NINJ1 mediated plasma membrane rupture is a two-step process requiring cell swelling

Plasma membrane rupture and the release of cytosolic contents 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 oligomerization and lesion opening remain unknown. Here, we combine cell biology and biophysical measurements to characterize the steps preceding NINJ1-induced PMR. 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 homolog NINJ2 oligomerizes with similar kinetics to NINJ1 but fails to form lesions during cell swelling. NINJ1-NINJ2 chimeras identify that the unstructured N-terminal region of NINJ1 controls lesion opening. In summary, our data establish a 2-step model for NINJ1-driven necrosis.