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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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Inhibition of gigantosome formation does not impair GzmB-induced apoptosis(a–b) HeLa cells transfected with EGFP-Rab5(WT) or EGFP-Rab5(S34N) dominant negative mutant (a, top row) were treated with buffer or sublytic rat PFN ± 100 nM native human GzmB, and apoptosis in EGFP+ cells was measured 2 h later by labeling with M30 mAb (which recognize a cytokeratin-18 epitope, revealed after caspase cleavage). Representative flow cytometry histograms (a) (MFI, mean fluorescence intensity) and mean ± s.d. of percentage of M30+ cells from three independent experiments (b) are shown. P values were determined by unpaired two-tailed student’s t-test. There was no significant (NS) difference in GzmB-mediated apoptosis in Rab5(S34N)-transfected cells relative to Rab5(WT)-transfected cells. (c) Analysis of procaspase-3 activation by immunoblot in HeLa cells transfected with EGFP-Rab5(WT) or EGFP-Rab5(S34N) and treated with buffer or sublytic rat PFN ± 50 nM native human GzmB for 30 min. Actin was a loading control. Data are representative of two independent experiments.
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Figure 1: Inhibition of gigantosome formation does not impair GzmB-induced apoptosis(a–b) HeLa cells transfected with EGFP-Rab5(WT) or EGFP-Rab5(S34N) dominant negative mutant (a, top row) were treated with buffer or sublytic rat PFN ± 100 nM native human GzmB, and apoptosis in EGFP+ cells was measured 2 h later by labeling with M30 mAb (which recognize a cytokeratin-18 epitope, revealed after caspase cleavage). Representative flow cytometry histograms (a) (MFI, mean fluorescence intensity) and mean ± s.d. of percentage of M30+ cells from three independent experiments (b) are shown. P values were determined by unpaired two-tailed student’s t-test. There was no significant (NS) difference in GzmB-mediated apoptosis in Rab5(S34N)-transfected cells relative to Rab5(WT)-transfected cells. (c) Analysis of procaspase-3 activation by immunoblot in HeLa cells transfected with EGFP-Rab5(WT) or EGFP-Rab5(S34N) and treated with buffer or sublytic rat PFN ± 50 nM native human GzmB for 30 min. Actin was a loading control. Data are representative of two independent experiments.

Mentions: We first verified that large EEA-1+ Lamp1− intracellular vesicles (gigantosomes) containing PFN and Gzms23, 24 formed after sublytic PFN treatment (Supplementary Fig. 1a,b)23, 24. These enlarged endosomes formed by homotypic fusion of early endosomes. (Supplementary Fig. 1c–e). Rab5 is a small GTPase that regulates fusion between endocytic vesicles and early endosomes, as well as the homotypic fusion between early endosomes31, 32, 33. Mutant Rab5(S34N), which has preferential affinity for GDP, acts as a dominant-negative inhibitor of Rab534. Gigantosomes formed within 10 min of sublytic PFN treatment of HeLa cells transfected with monomeric red fluorescent protein (mRFP)-EEA-1 and Rab5(WT), but not when the wild-type protein was replaced with enhanced green fluorescent protein (EGFP)-Rab5(S34N) (Supplementary Fig. 2). Thus gigantosome formation is Rab5-dependent. We next assessed whether gigantosome formation is required for induction of apoptosis by GzmB and PFN. PFN and GzmB-mediated apoptosis, assessed by caspase-3 and cytokeratin 18 cleavage and annexin-V–propidium iodide (PI) staining, was compared in HeLa cells transfected with Rab5(WT) or Rab5(S34N) (Fig. 1 and Supplementary Fig. 3). Apoptosis was similar in untransfected control cells and in cells expressing Rab5(WT) or Rab5(S34N). Thus gigantosome formation is dispensable for GzmB-mediated induction of apoptosis.


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)

Inhibition of gigantosome formation does not impair GzmB-induced apoptosis(a–b) HeLa cells transfected with EGFP-Rab5(WT) or EGFP-Rab5(S34N) dominant negative mutant (a, top row) were treated with buffer or sublytic rat PFN ± 100 nM native human GzmB, and apoptosis in EGFP+ cells was measured 2 h later by labeling with M30 mAb (which recognize a cytokeratin-18 epitope, revealed after caspase cleavage). Representative flow cytometry histograms (a) (MFI, mean fluorescence intensity) and mean ± s.d. of percentage of M30+ cells from three independent experiments (b) are shown. P values were determined by unpaired two-tailed student’s t-test. There was no significant (NS) difference in GzmB-mediated apoptosis in Rab5(S34N)-transfected cells relative to Rab5(WT)-transfected cells. (c) Analysis of procaspase-3 activation by immunoblot in HeLa cells transfected with EGFP-Rab5(WT) or EGFP-Rab5(S34N) and treated with buffer or sublytic rat PFN ± 50 nM native human GzmB for 30 min. Actin was a loading control. Data are representative of two independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3140544&req=5

Figure 1: Inhibition of gigantosome formation does not impair GzmB-induced apoptosis(a–b) HeLa cells transfected with EGFP-Rab5(WT) or EGFP-Rab5(S34N) dominant negative mutant (a, top row) were treated with buffer or sublytic rat PFN ± 100 nM native human GzmB, and apoptosis in EGFP+ cells was measured 2 h later by labeling with M30 mAb (which recognize a cytokeratin-18 epitope, revealed after caspase cleavage). Representative flow cytometry histograms (a) (MFI, mean fluorescence intensity) and mean ± s.d. of percentage of M30+ cells from three independent experiments (b) are shown. P values were determined by unpaired two-tailed student’s t-test. There was no significant (NS) difference in GzmB-mediated apoptosis in Rab5(S34N)-transfected cells relative to Rab5(WT)-transfected cells. (c) Analysis of procaspase-3 activation by immunoblot in HeLa cells transfected with EGFP-Rab5(WT) or EGFP-Rab5(S34N) and treated with buffer or sublytic rat PFN ± 50 nM native human GzmB for 30 min. Actin was a loading control. Data are representative of two independent experiments.
Mentions: We first verified that large EEA-1+ Lamp1− intracellular vesicles (gigantosomes) containing PFN and Gzms23, 24 formed after sublytic PFN treatment (Supplementary Fig. 1a,b)23, 24. These enlarged endosomes formed by homotypic fusion of early endosomes. (Supplementary Fig. 1c–e). Rab5 is a small GTPase that regulates fusion between endocytic vesicles and early endosomes, as well as the homotypic fusion between early endosomes31, 32, 33. Mutant Rab5(S34N), which has preferential affinity for GDP, acts as a dominant-negative inhibitor of Rab534. Gigantosomes formed within 10 min of sublytic PFN treatment of HeLa cells transfected with monomeric red fluorescent protein (mRFP)-EEA-1 and Rab5(WT), but not when the wild-type protein was replaced with enhanced green fluorescent protein (EGFP)-Rab5(S34N) (Supplementary Fig. 2). Thus gigantosome formation is Rab5-dependent. We next assessed whether gigantosome formation is required for induction of apoptosis by GzmB and PFN. PFN and GzmB-mediated apoptosis, assessed by caspase-3 and cytokeratin 18 cleavage and annexin-V–propidium iodide (PI) staining, was compared in HeLa cells transfected with Rab5(WT) or Rab5(S34N) (Fig. 1 and Supplementary Fig. 3). Apoptosis was similar in untransfected control cells and in cells expressing Rab5(WT) or Rab5(S34N). Thus gigantosome formation is dispensable for GzmB-mediated induction of apoptosis.

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