Domains or no domains? That is the question: insights from asymmetric bilayer models into plasma membrane lipid organization

Abstract The mammalian plasma membrane is characterized by its pronounced lipid asymmetry, including the exceptional enrichment of sphingomyelin (SM) in the exoplasmic leaflet. How this asymmetry influences cholesterol distribution and membrane organization remains a central and unresolved question in cell biology. The lipid raft hypothesis has long provided the prevailing framework, proposing that cholesterol and SM assemble into dynamic liquid-ordered nanodomains that compartmentalize proteins and regulate membrane trafficking, signaling, and host–pathogen interactions. More recently, a phospholipid (and cholesterol) imbalance model offers an alternative view, proposing that the exoplasmic leaflet contains substantially fewer phospholipids but is correspondingly enriched in cholesterol, potentially shifting its composition toward a uniformly liquid-ordered state. In this review, we examine these two conceptual frameworks in light of recent advances in membrane lipidomics and asymmetric model membranes. We discuss the experimental evidence supporting both models and emphasize that many observations attributed to a large phospholipid imbalance are not unique to that interpretation but can also be explained by membrane lateral lipid heterogeneity. We further highlight methodological considerations in enzymatic lipid digestion and cholesterol quantification, underscoring the need for additional experimental validation of leaflet-specific lipid composition. A central theme of this review is the role of interleaflet interactions. Experimental studies using asymmetric model membranes demonstrate that membrane domains in one leaflet can induce lateral organization in the opposing leaflet, even when the latter lacks the lipid composition required to form liquid-ordered domains independently. We suggest that interleaflet coupling provides a physical mechanism that reconciles lipid domain (lipid raft) formation with the compositional asymmetry of the plasma membrane. Rather than viewing the lipid raft and phospholipid imbalance models as mutually exclusive, we propose that their apparent differences depend critically on the magnitude of phospholipid and cholesterol asymmetry and on the strength of transbilayer coupling. Understanding how these factors collectively govern membrane organization will be essential for developing a unified physical description of the plasma membrane and for elucidating the mechanisms underlying cholesterol homeostasis, membrane protein regulation, and cellular signaling.