NINJ1 regulates plasma membrane fragility under mechanical tension

Plasma membrane integrity is vital not only for cell survival but also nearly all aspects of cell functioning1. Mechanical stress can cause plasma membrane damage2, but it is not known whether there are large molecules (proteins) that control plasma membrane integrity. Here we constructed a 384-well cellular stretch system that delivers precise, reproducible mechanical strain to adherent cells. Using the system, we screened 10,843 siRNAs targeting 2,726 multi-pass transmembrane proteins for stretch-induced membrane permeability changes. The screen identified NINJ1, a protein recently proposed to regulate pyroptosis and other lytic cell death^3,4, as the top hit. We demonstrate that NINJ1 is a critical regulator for mechanical force-induced plasma membrane rupture (PMR), without the need of stimulating any cell death programs. Low NINJ1 expression renders the membrane more resistant to stretching, while high expression of NINJ1 lowers the threshold of PMR under mechanical strain. NINJ1 level on the plasma membrane is inversely correlated to tension required to rupture the membrane. In the pyroptosis context, NINJ1 on its own is not sufficient to fully rupture the membrane, and additional mechanical stress is required for full PMR. Our work establishes that NINJ1 functions as a bona fide determinant of membrane biomechanical properties. Our study also suggests that PMR across tissues of distinct mechanical environments is subjected to fine tuning by differences in NINJ1 expression and external mechanical forces.