosomes. HeLa cells were treated with GzmB ± sublytic PFN, fixed at the indicated time and stained for EEA-1 and GzmB. Representative single spinning disk confocal sections from three independent experiments are shown. Percentage of cells with GzmB in gigantosomes or in the cytosol (bottom row) is indicated (mean ± s.d.). (b) HeLa cells were treated with native human GzmB ±sublytic rat PFN, fixed at the indicated times and stained for GzmB and DAPI. Images were acquired by 3D-capture widefield microscopy followed by iterative deconvolution and projection. Pictures are representative of three independent experiments. (c) HeLa cells were treated with A488-labeled GzmB ± sublytic PFN and fixed at the indicated times. After release, GzmB accumulates in and around the nucleus. Pictures are representative of two independent experiments. Color bars and associated numbers indicate fluorescence intensity levels. Scale bars, 5 μm (a), 10 μm (b,c). Dashed lines, plasma membrane.
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Figure 4: Endocytosed GzmB is released into the cytosol within ~10 min of PFN loading(a) Within 5–10 min of treatment with sublytic native rat PFN and native human GzmB, GzmB begins to be released from gigantosomes. HeLa cells were treated with GzmB ± sublytic PFN, fixed at the indicated time and stained for EEA-1 and GzmB. Representative single spinning disk confocal sections from three independent experiments are shown. Percentage of cells with GzmB in gigantosomes or in the cytosol (bottom row) is indicated (mean ± s.d.). (b) HeLa cells were treated with native human GzmB ±sublytic rat PFN, fixed at the indicated times and stained for GzmB and DAPI. Images were acquired by 3D-capture widefield microscopy followed by iterative deconvolution and projection. Pictures are representative of three independent experiments. (c) HeLa cells were treated with A488-labeled GzmB ± sublytic PFN and fixed at the indicated times. After release, GzmB accumulates in and around the nucleus. Pictures are representative of two independent experiments. Color bars and associated numbers indicate fluorescence intensity levels. Scale bars, 5 μm (a), 10 μm (b,c). Dashed lines, plasma membrane.

Mentions: To test our hypothesis that PFN pore formation in the endosomal membrane is responsible for Gzm release, we investigated by co-staining for EEA-1 and GzmB the timing of GzmB uptake and cytosolic release following treatment with PFN and GzmB. In the absence of PFN, cells did not efficiently take up GzmB (Fig. 4a). After exposure to sublytic PFN and GzmB, GzmB-containing EEA-1+ gigantosomes formed within 5 min. After ~10–15 min, GzmB was released from gigantosomes to the cytosol as the bright vesicular staining of the endocytosed cargo dispersed into a faintly detected haze in the cytosol. Within 20 min, the majority of the GzmB signal concentrated in the nucleus, as expected41, and gigantosomes were no longer detected (Fig. 4a,b). Uptake of Alexa488-GzmB into gigantosomes was also seen within 2 min of adding PFN. Cytosolic fluorescence began to be visible within 5 min, but by 15 min gigantosome staining had disappeared and GzmB became cytosolic and nuclear (Fig. 4c). Therefore the release of GzmB from gigantosomes in PFN treated cells within ~15 min coincided temporally with PFN pore formation as judged by the disappearance of Pf80 staining and PFN cross-linking.


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 GzmB is released into the cytosol within ~10 min of PFN loading(a) Within 5–10 min of treatment with sublytic native rat PFN and native human GzmB, GzmB begins to be released from gigantosomes. HeLa cells were treated with GzmB ± sublytic PFN, fixed at the indicated time and stained for EEA-1 and GzmB. Representative single spinning disk confocal sections from three independent experiments are shown. Percentage of cells with GzmB in gigantosomes or in the cytosol (bottom row) is indicated (mean ± s.d.). (b) HeLa cells were treated with native human GzmB ±sublytic rat PFN, fixed at the indicated times and stained for GzmB and DAPI. Images were acquired by 3D-capture widefield microscopy followed by iterative deconvolution and projection. Pictures are representative of three independent experiments. (c) HeLa cells were treated with A488-labeled GzmB ± sublytic PFN and fixed at the indicated times. After release, GzmB accumulates in and around the nucleus. Pictures are representative of two independent experiments. Color bars and associated numbers indicate fluorescence intensity levels. Scale bars, 5 μm (a), 10 μm (b,c). Dashed lines, plasma membrane.
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Figure 4: Endocytosed GzmB is released into the cytosol within ~10 min of PFN loading(a) Within 5–10 min of treatment with sublytic native rat PFN and native human GzmB, GzmB begins to be released from gigantosomes. HeLa cells were treated with GzmB ± sublytic PFN, fixed at the indicated time and stained for EEA-1 and GzmB. Representative single spinning disk confocal sections from three independent experiments are shown. Percentage of cells with GzmB in gigantosomes or in the cytosol (bottom row) is indicated (mean ± s.d.). (b) HeLa cells were treated with native human GzmB ±sublytic rat PFN, fixed at the indicated times and stained for GzmB and DAPI. Images were acquired by 3D-capture widefield microscopy followed by iterative deconvolution and projection. Pictures are representative of three independent experiments. (c) HeLa cells were treated with A488-labeled GzmB ± sublytic PFN and fixed at the indicated times. After release, GzmB accumulates in and around the nucleus. Pictures are representative of two independent experiments. Color bars and associated numbers indicate fluorescence intensity levels. Scale bars, 5 μm (a), 10 μm (b,c). Dashed lines, plasma membrane.
Mentions: To test our hypothesis that PFN pore formation in the endosomal membrane is responsible for Gzm release, we investigated by co-staining for EEA-1 and GzmB the timing of GzmB uptake and cytosolic release following treatment with PFN and GzmB. In the absence of PFN, cells did not efficiently take up GzmB (Fig. 4a). After exposure to sublytic PFN and GzmB, GzmB-containing EEA-1+ gigantosomes formed within 5 min. After ~10–15 min, GzmB was released from gigantosomes to the cytosol as the bright vesicular staining of the endocytosed cargo dispersed into a faintly detected haze in the cytosol. Within 20 min, the majority of the GzmB signal concentrated in the nucleus, as expected41, and gigantosomes were no longer detected (Fig. 4a,b). Uptake of Alexa488-GzmB into gigantosomes was also seen within 2 min of adding PFN. Cytosolic fluorescence began to be visible within 5 min, but by 15 min gigantosome staining had disappeared and GzmB became cytosolic and nuclear (Fig. 4c). Therefore the release of GzmB from gigantosomes in PFN treated cells within ~15 min coincided temporally with PFN pore formation as judged by the disappearance of Pf80 staining and PFN cross-linking.

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