The Effect of Ceramide Ratio on the Membrane Curvature of Mimetic Models of Matrix Vesicles

Abstract

The lipid composition of membrane systems plays a critical role in regulating their structural dynamics and curvature, particularly in the biological context of matrix vesicles (MVs) formation during bone mineralization. Recent evidence suggests that the lipid composition of MVs, particularly the balance between sphingomyelin (SM) and ceramide (CER), influences their curvature and stability. We report on the impact of SM and CER ratios on membrane curvature through surface pressure-area isotherm measurements and molecular dynamics (MD) simulations at atomistic and coarse-grained levels. Our findings reveal that increasing the CER content up to 25% significantly enhances membrane curvature, as demonstrated by changes in experimental compressibility moduli and lateral pressure profiles. The lateral pressure profiles and spontaneous bending moments calculated from MD simulations of osteoblast-mimetic membrane models suggest a strong propensity for curvature, particularly in asymmetrical bilayers. It also reveals the role of CER-rich domains in the stabilization of membrane curvature, potentially facilitating the budding processes critical for MVs formation in osteoblasts. These findings underscore the critical role of lipid composition in the mechanisms driving MVs biogenesis.

Keywords: Langmuir−Blodgett; ceramide; matrix vesicles; membrane curvature; molecular dynamic simulations; sphingomyelin; sphingomyelinase.

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Atomistic representations of bilayers with (a) 100% SM, (b) 50% SM and 50% CER, and (c) 100% CER. SM is shown in green, and CER in red. The nitrogen atom from the choline group of SM is highlighted in blue. (d) Coarse-grained representation of an asymmetrical bilayer mimicking the osteoblast membrane (CG₃). DPPC and DPPE are depicted in orange and yellow, SM in green, and CER in salmon. Both the molecular structure and coarse-grained beads are illustrated.

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(a) Average area per lipid A _L and (b) average membrane curvature S _C for binary mixtures of CER and SM, calculated for AT-simulated systems. The violin plots represent the data set distribution where the width of the violin indicates the data density at different values. The central line represents the median, and the box shows the interquartile range. The overall shape of the plot reveals the spread and skewness of the distribution. The averages were calculated over the final 100 ns of the simulations.

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(a) Excess molecular area (A _exc) obtained from experimental LB films (blue) and AT simulations at 303 K (dark blue) and (b) the excess Gibbs energy (ΔG _exc) calculated from experimental LB films for pure or binary mixtures of CER and SM.

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Experimental surface pressure isotherms versus molecular area for pure and SMase-converted binary mixtures of CER and SM. Isotherms for pure SM (green), pure CER (red), and the SM-CER ratio resulting from the SMase catalytic conversion of SM into CER (dark blue). The control isotherm (light blue) corresponds to a composition of 75% SM and 25% CER.

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(a) Area per lipid A _L and (b) average curvature S _C for CG simulations of the symmetrical membrane consisting of DPPC and DPPE (CG₁), the symmetrical membrane composed of SM and CER (CG₂), and the asymmetrical membrane composed of DPPC and DPPE in the inner leaflet and DPPC, DPPE, SM, and CER in the outer leaflet (CG₃). See Table for details on each system composition.

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Lateral pressure profile for simulated membranes CG₁, CG₂, and CG₃. The bilayer midplane is positioned at 0 nm. The negative region corresponds to the inner leaflet, while the positive region represents the outer leaflet. The symmetrical CG₁ system (red) is composed of DPPC and DPPE; the symmetrical CG₂ system (green) is composed of SM and CER; and the asymmetrical CG₃ system (blue) is composed of DPPC and DPPE in the inner leaflet, DPPC, DPPE, SM, and CER in the outer leaflet. See Table for the lipid composition of the system.

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Structure of the bilayer and semivesicle system through simulation. For clarity, DPPC and DPPE are shown in white, while CER is illustrated in salmon, and SM in green.