Sterol-lipid conjugation uncovers why membrane rigidity varies across scales

Lipid bilayers, the main scaffolds of cellular membranes, regulate a wide range of biological functions through precise dynamical processes. For instance, elastic bilayer fluctuations play key roles in modulating protein folding, enzyme activity, and endo/exocytosis. Yet, how the bilayer elastic properties change with cholesterol – an abundant component of mammalian cells – remains a topic of intense debate. For example, it has been shown that unsaturated DOPC membranes exhibit non-trivial stiffening with cholesterol on mesoscopic length and time scale, but not in the macroscopic regime. Recent molecular dynamics simulations suggest that this behavior could emanate from slow processes such as diffusion and flip-flop which are undetectable on mesoscopic scales. To test this hypothesis, we utilized chemically conjugated lipid-sterol hybrids which enable experimental tunability of diffusive and flip-flop dynamics. To explore the manifestation of such dynamic changes, we combined mesoscopic measurements of elastic moduli by neutron spin-echo spectroscopy with macroscopic observations from flickering spectroscopy. Our studies unequivocally demonstrate that – unlike cholesterol – sterol-lipid hybrids induce membrane stiffening on both mesoscopic and macroscopic scales. These results uncover the molecular origins of emergent elasticity and deepen our understanding of the design principles for advanced, tunable membrane systems.