Nanoscale Organization of Membrane Tension during Neutrophil Extracellular Trap Formation Revealed by Fluorescence Lifetime Imaging

Cells generate and respond to mechanical forces across compartments, with the plasma membrane acting as a nanoscale interface for sensing and transmitting tension. How intracellular forces translate into membrane tension during dynamic processes such as neutrophil extracellular trap (NET) formation remains unclear. Here, we combine the mechanosensitive fluorescent probe Flipper-TR with fluorescence lifetime imaging microscopy (FLIM) to map spatiotemporal plasma membrane tension changes in living cells. After validation in HeLa and dHL-60 cells under osmotic perturbation, we apply this approach to primary human neutrophils undergoing NETosis. Membrane tension transiently increases during chromatin decondensation and nuclear swelling within 60 min, followed by a marked decrease after membrane rupture. Prior to rupture, tension is spatially heterogeneous, indicating localized nanoscale mechanical regulation. Cholesterol depletion abolishes the transient increase and reduces heterogeneity without affecting NETosis kinetics. These findings establish the plasma membrane as a dynamic nanoscale reporter of intracellular mechanical stress during NETosis.