Designing lipids for selective partitioning into liquid ordered membrane domains

Abstract

Self-organization of lipid molecules into specific membrane phases is key to the development of hierarchical molecular assemblies that mimic cellular structures. While the packing interaction of the lipid tails should provide the major driving force to direct lipid partitioning to ordered or disordered membrane domains, numerous examples show that the headgroup and spacer play important but undefined roles. We report here the development of several new biotinylated lipids that examine the role of spacer chemistry and structure on membrane phase partitioning. The new lipids were prepared with varying lengths of low molecular weight polyethylene glycol (EGn) spacers to examine how spacer hydrophilicity and length influence their partitioning behavior following binding with FITC-labeled streptavidin in liquid ordered (Lo) and liquid disordered (Ld) phase coexisting membranes. Partitioning coefficients (Kp Lo/Ld) of the biotinylated lipids were determined using fluorescence measurements in studies with giant unilamellar vesicles (GUVs). Compared against DPPE-biotin, DPPE-cap-biotin, and DSPE-PEG2000-biotin lipids, the new dipalmityl-EGn-biotin lipids exhibited markedly enhanced partitioning into liquid ordered domains, achieving Kp of up to 7.3 with a decaethylene glycol spacer (DP-EG10-biotin). We further demonstrated biological relevance of the lipids with selective partitioning to lipid raft-like domains observed in giant plasma membrane vesicles (GPMVs) derived from mammalian cells. Our results found that the spacer group not only plays a pivotal role for designing lipids with phase selectivity but may also influence the structural order of the domain assemblies.

The mystery of membrane organization: composition, regulation and roles of lipid rafts

“…In the case of phospholipids, it is often challenging to preserve the natural physicochemical behaviour of the lipid after attaching a fluorophore 82,83 . In general, the least perturbing strategy is labelling the headgroup rather than the acyl chain, and adding a hydrophilic linker to ensure that the fluorophores (which are often membrane active) do not perturb the headgroups of the surrounding lipids 84 .…”

“…cell type or cell cylce. Altogether, to obtain reproducible results regarding raft formation and their biophysical properties it may be necessary to introduce fully synthetic probes (instead of semi-native labels) that exhibit validated affinities for ordered membrane domains 84 , and thus allow to careful correlations between ordered domain affinity and other experimental readouts 22 . Another approach that would minimize the experimental differences is the application of label-free detection of domains 72 .…”

A Tethered Vesicle Assay for High-Throughput Quantification of Membrane Fission

“…PIP 2 is incorporated to facilitate ENTH binding, as well as the negatively charged lipid 1,2-dioleoyl- sn -glycero-3-phosphoL-serine (DOPS) to further enhance ENTH recruitment via electrostatic interactions. The fluorescently labeled lipid Oregon Green 488 1,2dihexadecanoyl- sn -glycero-3-phosphoethanolamine (Oregon Green 488DHPE) is included to enable vesicle imaging, and the biotinylated lipid dipalmitoyl-decaethylene glycol-biotin (DP-EG10-biotin (Momin et al, 2015)) enables vesicle tethering. While the procedures described here focus on the ENTH domain, the membrane composition can readily be adapted to examine a wide variety of fission-driving proteins that bind to membrane surfaces.…”

Crowding-Induced Mixing Behavior of Lipid Bilayers: Examination of Mixing Energy, Phase, Packing Geometry, and Reversibility

“…1–4 These coexisting phases have distinct compositions and are able to selectively partition fluorescent probes and functionalized lipids. 6–7 The selective partitioning of fluorescent probes allows for identification of phases via fluorescence microscopy, while functionalized lipids allow for phase-specific protein targeting. Phase-specific adsorption and binding of proteins has been of interest for the development of biomaterials for various applications, such as small-scale fluidic devices, biosensors, and high-density arrays.…”