Plasma membrane integrity is vital for nearly all aspects of cell functioning1. Mechanical forces can cause plasma membrane damage2, but it is not known whether there are large molecules that regulate plasma membrane integrity under mechanical strain. Here we constructed a 384-well cellular stretch system that delivers precise, reproducible strain to cultured cells. Using the system, we screened 10,843 siRNAs targeting 2,726 multi-pass transmembrane proteins for strain-induced membrane permeability changes. The screen identified NINJ1, a protein recently proposed to regulate pyroptosis and other lytic cell death3,4, as the top hit. We demonstrate that NINJ1 is a critical regulator for mechanical strain-induced plasma membrane rupture (PMR), without the need of stimulating any cell death programs. NINJ1 level on the plasma membrane is inversely correlated to the amount of force required to rupture the membrane. In the pyroptosis context, NINJ1 on its own is not sufficient to fully rupture the membrane, and additional mechanical force 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 microenvironments is subjected to fine tuning by differences in NINJ1 expression and external forces.
