In Situ Determination of Structure and Fluctuations of Coexisting Fluid Membrane Domains

Peter Heftberger,Benjamin Kollmitzer,Alexander A Rieder,Heinz Amenitsch,Georg Pabst

doi:10.1016/j.bpj.2014.11.3488

Peter Heftberger, Benjamin Kollmitzer + Show 3 more

Open Access

https://doi.org/10.1016/j.bpj.2014.11.3488

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Abstract

Biophysical understanding of membrane domains requires accurate knowledge of their structural details and elasticity. We report on a global small angle x-ray scattering data analysis technique for coexisting liquid-ordered (Lo) and liquid-disordered (Ld) domains in fully hydrated multilamellar vesicles. This enabled their detailed analysis for differences in membrane thickness, area per lipid, hydrocarbon chain length, and bending fluctuation as demonstrated for two ternary mixtures (DOPC/DSPC/CHOL and DOPC/DPPC/CHOL) at different cholesterol concentrations. Lo domains were found to be ∼10 Å thicker, and laterally up to 20 Å2/lipid more condensed than Ld domains. Their bending fluctuations were also reduced by ∼65%. Increase of cholesterol concentration caused significant changes in structural properties of Ld, while its influence on Lo properties was marginal. We further observed that temperature-induced melting of Lo domains is associated with a diffusion of cholesterol to Ld domains and controlled by Lo/Ld thickness differences.

Highlights

Since the formulation of the raft model in 1997 by Simons and Ikonen [1] significant scientific efforts have been devoted to the characterization of physical properties of liquid-disordered Ld and liquid-ordered Lo domains [2,3,4,5,6,7,8,9,10,11]
We found that increased cholesterol concentrations reduce the thickness difference between Ld and Lo domains, which leads to a decrease of line tension and in turn promotes the temperature induced melting of Lo domains
DPPC, DSPC, and DOPC were purchased from Avanti Polar Lipids (Alabaster, AL), and cholesterol was obtained from Sigma-Aldrich (Vienna, Austria)

Summary

Introduction

Since the formulation of the raft model in 1997 by Simons and Ikonen [1] significant scientific efforts have been devoted to the characterization of physical properties of liquid-disordered Ld and liquid-ordered Lo domains [2,3,4,5,6,7,8,9,10,11]. Membrane rafts are thought to be small (nanoscopic) and highly dynamic platforms enriched in sphingolipids and cholesterol, enabling diverse cellular functions, but have so far escaped any direct visualization in live cells [5,12]. For example Frisz et al [13,14], using secondary ion mass spectrometry on fibroblasts, observed sphingolipid domains, in which cholesterol was evenly distributed throughout the membrane, challenging the standard raft hypotheses. In contrast to natural membranes, domains in lipid-only systems can grow up to several micrometers in size, enabling their detection (e.g., by optical microscopy [15]) and study with respect to the physics pertaining to their stability, size, or effect on protein sorting, to name but a few examples [8]. One of the parameters involved in, e.g., protein sorting, is the difference in thickness between the Lo and Ld domains and the corresponding match to the protein’s transmembrane region (see, e.g., Killian [16] and Pabst [17])

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