Fig. 4. Cleaved bGSDMs form membrane pores to elicit cell death.

(A) GFP was fused to the N-terminus of the Runella bGSDM. Cells expressing GFP-bGSDM alone (top) or with the caspase-like protease (bottom) are shown. GFP is colored green. Membrane dye (FM4-64) is in magenta. Scale bar, 1 μm. (B) Cleaved Runella gasdermin permeabilizes liposome membranes. Relative fluorescence units (RFU) were measured continuously from cleavage reactions of dioleoylphosphatidylcholine (DOPC) liposomes loaded with TbCl₃ with an external solution containing 20 μM dipicolonic acid (DPA). The top plot represents an example of time-course liposome leakage, whereas the bottom bar chart shows values for each condition at 60 min. Error bars represent the SEM of three technical replicates and statistical significance was determined by one-way ANOVA and Tukey multiple comparison test. n.s. ≥ 0.05; **P = 0.001–0.01; ****P < 0.0001. (C) Negative stain electron microscopy of Runella gasdermin pores in DOPC liposomes (left) and in mesh-like arrays (right). Scale bars, 50 nm. (D) Slices from representative tomogram (1 of 10) of Runella gasdermin pores in DOPC liposomes, at three different depths (Z). Yellow arrowheads indicate pores inserted within the liposome membrane. Scale bars, 50 nm. (E) Model of pyroptosis for bGSDMs and mammalian gasdermins.