Direct visualization of native GSDMD pores reveals lipid-driven stabilization during pyroptosis.

Gasdermin D (GSDMD) executes pyroptosis by forming membrane pores, yet how these structures assemble and are regulated in cells has remained technically inaccessible. We introduce polymer-supported plasma membranes (PSPMs), which preserve native PM properties while providing cytosolic access for nanoscopic imaging. Combining PSPMs with DNA-PAINT super-resolution microscopy, we visualize human and mouse GSDMD nanostructures directly at the PM of pyroptotic cells and uncover species-specific differences in pore size. Quantitative analyses reveal that GSDMD assembles into heterogeneous macromolecular architectures, including ring-shaped structures, which correlate with PM permeabilization. The palmitoylation-deficient C191A mutant retains minor membrane association but fails to form complete rings, indicating that ring assembly, more than membrane binding, determines pore activity. Last, we identify PI(3,4,5)P3 as a key regulator of pore stabilization. Its early increase during pyroptosis promotes growth of large rings, and mutations in PI(3,4,5)P3-interacting residues undermine assembly. These findings define the native architecture of GSDMD pores and reveal lipid-dependent stabilization as a central mechanism regulating pyroptotic membrane permeabilization.