Dynamic dissection of NINJ1-mediated plasma membrane rupture reveals a multi-stage process

Ninjurin1 (NINJ1) serves as a pivotal mediator of plasma membrane rupture (PMR) during pyroptosis, post-apoptotic lysis, necrosis, and related cell death pathways. While recent structural studies revealed near-atomic resolution architectures of NINJ1 in both inactive and active states, its activation mechanisms and membrane remodeling dynamics remain elusive. Through combined methodologies employing atomic force microscopy (AFM), proteoliposome systems, and cellular experiments, we identified negatively charged phospholipids—particularly PS—as critical cofactors in promoting NINJ1 activation. Using membrane-reconstituted NINJ1 with high-speed AFM (HS-AFM) real-time imaging, we captured the PMR stages: Membrane-bound NINJ1 transitions from metastable monomers/dimers/tetramers to arc-shaped oligomers, nucleating ~34-nm pores. Subsequent curvature-driven pore expansion generates paired filaments and irregular membrane fragments. Furthermore, our work integrates previous structural models with dynamic assembly data to propose a unified mechanistic framework that reconciles multiple aspects of NINJ1 function in membrane disruption.