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

ABSTRACT 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)P 3 concentration as a key regulatory element of PM permeabilization. Increase in PI(3,4,5)P 3 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)P 3 stabilizes the GSDMD assembly.