Giant unilamellar vesicles composed of dioleoylphosphatidylcholine (DOPC) with sphingomyelin (SM), with or without cholesterol (Chol), exhibit fluid, disordered nanodomains of roughly 10 nm containing a few hundred lipids at compositions that published phase diagrams place firmly inside the single-phase region. Detected jointly by Monte-Carlo-modeled Förster resonance energy transfer (MC-FRET), solid-state NMR, and fluorescence correlation spectroscopy, these nanodomains are enriched in SM yet remain liquid-disordered, and their lifetimes are bounded only broadly to the microsecond-to-millisecond range. The microscopic mechanism that forms and stabilizes them against mixing entropy far from any phase boundary is unresolved: packing mismatch between DOPC and SM, preferential SM-Chol association, hydrogen-bond networks involving SM, and thermal or compositional fluctuations all remain candidate explanations, none discriminated for this system. The problem is to determine the dominant formation and stabilization mechanism, to measure directly the kinetics (lifetime distribution and its composition dependence) of these nanodomains, and to settle the probe-partition assumptions on which their detection and phase assignment rest.

Background

A long-standing question in membrane biophysics is whether lipids alone can organize a fluid membrane into nanoscale compartments. The lipid raft hypothesis proposes that sphingomyelin (SM, a saturated-chain phospholipid with a ceramide backbone and phosphocholine headgroup) and cholesterol (Chol) segregate with specific proteins into functional nanodomains in the plasma membrane; whether and how such nanodomains form remains contested (Sezgin et al., 2017). In model membranes made of a low-melting lipid such as DOPC (dioleoyl-phosphatidylcholine, an unsaturated phosphatidylcholine), a high-melting lipid such as SM, and Chol, the well-established paradigm is macroscopic liquid-liquid phase separation: a liquid-ordered (LoL_o) phase, rich in SM and Chol with tightly packed but mobile chains, coexists with a liquid-disordered (LdL_d) phase rich in DOPC. Miscibility phase diagrams, for example the DOPC/palmitoyl-SM/Chol diagram constructed by Nyholm et al. (2011) from the fluorescence of trans-parinaric acid (a fluorescent fatty-acid probe whose emission reports local lipid packing order), map which compositions are homogeneous and which phase-separate. In living cells, by contrast, STED-FCS (stimulated emission depletion microscopy combined with fluorescence correlation spectroscopy, which measures diffusion in fluorescent spots smaller than the diffraction limit) revealed that sphingolipids are transiently trapped in nanocompartments of roughly 20 nm on sub-millisecond timescales (Eggeling et al., 2009), and super-resolution studies indicate sub-resolution heterogeneity in the plasma membrane; the physical origin of these nanocompartments is unknown.

Koukalová et al. (2017) connected these threads with an unexpected model-membrane result. Using MC-FRET — time-resolved Förster resonance energy transfer (FRET, the distance-dependent energy transfer between donor and acceptor fluorophores, here interpreted through Monte Carlo simulations of probe distributions on model membranes) — together with solid-state wide-line and magic-angle-spinning NMR (which reports lipid headgroup and chain order and mobility) and z-scan FCS (fluorescence correlation spectroscopy calibrated by scanning the confocal volume through the membrane, which detects nanodomain-confined diffusion), they found nanodomains of approximately 10 nm (about 400-500 lipids, covering roughly 37-55% of the bilayer area) in binary DOPC/SM at 10-15 mol% SM and in ternary DOPC/Chol/SM at 25 mol% Chol and 5-10 mol% SM. Crucially, these compositions lie inside the single-phase region of the published phase diagrams, far from phase boundaries, so the domains cannot be ordinary phase separation or near-critical composition fluctuations (which require proximity to a miscibility critical point). The nanodomains are enriched in SM but retain enough DOPC to remain fluid and disordered (LdL_d-like), and later MC-FRET work showed they are registered across the two membrane leaflets. NMR averaging and FCS entrapment jointly constrain nanodomain lifetimes only to the window between microseconds and several milliseconds.

The study left open which microscopic mechanism forms and stabilizes these domains. The candidate factors it lists are: geometrical packing differences between the cone-shaped, unsaturated DOPC and the cylindrical saturated SM; preferential SM-Chol interactions; hydrogen bonding between the SM amide group and the Chol hydroxyl and between SM and DOPC; and thermal or concentration fluctuations. It also relies on an untested interpretive assumption: probe distribution constants (KDK_D, the equilibrium ratios describing how fluorescent lipid analogues partition between coexisting phases) measured for microscopic LoL_o domains are assumed to apply to nanoscopic domains of slightly different composition. No direct measurement of a nanodomain lifetime distribution, or of its dependence on SM and Chol fractions, exists.

Problem Statement

Determine the physical origin and dynamics of the fluid, disordered, sphingomyelin-enriched nanodomains of approximately 10 nm that form in DOPC/SM and DOPC/Chol/SM bilayers at compositions inside the single-phase region of published phase diagrams. A complete answer must (1) identify which microscopic mechanism dominates their formation and stabilization against mixing entropy far from phase boundaries — discriminating among geometrical packing mismatch between DOPC and SM, preferential SM-Chol association, hydrogen-bond networks involving SM (with Chol and with DOPC), and thermal or concentration fluctuations — and account quantitatively for the observed nanodomain size, area fraction, liquid-disordered character, and composition thresholds; (2) determine, rather than merely bound, the kinetics of the nanodomains, i.e., their lifetime or exchange-time distribution and its dependence on SM and Chol composition within the nanodomain-forming region; and (3) establish, or correct, the interpretive assumptions underlying their detection — in particular whether probe distribution constants measured for microscopic domains apply to nanoscopic domains of slightly different compositions — so that the mechanistic and kinetic conclusions do not rest on unvalidated probe-partition assumptions. The suggestion that these nanodomains serve as a model for cellular plasma-membrane nanocompartments is the motivating significance of the question, not a required deliverable.

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