Reorganization of lipid domain distribution in giant unilamellar vesicles upon immobilization with different membrane tethers

Abstract

Characterization of phase coexistence in biologically relevant lipid mixtures is often carried out through confocal microscopy of giant unilamellar lipid vesicles (GUVs), loaded with fluorescent membrane probes. This last analysis is generally limited to the vesicle hemisphere further away from the coverslip, in order to avoid artifacts induced by the interaction with the solid surface, and immobilization of vesicles is in many cases required in order to carry out intensity, lifetime or single-molecule based microscopy. This is generally achieved through the use of membrane tethers adhering to a coverslip surface. Here, we aimed to determine whether GUV immobilization through membrane tethers induces changes in lipid domain distribution within liposomes displaying coexistence of lipid lamellar phases. Confocal imaging and a Förster resonance energy transfer (FRET) methodology showed that biotinylated phospholipids present significantly different membrane phase partition behavior upon protein binding, depending on the presence or absence of a linker between the lipid headgroup and the biotinyl moiety. Membrane phases enriched in a membrane tether displayed in some cases a dramatically increased affinity for the immobilization surface, effectively driving sorting of lipid domains to the adherent membrane area, and in some cases complete sequestering of a lipid phase to the interaction surface was observed. On the light of these results, we conclude that tethering of lipid membranes to protein surfaces has the potential to drastically reorganize the distribution of lipid domains, and this reorganization is solely dictated by the partition properties of the protein-tether complex.

Accurate quantification of inter-domain partition coefficients in GUVs exhibiting lipid phase coexistence

“…Quantification of lipid and lipid analogue stock concentration was carried out as described elsewhere 13 . For preparation of multilamellar vesicles (MLVs), lipid mixtures composed of the adequate amount of lipids were prepared in chloroform to a final concentration of 0.25 mM.…”

“…This method has been extensively applied to the quantification of the partition of molecules between gel and fluid as well as between lo and ld lipid phases. Its application has focused on large unilamellar or multilamelar vesicles through fluorescence spectroscopy [9][10][11] , and supported membranes 12 or giant unilamellar vesicles (GUV's) through fluorescence microscopy 13,14 .…”

“…This eliminates the possibility of erroneous interpretation of data obtained from an ensemble measurement with heterogeneous populations of vesicles. Additionally, in lipid membranes with phase coexistence, molecules in some cases show preferential incorporation into domain interfaces 13 , where lipid packing defects are much higher. Although thermodynamically the domain interfaces are not a formal phase, they are for practical reasons a third "pseudo"-phase, which competes for the incorporation of the fluorescent molecule.…”

Phase Composition Control in Microsphere-Supported Biomembrane Systems

“…Phase separation is displayed by non-uniform membrane labeling of QD705-Streptavidin bound to DOPE-cap-biotin lipids known to partition to the liquid-disordered (L d ) phase enriched by DOPC. 35 In labeling scheme III, K 3 A 4 L 2 A 7 L 2 A 3 K-Lys(FITC) is partitioned into the L o and L d phases and the L d phase is preferentially stained by the C-12 DiI lipid tracer.…”

“…17, 35, 36 Phase composition-based sorting of individual PLB assemblies would therefore be an ideal way of both controlling and probing membrane protein microenvironment in PLB-based assays. Alternatively, this method could provide a route to isolate assemblies for biomimetic ligand display.…”