Biology subjects

Winnikoff, J. R.

Publications and source records attributed to Winnikoff, J. R..

3 recordsLinked to original sources

bioRxiv2026

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.

biophysics↗

bioRxiv2026

Ether linkages in phospholipids provide modular control of membrane mechanics

The structures of phospholipid headgroups and chains are well-established determinants of membrane elastic properties, but functions for different chemistries that join these moieties together are poorly understood. While common phospholipids feature ester linkages, alkyl ether- and plasmenyl-linked species emerged in prokaryotes, are highly abundant in metazoans, and have been implicated in neurodegeneration and aging. Multiple pathways for ether lipid synthesis evolved convergently, suggesting conserved functions for ether linkage chemistry in the structure of cell membranes. Here we combine experiments and molecular simulations to show that backbone linkage chemistry provides modular control of membrane mechanics and topology through a set of discrete drivers. Alkyl linkages provide the first of these by additively promoting negative intrinsic curvature, which destabilizes bilayers. Ethers also decouple membrane stiffness from viscosity, softening membranes while maintaining packing in the hydrophobic core. The plasmenyl C=C bond represents a second, distinct driver that stabilizes the inverted hexagonal phase by relieving interstitial packing frustration. These results explain the fusogenicity of ether lipids, show how they regulate membrane topology through multiple physical mechanisms, and provide a rationale for convergent evolution of the complex plasmenyl linkage moiety.

biophysics↗

bioRxiv2025

Homeostatic regulation of intrinsic lipid curvature in eukaryotic cells

Cell membranes are composed of both bilayer-supporting and non-bilayer phospholipids, with the latters negative intrinsic curvature aiding in membrane trafficking and the dynamics of membrane proteins. Phospholipid metabolism has long been recognized to maintain membrane fluidity, but whether it also acts to maintain the function of high-curvature lipids is not resolved. Here, we find that cells grown under hydrostatic pressure - used to artificially reduce lipid curvature - maintain lipidome curvature through metabolic acclimation. We first observed that manipulation of the lipidome curvature via the phosphatidylethanolamine (PE) to phosphatidylcholine (PC) ratio affects high-pressure growth and viability of yeast independently of membrane fluidity. In wild-type cells, X-ray scattering measurements revealed an increased propensity for lipid extracts to form non-lamellar phases after extended pressure incubations. Unexpectedly, this change in phase behavior was not due to increased levels of PE, but of phosphatidylinositol (PI), the only major phospholipid class whose curvature had not been previously characterized. We found that PI is a non-bilayer lipid, with a negative curvature intermediate to that of PE and PC. Accounting for PI, mean lipidome curvature was defended in response to pressure by two distantly related yeasts. Lipidome curvature also responded to pressure in a human cancer cell line through ether phospholipid metabolism and chain remodeling, but not in bacterial cells. These findings indicate that eukaryotic phospholipid metabolism uses diverse mechanisms to maintain curvature frustration in cell membranes.

biophysics↗