(179c) Elucidating Myelin Assembly: How Cerebroside Shapes the Structure and Mechanics of Lipid Monolayers

The myelin sheath, a specialized glial cell extension, forms a multilayered dielectric structure essential for rapid nerve impulse conduction. Its function relies critically on its lipid composition, comprising approximately 80% lipid by weight (typically ~40% cholesterol, ~40% phospholipids, ~20% glycolipids), which deviates significantly from typical plasma membranes. While protein components such as Myelin Basic Protein (MBP) have been studied extensively for their role in compacting cytoplasmic leaflets, the specific contributions of key lipid constituents, particularly the abundant glycolipid cerebroside, to the fundamental physical properties governing myelin assembly and stability remain less defined.

This study employs Langmuir monolayers at the air-water interface as a controlled model system to quantitatively investigate the impact of varying total cerebroside concentration on the physicochemical properties of myelin-mimetic lipid films. We utilized surface pressure-area (Π-A) isotherm measurements to characterize the thermodynamic phase behavior of monolayers, yielding critical parameters such as limiting molecular area (A₀), collapse pressure (Π_col) and isothermal compressibility modulus. These parameters provide insights into molecular packing efficiency and intermolecular cohesive forces modulated by cerebroside content. Furthermore, the thermodynamic data was relied on to calculate the excess Gibbs free energy of mixing (ΔG_mix), allowing quantification of deviations from ideal mixing behavior and assessing the influence of cerebroside on lipid miscibility and specific molecular interactions within the monolayer.

Combination with isotherm measurements, fluorescence microscopy visualized the mesoscopic structure, specifically the formation, morphology, and coexistence of distinct lipid phases (e.g., liquid-ordered and liquid-disordered. Quantitative image analysis techniques were applied to determine the line tension (λ) and dipole density difference (Δm) at the boundaries between these domains, revealing the energetic cost associated with maintaining phase separation, and the difference in normal dipole density across phase boundaries, probing the effect of cerebroside on the interfacial electrostatic environment. To characterize the response of monolayer to dynamic deformation, interfacial dilational rheology was performed, measuring the frequency-dependent complex viscoelastic modulus. These measurements directly probe how cerebroside concentration impacts the monolayer's elasticity (storage modulus, E') and viscosity (loss modulus, E''), critical determinants of membrane mechanical stability and fluidity. Overall, we demonstrate how cerebroside systematically alters molecular packing, phase separation energetics, interfacial electrostatics, and the viscoelastic response of myelin model membranes, thereby contributing fundamentally to the structural integrity and material properties essential for myelin function.