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

Membrane fusion is a crucial cellular process that relies on a careful balance of lipid composition to enable the necessary topological changes. During fusion, lipids on opposing membranes must invert and contend with energy barriers in the formation of highly curved stalk and pore intermediates. Though theoretical models have attempted to explain how lipid intrinsic curvature impacts stalk formation, experimental validation has been difficult due to the simultaneous alteration of other fusion-influencing properties when attempting to change lipid curvature.

To overcome this challenge, researchers employed hydrostatic pressure to independently modulate lipid intrinsic curvature, without affecting other chemical characteristics. By utilizing high-pressure stopped-flow fluorimetry, scientists were able to measure the rates of calcium-mediated lipid mixing between vesicle populations, a process that is significantly impeded by pressure.

To further understand the relationship between lipid curvature and fusion, the team incorporated small-angle x-ray scattering measurements to determine mean lipid intrinsic curvature for each vesicle component. Their analysis revealed that lipid mixing rates, serving as a proxy for hemifusion, across various compositions and pressure levels are determined by alterations in lipid spontaneous curvature.

Aligning with earlier theoretical models, the researchers found a linear correlation between lipid intrinsic curvature and the energy required for hemifusion stalk formation, providing compelling experimental evidence supporting the stalk hypothesis.