Biophysical membrane properties are altered during ferroptosis execution

Ferroptosis is a form of regulated necrosis characterized by the iron-dependent accumulation of lipid peroxides in cellular membranes; however, the specific biophysical membrane properties affected during this process and how these alterations integrate into the molecular machinery of ferroptosis remain key unanswered questions. To address this, we combined quantitative live-cell confocal microscopy with several complementary biophysical methods to systematically investigate changes in membrane biophysics upon ferroptosis induction. Our data reveal that ferroptosis execution is a multi-step process driven by a defined sequence of biophysical alterations. We delineated a precise timeline in which initial lipid peroxidation directly increases water permeation into the hydrophobic membrane core, leading to membrane solvation and calcium influx into the cytosol. This osmotic influx drives a cascade of secondary events, including cell rounding and phosphatidylserine externalization. The plasma membrane subsequently detaches from the underlying actin cortex, resulting in the formation of a single massive swelling bleb that is a morphological hallmark of ferroptosis. This stage is accompanied by a reorganization of membrane components into immobile fractions, a reduction in lipid packing density specifically within bleb-associated membrane regions, and a decrease in the mechanical resistance of the plasma membrane to breakthrough. The process culminates in complete cellular collapse, characterized by the irreversible loss of lipid asymmetry and membrane integrity. We propose that this stepwise disruption of membrane integrity is the fundamental driver of the loss of membrane functionality that ultimately defines ferroptotic death.