How to minimize dye-induced perturbations while studying biomembrane structure and dynamics: PEG linkers as a rational alternative

Edouard Mobarak,Matti Javanainen,Waldemar Kulig,Alf Honigmann,Erdinc Sezgin,Noora Aho,Christian Eggeling,Tomasz Rog,Ilpo Vattulainen

doi:10.1016/j.bbamem.2018.07.003

Abstract

Organic dye-tagged lipid analogs are essential for many fluorescence-based investigations of complex membrane structures, especially when using advanced microscopy approaches. However, lipid analogs may interfere with membrane structure and dynamics, and it is not obvious that the properties of lipid analogs would match those of non-labeled host lipids. In this work, we bridged atomistic simulations with super-resolution imaging experiments and biomimetic membranes to assess the performance of commonly used sphingomyelin-based lipid analogs. The objective was to compare, on equal footing, the relative strengths and weaknesses of acyl chain labeling, headgroup labeling, and labeling based on poly-ethyl-glycol (PEG) linkers in determining biomembrane properties. We observed that the most appropriate strategy to minimize dye-induced membrane perturbations and to allow consideration of Brownian-like diffusion in liquid-ordered membrane environments is to decouple the dye from a membrane by a PEG linker attached to a lipid headgroup. Yet, while the use of PEG linkers may sound a rational and even an obvious approach to explore membrane dynamics, the results also suggest that the dyes exploiting PEG linkers interfere with molecular interactions and their dynamics. Overall, the results highlight the great care needed when using fluorescent lipid analogs, in particular accurate controls.

Highlights

There is increasing proof that cellular signaling is modulated and highly dependent on heterogeneous lateral organization of the plasma membrane [1,2,3]
We studied the lipid analogs as 8 different scenarios with the dye attached to the headgroup (HEAD-Lo, headgroup of ceramide phosphoethanolamine d17:1/12:0 (HEAD)-Ld, HEAD-PEG10-Lo, HEAD-PEG10-Ld, HEAD-PEG50-Lo, HEAD-PEG50-Ld) or to an acyl chain (TAIL-Lo, TAILLd), with three different dyes (Table 1)
Using atomistic Molecular dynamics (MD) simulations and microscopy experiments, we here showed the influence of an organic dye-tag on the structural characteristics, bilayer perturbations, membrane order preferences, and mobility of fluorescent lipid analogs

Summary

Introduction

There is increasing proof that cellular signaling is modulated and highly dependent on heterogeneous lateral organization of the plasma membrane [1,2,3]. Such heterogeneity is multi-fold, including a variety of distinct sub-compartments that differ in their biophysical properties and composition [2]. It has appeared that techniques for observing such heterogeneity require strong specificity and high spatio-temporal resolution, since the involved sub-compartments are distinct, appear on very small spatial scales, and are transient. Using fluorescent analogs of specific lipids (i.e., saturated and unsaturated forms of phosphoethanolamine, sphingomyelin (SM), or gangliosides tagged with an organic dye), STED-FCS observations of the diffusion dynamics of these fluorescent lipid analogs revealed transient interactions with other slowly moving entities in systems with cholesterol, and including actin

Objectives

Methods

Results

Conclusion