Biological membranes are partitioned across a variety of length scales from multi-component molecular complexes to micron-scale domains in polarized cells. Between these regimes is the mesoscale organization of cellular membranes into lipid-driven domains termed lipid rafts. This ultrastructural organization has fascinated and baffled a generation of membrane biologists, with persistent controversy about the nature and functions of rafts in vivo.

The raft concept proposes that cell membranes have the capacity for spontaneous self-organization into structurally and functionally distinct fluid domains, driven by subtle preferences between certain lipid classes. This concept was built on two experimental pillars. The first is the fact that biochemical fractionation of cells or tissues after lysis with non-ionic detergents yields ‘detergent-resistant membrane’ residues that consistently enrich for specific protein and lipid classes, implying intrinsic heterogeneity of cellular membranes. The second stems from observations that synthetic membranes composed of biomimetic lipid mixtures form beautiful, macroscopic fluid domains, owing to phase separation between ordered and disordered lipids.

Individually, these lines of evidence fell short of being truly convincing: detergent lysis creates the potential for artefactual assemblies never present in intact cells, while synthetic membranes lack the complexity and protein content of living cells. These shortcomings created confusion and doubt about the validity and value of the raft hypothesis that persists to this day. What good is all this intricate biochemistry and biophysics if it can never properly describe living membranes?

The turning point that reinvigorated the field was a remarkable paper by Baumgart et al. in 2007 showing that mammalian plasma membranes — isolated as giant plasma membrane vesicles (GPMVs) — can spontaneously separate into co-existing liquid phases that organize biomembranes in striking concordance with their detergent resistance. This breakthrough was enabled by advances in confocal microscopy (Baumgart et al., 2003) applied to plasma membrane blebs isolated intact from cultured mammalian cells.

Personally, seeing these images was a moment of epiphany: I had studied similar organization in synthetic membranes for years, wondering whether it had anything to do with cells, and here it was in living colour! The synthesis of these observations with the biochemical and biophysical pillars underlying the raft concept seems obvious in hindsight. Subsequent work with GPMVs showed that the ‘raft phase’ is more tightly packed, more viscous, rich in saturated lipids, sterol analogues and lipidated proteins (Sezgin et al., 2016), validating the principle that ordered, lipid-driven phases can organize biomembranes. Equally important, this paper demonstrated a straightforward methodology to investigate the properties and compositions of raft domains in intact cell membranes, allowing quantitative correlations between proteins’ raft affinity and their behaviour (Komura et al., 2016) and functions (Diaz-Rohrer et al., 2017) in living cells.