Energetics of stalk intermediates elucidated from hydrostatic pressure effects on membrane fusion

Membrane fusion is an essential process in cells that requires a balance of lipid composition to establish biophysical properties conducive to topology changes. During fusion, lipids of opposing membranes must invert and overcome energy barriers associated with forming highly curved stalk and pore intermediates. While theoretical work has modelled the effect of lipid intrinsic curvature on stalk formation, quantifying the relationship experimentally has proven to be a challenge due to the inability to vary lipid curvature without concomitantly changing other properties that affect fusion. Here we address this hurdle by using hydrostatic pressure to modulate lipid intrinsic curvature independently of chemical composition. Using high-pressure stopped-flow fluorimetry, we measured rates of calcium-mediated lipid mixing between populations of vesicles, a process that is strongly inhibited by pressure. We correlated mean lipid intrinsic curvature across pressure with lipid mixing rates by incorporating complementary small-angle x-ray scattering measurements for each individual lipid component. This analysis showed that lipid mixing rates, a proxy for hemifusion, across compositional and pressure regimes are determined by changes in lipid spontaneous curvature. Consistent with previous theoretical models, we find a linear relation between lipid intrinsic curvature and the hemifusion stalk formation energy, offering direct experimental support for the stalk hypothesis. Significance statementMembrane fusion proceeds through a hemifusion stalk intermediate whose formation energy depends on lipid intrinsic curvature, a central prediction of the stalk hypothesis that has lacked direct experimental support. Previous tests relied on changes in lipid composition that affect multiple membrane properties, confounding the contribution of curvature alone. Here we use hydrostatic pressure to tune lipid curvature independently of chemical composition and calibrate its effects with high-pressure SAXS. Hemifusion rates across three lipid compositions and four pressures collapse into a single exponential dependence on mean spontaneous curvature, yielding a linear relation between curvature and energy consistent with continuum elastic theory. This work quantifies how lipid composition tunes fusion kinetics, suggesting that small changes in lipid curvature may strongly affect fusogenicity.