Fig. 5

Perforin forms non-porating clusters on phosphatidylserine membranes. a AFM images of DOPC and DOPS supported lipid bilayers after incubation with TMH1-PRF without or with glutaraldehyde (+GA) fixation, with unlocked (+DTT) TMH1-PRF, or with WT-PRF. Height profiles were plotted as acquired along the dashed lines in the AFM image. On DOPC, prepore locked TMH1-PRF, and TMH1-PRF +DTT and WT-PRF assemblies all extend 10–11 nm above the membrane surface, in agreement with previous observations^5,6,19. On DOPS, fixed (and locked) TMH1-PRF assemblies have the same height, but the clusters of unlocked TMH1-PRF (+DTT) and WT-PRF are distinctly taller, about 15 nm above the membrane. b AFM images of DOPS supported lipid bilayers incubated with WT-PRF or TMH-PRF +GA, before and after Ca²⁺ chelation by washing the membrane with 5 mM EGTA (±EGTA). Clusters formed by WT-PRF are not removed or visibly affected by washing the membrane with EGTA. In contrast, cross-linked plaques of TMH1-PRF +GA are removed after the EGTA wash. This is consistent with failure of TMH1-PRF to insert into the membrane⁶ and suggests that WT-PRF partially inserts into the DOPS membrane. All samples were incubated at 37 °C and imaged at room temperature. Colour (height) scale for all panels as in Fig. 3b. c AFM images of supported DOPC bilayers mixed with 0–100% DOPS, after incubation with 150 nM WT-PRF. The images show decreased formation of arc- and ring-shaped perforin pores upon increased DOPS content, and an increased amount of clustering. Bar graph on the right shows quantification (see Methods; mean ± standard deviation) of pore formation on DOPS containing membranes: for 60% DOPS, there is a significant decrease in the number of pores, and at 100% DOPS there are no unambiguous pore features found. Statistical significance was assessed using ANOVA with Dunnett’s post-hoc analysis, where ‘ns’–not significant, ****p < 0.001. Scale bars, 200 nm.