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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We thank Y. Jones (University of Oxford) for the mammalian expression vector pHLseq; G.M. Griffiths (Oxford University) for 2d4 mouse antibody to human perforin; and E. Marino for assistance with microscopy and image analysis. Supported by the US National Institutes of Health (AI063430 to J.L. and GM075252 to T.K.), the Canadian Institutes of Health Research (R.C.B.), the Canadian Cancer Society (RCB), the Stiefel-Zangger Foundation (M.W.), the Human Frontier Science Program Organization (E.B.), the Harvard Digestive Diseases Center (S.B.) and the Immune Disease Institute and GlaxoSmithKline Alliance (S.B.).
The authors declare no competing financial interests.
Supplementary Figures 1–7 and Supplementary Methods (PDF 941 kb)
PFN-mediated release of endocytosed dextran from a gigantosome. Representative EGFP-EEA-1+ (green) gigantosomes from transfected HeLa cells treated with TR-Dextran (red) and sublytic rat PFN. Live images were acquired by spinning disk confocal microscopy starting 10 min after addition of TR-Dextran and PFN (duration 5 min, 2.5 sec/frame). Selected static individual frames from these movies are shown in Figure 5c. (MOV 2009 kb)
PFN-mediated release of endocytosed dextran from a gigantosome. Representative EGFP-EEA-1+ (green) gigantosomes from transfected HeLa cells treated with TR-Dextran (red) and sublytic rat PFN. Live images were acquired by spinning disk confocal microscopy starting 10 min after addition of TR-Dextran and PFN (duration 6 min, 10 sec/frame). Selected static individual frames from these movies are shown in Supplementary Figure 6a. (MOV 113 kb)
PFN-mediated release of endocytosed dextran from a gigantosome. Representative EGFP-EEA-1+ (green) gigantosomes from transfected HeLa cells treated with TR-Dextran (red) and sublytic rat PFN. Live images were acquired by spinning disk confocal microscopy starting 10 min after addition of TR-Dextran and PFN (duration 13.5 min, 10 sec/frame). Selected static individual frames from these movies are shown in Supplementary Figure 6b. (MOV 461 kb)
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Thiery, J., Keefe, D., Boulant, S. et al. Perforin pores in the endosomal membrane trigger the release of endocytosed granzyme B into the cytosol of target cells. Nat Immunol 12, 770–777 (2011). https://doi.org/10.1038/ni.2050
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