Complex Supported Lipid Bilayers with High Cholesterol Content formed by α-Helical Peptide-Induced Vesicle Fusion

AbstractNeuronal fusion mediated by soluble N-ethylmaleimide-sensitive-factor attachment protein receptors (SNAREs) is a fundamental cellular process by which two initially distinct membranes merge resulting in one interconnected structure to release neurotransmitters into the presynaptic cleft. To get access to the different stages of the fusion process, several in vitro assays have been developed. In this review, we provide a short overview of the current in vitro single vesicle fusion assays. Among those assays, we developed a single vesicle assay based on pore-spanning membranes (PSMs) on micrometre-sized pores in silicon, which might overcome some of the drawbacks associated with the other membrane architectures used for investigating fusion processes. Prepared by spreading of giant unilamellar vesicles with reconstituted t-SNAREs, PSMs provide an alternative tool to supported lipid bilayers to measure single vesicle fusion events by means of fluorescence microscopy. Here, we discuss the diffusive behaviour of the reconstituted membrane components as well as that of the fusing synthetic vesicles with reconstituted synaptobrevin 2 (v-SNARE). We compare our results with those obtained if the synthetic vesicles are replaced by natural chromaffin granules under otherwise identical conditions. The fusion efficiency as well as the different fusion states observable in this assay by means of both lipid mixing and content release are illuminated.

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Single-lipid dynamics in phase-separated supported lipid bilayers

10.1101/2020.05.28.121830 ◽

2020 ◽

Cited By ~ 1

Author(s):

Xinxin Woodward ◽

Christopher V. Kelly

Keyword(s):

Lipid Bilayers ◽

Fluorescence Correlation Spectroscopy ◽

Cholesterol Content ◽

Single Particle Tracking ◽

Correlation Spectroscopy ◽

Supported Lipid Bilayers ◽

Membrane Composition ◽

Fluorescence Correlation ◽

Diffusion Rates ◽

Lipid Diffusion

ABSTRACTPhase separation is a fundamental organizing mechanism on cellular membranes. Lipid phases have complex dependencies on the membrane composition, curvature, tension, and temperature. Single-molecule diffusion measures a key characteristic of membrane behavior and relates to the effective membrane viscosity. Lipid diffusion rates vary by up to ten-fold between liquid-disordered (Ld) and liquid-ordered (Lo) phases depending on the membrane composition, measurement technique, and the surrounding environment. This manuscript reports the lipid diffusion on phase-separated supported lipid bilayers (SLBs) with varying temperature, composition, and lipid phase. Lipid diffusion is measured by single-particle tracking (SPT) and fluorescence correlation spectroscopy (FCS) via custom data acquisition and analysis protocols that apply to diverse membranes systems. We demonstrate agreement between FCS and SPT analyses with both the single-step length distribution and the mean squared displacement of lipids with significant immobile diffusers. Traditionally, SPT is sensitive to diffuser aggregation, whereas FCS largely excludes aggregates from the reported data. Protocols are reported for identifying and culling the aggregates prior to calculating diffusion rates via SPT. With aggregate culling, all diffusion measurement methods provide consistent results. With varying membrane composition and temperature, we demonstrate the importance of the tie-line length that separates the coexisting lipid phases in predicting the differences in diffusion between the Ld and Lo phases.HIGHLIGHTSLipid diffusion varies with the lipid phases, temperature, and aggregationAggregate culling yields consistent measurements from single-particle tracking and fluorescence correlation spectroscopyMembrane with higher cholesterol content or at low temperature have more aggregatesA more variation in the diffusion rates occurred between the coexisting lipid phases at low temperatures and low cholesterol content

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