Exploring the raft-hypothesis by probing planar bilayer patches of free-standing giant vesicles at nanoscale resolution, with and without Na,K-ATPase

Abstract

The structure of functional lipid domains (rafts) in biological membranes has for long time been unresolved due to their small length scales and transient nature. These cooperative properties of the lipid bilayer matrix are modelled by free-standing giant unilammellar vesicles (GUVs) with well-characterized lipid composition. We review a series of recent advances in preparation and analysis of GUVs, which allows for characterization of small domains by high-resolution imaging techniques. These includes a new GUV preparation method with a desired overall lipid composition achieved by mixing small unilammellar vesicles (SUVs), test of the lipids compositional uniformity in GUVs and swift adsorption of GUVs to solid support by kinetically arresting the lateral structure of membrane prior to collapse for subsequent imaging. The techniques are applied to the analysis of membrane domains in GUVs formed from mixtures of DOPC/DPPC/cholesterol with and without Na,K-ATPase (NKA), a transmembrane protein known to be associated with rafts. Two mechanisms of domain formation are revealed: 1) close to l/l phase coexistence, domains in size up to 100nm appear as thermally induced droplet fluctuations, 2) NKA shows interfacial activity and cluster in l/l micro-emulsion droplets. Some perspectives for the application of the techniques and the understanding of the nature of raft domains are outlined.

Cholesterol-rich naked mole-rat brain lipid membranes are susceptible to amyloid beta-induced damage in vitro

“…The origin of the islands of DPPC is due to the packing arrangement of DPPC chains, resulting in a height difference of around 1.5 nm to the surrounding DOPC (Figure 4B). Such spontaneous phase separation behaviour in model systems has been used as a rationale for the existence of lipid rafts in cells [25]. After a 2-hour exposure to 8 μM of human Aβ the bilayer remained completely intact, but with AGING clearly visible features due to the Aβ peptide in between the DPPC gel phase domains, which are absorbed on top of the DOPC liquid disordered phase (highlighted by blue circle in Figure 4C).…”

Lipid membranes from naked mole-rat brain lipids are cholesterol-rich, highly phase-separated, and sensitive to amyloid-induced damage

“…The origin of the islands of DPPC is due to the packing arrangement of DPPC chains, resulting in a height difference of around 1.5 nm to the surrounding DOPC ( Figure 4B). Such spontaneous phase separation behaviour in model systems has been used as a rationale for the existence of lipid rafts in cells 25 . After 2 hours exposure to 8 µM of human amyloid beta the bilayer remained completely intact, but with clearly visible features in between the DPPC gel phase domains, that are absorbed on top of the DOPC liquid disordered phase (highlighted by blue circle in Figure 4C).…”

Interactions between lipids and proteins are critical for organization of plasma membrane-ordered domains in tobacco BY-2 cells

“…With this model in mind, we noticed a remarkable limitation in the size of ordered domains within GVPMs, which could undoubtedly be attributed to the presence of proteins. Indeed, GVPMs and living cells both exhibit similar small ordered domains, and this small size originates from the ability of proteins to emulsify ordered domains ( Bhatia et al , 2016 ). This PM organization should be based on hydrogen bonds and van der Waals interactions operating on a time scale representative of what we observed, and should show, in a coherent manner, an insensitivity to protein kinase inhibitors.…”