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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.

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Figure 1: Membrane perforation by WT perforin, as imaged by AFM after completion of membrane pore formation.
Figure 2: TMH1-lock mutant perforin forms prepores and pores on lipid membranes.
Figure 3: Evolution of perforin assembly on egg PC:cholesterol monolayers for TMH1-lock mutant perforin, compared to WT perforin pores.
Figure 4: Effect of TMH1-lock mutant on pore formation by WT perforin.
Figure 5: AFM of growing assemblies of WT perforin incubated and imaged at 27 °C to slow pore formation compared with 37 °C.


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This work was funded by the BBSRC (BB/J005932/1, BB/J006254/1 and BB/N015487/1), the ERC (advanced grant no. 294408), the Wellcome Trust (no. 079605/2/06/02), NHMRC Fellowship (1059126), Project (1062706) and Program (1013667) grants, and the Sackler Foundation. The authors thank N. Nand Gosvami and J. Pegman for assistance with early experiments and analysis, and R. Thorogate, A. Ciccone and S. Verschoor for technical support.

Author information




C.L. designed, performed and analysed AFM experiments and co-wrote the paper. A.W.H. designed, performed and analysed AFM and FRAP experiments and co-wrote the paper. A.J.B. expressed mutant perforin, developed the TMH1-lock mutant and carried out haemolysis experiments. N.L. carried out and analysed electron microscopy experiments and co-wrote the paper. S.T. performed and analysed haemolysis experiments. C.M.H. performed mass spectrometric analysis and co-wrote the paper. S.C.K., J.C.W. and M.A.D. contributed to the TMH1-lock mutant design. J.A.T. designed in vitro experiments. I.V., H.R.S. and B.W.H. analysed the data, led the research and co-wrote the paper. All authors read and commented on the manuscript.

Corresponding authors

Correspondence to Ilia Voskoboinik or Helen R. Saibil or Bart W. Hoogenboom.

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The authors declare no competing financial interests.

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Leung, C., Hodel, A., Brennan, A. et al. Real-time visualization of perforin nanopore assembly. Nature Nanotech 12, 467–473 (2017).

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