Order of lipid phases in model and plasma membranes

Abstract

Lipid rafts are nanoscopic assemblies of sphingolipids, cholesterol, and specific membrane proteins that contribute to lateral heterogeneity in eukaryotic membranes. Separation of artificial membranes into liquid-ordered (Lo) and liquid-disordered phases is regarded as a common model for this compartmentalization. However, tight lipid packing in Lo phases seems to conflict with efficient partitioning of raft-associated transmembrane (TM) proteins. To assess membrane order as a component of raft organization, we performed fluorescence spectroscopy and microscopy with the membrane probes Laurdan and C-laurdan. First, we assessed lipid packing in model membranes of various compositions and found cholesterol and acyl chain dependence of membrane order. Then we probed cell membranes by using two novel systems that exhibit inducible phase separation: giant plasma membrane vesicles [Baumgart et al. (2007) Proc Natl Acad Sci USA 104:3165-3170] and plasma membrane spheres. Notably, only the latter support selective inclusion of raft TM proteins with the ganglioside GM1 into one phase. We measured comparable small differences in order between the separated phases of both biomembranes. Lateral packing in the ordered phase of giant plasma membrane vesicles resembled the Lo domain of model membranes, whereas the GM1 phase in plasma membrane spheres exhibited considerably lower order, consistent with different partitioning of lipid and TM protein markers. Thus, lipid-mediated coalescence of the GM1 raft domain seems to be distinct from the formation of a Lo phase, suggesting additional interactions between proteins and lipids to be effective.generalized polarization value ͉ giant unilamellar vesicle ͉ membrane organization ͉ lipid sorting ͉ lipid raft T he lipid raft hypothesis postulates that selective interactions among sphingolipids, cholesterol, and membrane proteins contribute to lateral membrane heterogeneity (1). A tenet of the model is that small, dynamic cholesterol-sphingolipid-enriched assemblies can be induced to coalesce into larger, more stable structures through clustering of domain components (2). Although experimental data support cholesterol-dependent nano-scale membrane heterogeneity (3-8) and selective domain formation upon raft cross-linking (9-12), the mechanisms that govern such associations in cell membranes remain unclear.On the molecular level, a key feature that is thought to contribute to raft assembly is the propensity of cholesterol to pack tightly with saturated acyl chains of lipids causing them to adopt an extended conformation (13,14). In multi-component model membranes (n Ͼ 2), this interaction can lead to microscopically separate fluid membrane phases: the liquid-ordered (Lo) phase, enriched in saturated (sphingo-)lipids and cholesterol in a highly condensed state, and the liquid-disordered (Ld) phase, enriched in unsaturated glycerophospholipid in a disordered state (15)(16)(17).Several features of the Lo phase in model membranes correspond to the predicted properties of lipid rafts in cel...

Mammalian phospholipid homeostasis: Homeoviscous adaptation deconstructed by lipidomic data driven modelling

“…One of the limitations of this methodology is that the membrane order of individual lipids is often dependent on the other lipids in the system. For example in model membranes cholesterol increases the membrane order of phospholipid membranes as reported (Kaiser et al, 2009).…”

“…Additionally, work over the last decade has suggested that, rather than being a homogenous bilayer mixture, cell membranes are heterogeneous and contain microdomains of cholesterol, sphingomyelin, proteins and to a lesser extent saturated lipids (Simons and Sampaio, 2011). These 'lipid rafts' are widely accepted to be analogous to the disordered (L d ) and ordered (L o ) lipid phases observed in binary and tertiary lipid mixtures containing cholesterol (Kaiser et al, 2009). A number of other experimental factors further complicate the interpretation of HVA phenomena.…”

“…Given all the evidence from model membranes, we would expect a contribution to membrane order from the lipid membrane components we have not measured, such as, the protein, cholesterol and sphingomyelin. However since the phospholipids we have quantified in our datasets are predominantly associated with the proposed liquid disordered phase, and other lipids like sphingomyelin are associated with the cholesterol-rich ordered domains (Kaiser et al, 2009) the parameter sets in Table 1 could be considered a proxy measure of the liquid disordered phases regions of the cell bilayer (Wu et al, 2013). In this instance the concentration of cholesterol and sphingolipids would be much lower than that observed in whole cell extracts and thus the membrane order induced by cholesterol would not be as significant.…”

Limited cholesterol depletion causes aggregation of plasma membrane lipid rafts inducing T cell activation

“…and at physiological temperature [34,45] as well as in giant unilamellar vesicles isolated from plasma membranes [45,46]. A common feature of these systems is that they can reach equilibrium, since they are no longer parts of viable cells, which suggests that phase separation in the plasma membrane is prevented because viable cells are not at equilibrium.…”

Membrane raft association is a determinant of plasma membrane localization

“…GPMVs are cell-detached PM blebs whose protein (26) and lipid (27) diversity mirrors that of the native PM. These PM vesicles separate into coexisting liquid phases (4) with different order (28), which recruit membrane components in accordance with their predicted raft affinity, i.e., saturated lipids, glycosphingolipids (29), glycosylphosphatidyl inositol-anchored proteins (4), and palmitoylated proteins (30) partition to the ordered phase, denoted…”