Structure and regulation of GSDMD pores at the plasma membrane of pyroptotic cells

Gasdermin D (GSDMD) executes inflammatory cell death pyroptosis by permeabilizing the plasma membrane (PM). We introduce polymer-supported PM (PSPM) to gain access to the cytoplasmic side of the PM with imaging techniques while preserving the native PM complexity and lipid microenvironment. By combining PSPM with DNA-PAINT super-resolution microscopy we visualized, for the first time, GSDMD nanostructures directly at the PM of pyroptotic cells. We resolved diverse macromolecular architectures with ring-and arc-shaped GSDMD oligomers that enable PM permeabilization. The pyroptotically-inactive mutant GSDMD-C192A (human C191A) still interacts with the PM however fails to form pores. GSDMD expression levels affect pore density but not permeabilization ability. Finally, we identified the local PI(3,4,5)P3 concentration as a key regulatory element of PM permeabilization. Increase in PI(3,4,5)P3 levels in the PM during pyroptosis facilitates growth into large ring-shaped pores. Using molecular dynamics (MD) simulations, we identified the mechanism by which PI(3,4,5)P3 stabilizes the GSDMD assembly.