Real-time visualization of perforin nanopore assembly.
Perforin is a key protein of the vertebrate immune system. Secreted by cytotoxic lymphocytes as soluble monomers, perforin can self-assemble into oligomeric pores of 10-20 nm inner diameter in the membranes of virus-infected and cancerous cells. These large pores facilitate the entry of pro-apoptotic granzymes, thereby rapidly killing the target cell. To elucidate the pathways of perforin pore assembly, we carried out real-time atomic force microscopy and electron microscopy studies. Our experiments reveal that the pore assembly proceeds via a membrane-bound prepore intermediate state, typically consisting of up to approximately eight loosely but irreversibly assembled monomeric subunits. These short oligomers convert to more closely packed membrane nanopore assemblies, which can subsequently recruit additional prepore oligomers to grow the pore size.
London Centre for Nanotechnology, University College London, London WC1H 0AH, UK.|Department of Crystallography/Biological Sciences, Institute of Structural and Molecular Biology, Birkbeck College, London WC1E 7HX, UK.
Carl
C
Leung
London Centre for Nanotechnology, University College London, London WC1H 0AH, UK.|Institute of Structural and Molecular Biology, University College London, London WC1E 6BT, UK.
Adrian W
AW
Hodel
Killer Cell Biology Laboratory, Peter MacCallum Cancer Centre, East Melbourne, Victoria 3000, Australia.
Amelia J
AJ
Brennan
Department of Crystallography/Biological Sciences, Institute of Structural and Molecular Biology, Birkbeck College, London WC1E 7HX, UK.
Natalya
N
Lukoyanova
Killer Cell Biology Laboratory, Peter MacCallum Cancer Centre, East Melbourne, Victoria 3000, Australia.
Sharon
S
Tran
Cancer Cell Death Laboratory, Peter MacCallum Cancer Centre, East Melbourne, Victoria 3000, Australia.
Colin M
CM
House
Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Victoria 3800, Australia.
Stephanie C
SC
Kondos
Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Victoria 3800, Australia.|The ARC Centre of Excellence in Advanced Molecular Imaging, Monash University, Melbourne, Victoria 3800, Australia.
James C
JC
Whisstock
Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Victoria 3800, Australia.|The ARC Centre of Excellence in Advanced Molecular Imaging, Monash University, Melbourne, Victoria 3800, Australia.|Department of Microbiology, Monash University, Melbourne, Victoria 3800, Australia.
Michelle A
MA
Dunstone
Cancer Cell Death Laboratory, Peter MacCallum Cancer Centre, East Melbourne, Victoria 3000, Australia.|Sir Peter MacCallum Department of Oncology, University of Melbourne, Melbourne, Victoria 3010, Australia.
Joseph A
JA
Trapani
Killer Cell Biology Laboratory, Peter MacCallum Cancer Centre, East Melbourne, Victoria 3000, Australia.|Sir Peter MacCallum Department of Oncology, University of Melbourne, Melbourne, Victoria 3010, Australia.
Ilia
I
Voskoboinik
Department of Crystallography/Biological Sciences, Institute of Structural and Molecular Biology, Birkbeck College, London WC1E 7HX, UK.
Helen R
HR
Saibil
London Centre for Nanotechnology, University College London, London WC1H 0AH, UK.|Institute of Structural and Molecular Biology, University College London, London WC1E 6BT, UK.|Department of Physics and Astronomy, University College London, London WC1E 6BT, UK.
Bart W
0000-0002-8882-4324
BW
Hoogenboom
D000818:Animals; D004910:Erythrocyte Membrane / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D051379:Mice; D018625:Microscopy, Atomic Force; D008854:Microscopy, Electron; D058608:Nanopores / Q000648:ultrastructure*; D052899:Pore Forming Cytotoxic Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012756:Sheep
D052899:Pore Forming Cytotoxic Proteins; C483886:perforin, mouse