Ninjurin-1 mediated plasma membrane rupture is a two-step process requiring cell swelling

Plasma membrane rupture (PMR) and the release of cytosolic content are hallmarks of necrotic cell death. The plasma membrane protein ninjurin-1 (NINJ1) actively promotes cell lysis by polymerizing into membrane embedded filaments that induce the formation of large plasma membrane lesions. Yet the signals controlling NINJ1 polymerization and the opening of membrane lesions remain unknown. Here we combine cell biology and biophysical measurements to characterize the steps preceding NINJ1-induced PMR in cells undergoing ferroptosis. Our results show that NINJ1 lesions form through a 2-step mechanism requiring oligomerization, which is triggered by signal 1, and subsequent lesion opening driven by signal 2, which we determine to be cell swelling without a concomitant increase in intracellular pressure. The close homologue NINJ2 polymerizes with similar kinetics to NINJ1 but fails to form lesions during cell swelling, further highlighting that oligomerization and PMR are distinct events. Chimeras between NINJ1 and NINJ2 show that the unstructured N-terminal region of NINJ1 controls lesion opening in response to cell swelling. In summary, our data establish a new 2-step model for NINJ1-driven necrosis in which initial oligomerization causes the formation of NINJ1 filaments that serve as ‘weak links’ in the plasma membrane and can break once cell swelling causes a rise in membrane tension and mechanical stress.