Cholesterol-Dependent Nanomechanical Stability of Phase-Segregated Multicomponent Lipid Bilayers

Abstract

Cholesterol is involved in endocytosis, exocytosis, and the assembly of sphingolipid/cholesterol-enriched domains, as has been demonstrated in both model membranes and living cells. In this work, we explored the influence of different cholesterol levels (5-40 mol%) on the morphology and nanomechanical stability of phase-segregated lipid bilayers consisting of dioleoylphosphatidylcholine/sphingomyelin/cholesterol (DOPC/SM/Chol) by means of atomic force microscopy (AFM) imaging and force mapping. Breakthrough forces were consistently higher in the SM/Chol-enriched liquid-ordered domains (Lo) than in the DOPC-enriched fluid-disordered phase (Ld) at a series of loading rates. We also report the activation energies (DeltaEa) for the formation of an AFM-tip-induced fracture, calculated by a model for the rupture of molecular thin films. The obtained DeltaEa values agree remarkably well with reported values for fusion-related processes using other techniques. Furthermore, we observed that within the Chol range studied, the lateral organization of bilayers can be categorized into three distinct groups. The results are rationalized by fracture nanomechanics of a ternary phospholipid/sphingolipid/cholesterol mixture using correlated AFM-based imaging and force mapping, which demonstrates the influence of a wide range of cholesterol content on the morphology and nanomechanical stability of model bilayers. This provides fundamental insights into the role of cholesterol in the formation and stability of sphingolipid/cholesterol-enriched domains, as well as in membrane fusion.

The temperature-dependent physical state of polar lipids and their miscibility impact the topography and mechanical properties of bilayer models of the milk fat globule membrane

“…MSM/dioleylphosphatidylcholine (DOPC) at 25°C (i.e., below Tm of MSM [16]) or with that of other SM species in model bilayers [37][38][39][40]. The characterization of high-Tm lipid rich domains below 35°C is also in agreement with previous studies performed with dried Langmuir-Blodgett monolayers [8,9].…”

“…Previous reports have shown that this approach successfully scales high features of ordered lipid domains as significantly more rigid than the l d phase at ambient temperature [17,21,40,56]. To the authors' knowledge, except for the DPPC/cholesterol study by Redondo-Morata et al [42] no previous AFM investigation of the temperature-dependence of the mechanical properties had been reported so far on multiple-component lipid bilayers.…”

“…However, experimental conditions yet need to be standardized to avoid bias due to ions [28,55], loading rate [40,54] or tip geometry [46]. Previous reports have shown that this approach successfully scales high features of ordered lipid domains as significantly more rigid than the l d phase at ambient temperature [17,21,40,56].…”

Stiffened lipid platforms at molecular force foci

“…This result can be observed in simulations (75). The rigidity of the platform should reduce such variations by increasing the elastic moduli of lipids and thereby suppressing bilayer geometry fluctuations (76)(77)(78)(79). The third potential source of noise for a channel comes from encounters with other proteins in the bilayer; this can be effectively reduced by the stiffened platform because not all membrane proteins are allowed in.…”

Novel biomedical applications of supported lipid bilayers

“…52,53,61 Scanning probe techniques such as AFM can be used to measure the thickness of SLBs [110][111][112] and the rigidity of pore spanning SLBs and to map the surface topography to, for example, visualize lipid rafts (Figure 1.7d). 96,113 The use of AFM in studying phase transitions in SLBs have recently been reviewed. 114 Combinations of QCM-D and AFM have been adopted to explore the intermediate states leading to SLB formation.…”