Adaptive lipid packing and bioactivity in membrane domains

Abstract

Lateral compositional and physicochemical heterogeneity is a ubiquitous feature of cellular membranes on various length scales, from molecular assemblies to micrometric domains. Segregated lipid domains of increased local order, referred to as rafts, are believed to be prominent features in eukaryotic plasma membranes; however, their exact nature (i.e. size, lifetime, composition, homogeneity) in live cells remains difficult to define. Here we present evidence that both synthetic and natural plasma membranes assume a wide range of lipid packing states with varying levels of molecular order. These states may be adapted and specifically tuned by cells during active cellular processes, as we show for stimulated insulin secretion. Most importantly, these states regulate both the partitioning of molecules between coexisting domains and the bioactivity of their constituent molecules, which we demonstrate for the ligand binding activity of the glycosphingolipid receptor GM1. These results confirm the complexity and flexibility of lipid-mediated membrane organization and reveal mechanisms by which this flexibility could be functionalized by cells.

Fig 1. Various lipid mixtures yield a range of lipid packing states.

(a) Natural PC mixtures (brain or liver PC extracts) yield liposomes with higher GP than synthetic DOPC bearing two unsaturated acyl chain. (b) Mixtures of cholesterol with natural SM (egg SM extract) and DPPC yield liposomes with lower order than cholesterol with pure SM 18:0. (c) Lipid packing of DOPC liposomes increases monotonically with increasing cholesterol. (d) Quantification of the effect of compositional variation on membrane order in one-phase liposomes.

Fig 2. Tuning lipid packing in coexisting domains of GUVs and GPMVs.

GP imaging of ordered and disordered phases in (a) DOPC:SSM:Chol GUVs with equimolar DOPC and SSM and varying Chol; (b) 2:2:1 GUVs with various ordering and disordering components; and (c) various GPMV preparations. In GPMVs, 0.5mM DCA lowers the GP of the disordered phase; [DTT] increases GP of the ordered and lowers GP of disordered. (d) Lipid packing (GP) of individual membrane domains and the differences (ΔGP) between ordered and disordered phases in phase separated membranes.

Fig 3. Membrane order is modulated in a domain-specific way by cellular activity.

(a) Confirmation of insulin secretion induced by glucose stimulation. (b) Time course of overall PM order during glucose stimulation of beta cells. (c) C-Laurdan microscopy of GPMVs shows domain-specific changes to membrane order during the insulin stimulation time-course (p values determined with unpaired t-test). (d) Changes to PM order are driven by changes in membrane composition, with PM cholesterol (normalized to membrane PLs) increasing with glucose stimulation, and decreasing during recovery.

Fig 4. Relative lipid packing between domains correlates with BD-GM1 partitioning.

a) GP, BD-GM1 (green) and the disordered marker FAST-DiI (red) imaged in various membrane preparations. BD-GM1 and FAST-DiI are imaged in the same vesicle; GP is shown form a different vesicle representative of the preparation. b) The quantification of BD-GM1 ordered phase partitioning versus ΔGP.

Fig 5. Membrane packing affects receptor-ligand binding.

(a) CTxB binds (magenta) almost exclusively to the disordered domain pool of BD-GM1 (green) in phase separated GUVs and GPMVs where disordered phase is marked by FastDiI (red). (b) This behavior is quantified by the “CTxB binding to ordered domain”, the ratio between the phase localization of CTxB (K_p,CTxB) and its receptor BD-GM1 (K_p,GM1). (c) At high ΔGP, CTxB essentially does not recognize BD-GM1 present in the ordered domain; as the order difference between phases is reduced, recognition of CTxB in ordered phase increases. CTxB ordered binding is also well-correlated with disordered phase GP but not ordered phase GP. (d) Disordered domain binding of CTxB is preferential, but not exclusive, as ordered phase binding increases with increasing CTxB concentration for lower values of ΔGP.