title: Perforin oligomers form arcs in cellular membranes: a locus for intracellular delivery of granzymes.
abstract: Perforin-mediated cytotoxicity is an essential host defense, in which defects contribute to tumor development and pathogenic disorders including autoimmunity and autoinflammation. How perforin (PFN) facilitates intracellular delivery of pro-apoptotic and inflammatory granzymes across the bilayer of targets remains unresolved. Here we show that cellular susceptibility to granzyme B (GzmB) correlates with rapid PFN-induced phosphatidylserine externalization, suggesting that pores are formed at a protein-lipid interface by incomplete membrane oligomers (or arcs). Supporting a role for these oligomers in protease delivery, an anti-PFN antibody (pf-80) suppresses necrosis but increases phosphatidylserine flip-flop and GzmB-induced apoptosis. As shown by atomic force microscopy on planar bilayers and deep-etch electron microscopy on mammalian cells, pf-80 increases the proportion of arcs which correlates with the presence of smaller electrical conductances, while large cylindrical pores decline. PFN appears to form arc structures on target membranes that serve as minimally disrupting conduits for GzmB translocation. The role of these arcs in PFN-mediated pathology warrants evaluation where they may serve as novel therapeutic targets.
authors.affiliation: NorthShore University Health Systems Research Institute and University of Chicago, Evanston, IL, USA.
authors.forename: S S
authors.initials: SS
authors.lastname: Metkar
authors.affiliation: 1] Consiglio Nazionale delle Ricerche, Istituto di Biofisica & Fondazione Bruno Kessler Via alla Cascata 56/C, Trento 38123, Italy [2] Center for Integrative Biology CIBIO, University of Trento Via delle Regole, 101 Mattarello (Trento) 38123, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Marchioretto
authors.affiliation: Consiglio Nazionale delle Ricerche, Istituto di Biofisica & Fondazione Bruno Kessler Via alla Cascata 56/C, Trento 38123, Italy.
authors.forename: V
authors.initials: V
authors.lastname: Antonini
authors.affiliation: Consiglio Nazionale delle Ricerche, Istituto di Biofisica & Fondazione Bruno Kessler Via alla Cascata 56/C, Trento 38123, Italy.
authors.forename: L
authors.initials: L
authors.lastname: Lunelli
authors.affiliation: NorthShore University Health Systems Research Institute and University of Chicago, Evanston, IL, USA.
authors.forename: B
authors.initials: B
authors.lastname: Wang
authors.affiliation: Division of Structural Biology, Henry Wellcome Building for Genomic Medicine, Oxford, UK.
authors.forename: R J C
authors.initials: RJ
authors.lastname: Gilbert
authors.affiliation: Laboratory of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Hajdrihova 19, Ljublana, Slovenia.
authors.forename: G
authors.initials: G
authors.lastname: Anderluh
authors.affiliation: Department of Cell Biology and Physiology, Washington University in Saint Louis, 660 S. Euclid Avenue, St Louis, MO, USA.
authors.forename: R
authors.initials: R
authors.lastname: Roth
authors.affiliation: Institute of Molecular and Cell Biology, University of Tartu, Riia Str 23, Tartu, Estonia.
authors.forename: M
authors.initials: M
authors.lastname: Pooga
authors.affiliation: Department of Biochemistry and Molecular and Cell Biology, Biomedical Research Centre of Aragon (CIBA), IIS Aragon and Nanoscience Institute of Aragon (INA), University of Zaragoza/ARAID Foundation, 50009 Zaragoza, Spain.
authors.forename: J
authors.initials: J
authors.lastname: Pardo
authors.affiliation: Department of Cell Biology and Physiology, Washington University in Saint Louis, 660 S. Euclid Avenue, St Louis, MO, USA.
authors.forename: J E
authors.initials: JE
authors.lastname: Heuser
authors.affiliation: Consiglio Nazionale delle Ricerche, Istituto di Biofisica & Fondazione Bruno Kessler Via alla Cascata 56/C, Trento 38123, Italy.
authors.forename: M D
authors.initials: MD
authors.lastname: Serra
authors.affiliation: NorthShore University Health Systems Research Institute and University of Chicago, Evanston, IL, USA.
authors.forename: C J
authors.initials: CJ
authors.lastname: Froelich
mesh_terms: D057134:Antibodies, Neutralizing / Q000737:chemistry; D017209:Apoptosis; D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism; D002463:Cell Membrane Permeability; D053804:Granzymes / Q000737:chemistry*; D006801:Humans; D019169:Jurkat Cells; D046912:Multiprotein Complexes / Q000737:chemistry*; D009336:Necrosis / Q000378:metabolism; D054353:Perforin / Q000737:chemistry*; D021381:Protein Transport
chemical_list: D057134:Antibodies, Neutralizing; D046912:Multiprotein Complexes; C510468:PRF1 protein, human; D054353:Perforin; C509213:GZMB protein, human; D053804:Granzymes