Gasdermin D (GSDMD) plays a pivotal role in pyroptosis, a form of inflammatory cell death that releases pro-inflammatory cytokines like IL-1β via GSDMD membrane pore formation. While prior studies using bulk assays and static imaging techniques such as electron microscopy have outlined pore formation steps, including membrane binding, oligomerization, and insertion, key mechanistic details remain unresolved due to the lack of spatiotemporal resolution at the single-pore level. To address this gap, we developed a real-time fluorescence-based assay combining TIRF microscopy with polar lipid containing liposomes to dissect GSDMD pore formation dynamics.
Fluorescently labelled GSDMD was combined with dye-loaded vesicles to enable simultaneous visualization of protein binding kinetics and pore insertion. Initially, transient monomer binding is observed prior to the formation of a stable dimeric nucleus. Oligomerization is then observed as subsequent increases in the intensity of membrane bound GSDMD. Finally, by tracking fluorescent dye release as a proxy for pore formation, we determined the molecular threshold required for membrane permeabilization. These results provide the first quantitative insights into GSDMD pore formation at single-particle resolution, revealing a distinct mechanism compared to other pore-forming proteins.