Giant plasma membrane vesicles (GPMVs) blebbed from mammalian cells display Förster-resonance-energy-transfer (FRET)-detected ordered lipid nanodomains that persist roughly 15-25 degrees C above the large-scale phase-separation temperature, i.e. up to physiological temperature, and the thermal stability of these nanodomains is modulated by cyclodextrin-catalyzed lipid exchange in the intact cells from which the vesicles are derived (Li, Wang, Kakuda, and London, 2020). Because GPMV preparation partially destroys the native transbilayer lipid asymmetry and may sample a biased subset of plasma membrane lipids, these observations leave unproven whether equivalent ordered nanodomains exist in the plasma membrane of intact, living cells under the same conditions, and leave unexplained the physical mechanism (low line tension, proximity to critical points, interleaflet coupling, or protein/cytoskeletal interactions) that prevents such nanodomains from coalescing into micrometer-scale domains in natural membranes. Adjacent progress - asymmetry-loss induction of ordered domains, super-resolution imaging of cholesterol heterogeneity, stimulus-stabilized ordered regions around receptor clusters, microemulsion and criticality theories, and refined FRET analysis of nanoscopic miscibility transitions in model bilayers - has yielded no decisive demonstration in unperturbed intact cells and no experimental discrimination among the candidate size-limiting mechanisms, so the problem remains open.

Background

The lipid raft hypothesis posits that preferential interactions among sterols (e.g. cholesterol), sphingolipids, and saturated phospholipids laterally organize the plasma membrane into domains whose packed, ordered structure differs from the surrounding membrane. In ternary model membranes such mixtures demix into a liquid-ordered (Lo) phase - a fluid but tightly packed phase rich in cholesterol and saturated lipids - coexisting with a liquid-disordered (Ld) phase rich in unsaturated lipids. Giant plasma membrane vesicles (GPMVs) are micrometer-scale vesicles blebbed directly from the plasma membrane of living cells; they retain much of the native lipid and protein complexity and macroscopically separate into coexisting Lo and Ld phases, but this large-scale phase separation is observed by light microscopy only below a miscibility transition temperature that is typically well below 37 degrees C. Many GPMV compositions lie near thermodynamic critical points, the conditions at which the two phases become identical and composition fluctuations of tens of nanometers are expected to persist above the transition. Förster resonance energy transfer (FRET) is the nonradiative transfer of excitation energy between a donor and an acceptor fluorophore separated by roughly 1-10 nm (the characteristic Förster radius is about 4 nm for the probes used); because FRET reports molecular proximity far below the optical diffraction limit, a donor/acceptor pair that partitions differently between ordered and disordered regions detects coexisting lipid phases (nanodomains) of only a few nanometers. Using this approach, Li, Wang, Kakuda, and London (2020, Journal of Lipid Research 61:758-766) showed that FRET-detected ordered nanodomains in GPMVs persist up to about 20 degrees C above the large-scale miscibility transition - that is, up to physiological temperature - consistent with predictions for domains above a critical point, and that substituting plasma membrane lipids in the intact cells (by cyclodextrin-catalyzed lipid exchange, in which cyclodextrin carrier molecules swap lipids between the cell membrane and exogenous lipids) raises or lowers nanodomain thermal stability in GPMVs in the same direction that the substituted lipids stabilize or destabilize ordered domains in artificial asymmetric vesicles. The authors emphasized, however, that GPMV preparation perturbs the native plasma membrane - at least some transbilayer lipid asymmetry (the different lipid composition of the outer and inner leaflets, e.g. the exclusive cytoplasmic exposure of phosphatidylserine in live cells) is lost, and GPMV lipids may represent a biased subset of plasma membrane lipids - so persistence of nanodomains to 37 degrees C in GPMVs does not establish their presence in intact, living cells. In addition, why nanodomains do not merge into large-scale domains was already a much-discussed question without consensus: candidate explanations include low line tension (the energy per unit length of the boundary between Lo and Ld domains, proposed to suppress coalescence when it falls below about 0.3 pN), proximity to critical points, interleaflet coupling (the structural influence of one bilayer leaflet on the other, which can correlate domains across the bilayer), and protein or cortical-cytoskeleton interactions. More broadly, whether raft-like ordered domains exist in living cells at all has been called the pressing open question of the field (Levental, Levental, and Heberle, 2020), so the GPMV-to-intact-cell extrapolation is a specific, experimentally resolvable instance of that central issue.

Problem Statement

Establish whether the ordered lipid nanodomains that FRET detects up to physiological temperature in giant plasma membrane vesicles exist in the plasma membrane of intact, living cells under the same conditions, and determine the physical mechanism that keeps such nanodomains from coalescing into large-scale domains in natural membranes. Concretely, the problem comprises one overarching objective - establishing the existence and the physical basis of physiological-temperature ordered nanodomains in native plasma membranes - whose jointly supporting parts are: (1) direct, nanoscale, order-sensitive detection (or calibrated exclusion) of ordered nanodomains in the plasma membrane of unperturbed, living cells at physiological temperature, distinguishing lipid-driven order from protein-cluster, cortical-actin, and endocytic heterogeneity, and comparing like-for-like with the GPMV FRET observations; and (2) experimental discrimination among the candidate mechanisms proposed to limit nanodomain size - line tension at ordered/disordered boundaries falling below roughly 0.3 pN, proximity to a critical point, interleaflet coupling, and protein/cytoskeletal interactions - in natural or cell-derived membranes. The source itself notes that experiments using milder methods to prepare GPMVs and experiments in intact cells will be needed to further investigate the asymmetry-loss and biased-lipid-sampling caveats, and that differing extents of exchange for different sterols complicate quantitative comparison of lipid-substitution experiments with artificial vesicles of well-defined composition; such enabling measurements (for example, asymmetry-preserving GPMV protocols or better accounting of post-exchange sterol content) would facilitate resolving parts (1) and (2), but they are context for the objective rather than additional goals in their own right. An answer that demonstrated only induction of ordered domains by destroying asymmetry, by protein crosslinking, or by other perturbations would not resolve the problem; the question concerns the unperturbed, native state of the living-cell plasma membrane at physiological temperature.

References

  1. Li G, Wang Q, Kakuda S, London E (2020). Nanodomains can persist at physiologic temperature in plasma membrane vesicles and be modulated by altering cell lipids. Journal of Lipid Research 61(5):758-766. DOI: 10.1194/jlr.ra119000565. https://doi.org/10.1194/jlr.ra119000565
  2. Levental I, Levental KR, Heberle FA (2020). Lipid rafts: controversies resolved, mysteries remain. Trends in Cell Biology 30(5):341-353. DOI: 10.1016/j.tcb.2020.01.009. https://doi.org/10.1016/j.tcb.2020.01.009
  3. St. Clair JW, Kakuda S, London E (2020). Induction of ordered lipid raft domain formation by loss of lipid asymmetry. Biophysical Journal 119(3):483-492. DOI: 10.1016/j.bpj.2020.06.030. https://doi.org/10.1016/j.bpj.2020.06.030
  4. Kakuda S, Suresh P, Li G, London E (2022). Loss of plasma membrane lipid asymmetry can induce ordered domain (raft) formation. Journal of Lipid Research 63(1):100155. DOI: 10.1016/j.jlr.2021.100155. https://doi.org/10.1016/j.jlr.2021.100155
  5. Kakuda S, London E (2025). Effect of a scramblase activator upon lipid and probe scrambling and membrane domain formation in HEK 293T cells. Faraday Discussions 259:26-44. DOI: 10.1039/d4fd00211c. https://doi.org/10.1039/d4fd00211c
  6. Sezgin E (2022). Giant plasma membrane vesicles to study plasma membrane structure and dynamics. Biochimica et Biophysica Acta - Biomembranes 1864(4):183857. DOI: 10.1016/j.bbamem.2021.183857. https://doi.org/10.1016/j.bbamem.2021.183857
  7. Veatch SL, Rogers N, Decker A, Shelby SA (2023). The plasma membrane as an adaptable fluid mosaic. Biochimica et Biophysica Acta - Biomembranes 1865(3):184114. DOI: 10.1016/j.bbamem.2022.184114. https://doi.org/10.1016/j.bbamem.2022.184114
  8. Shaw TR, Ghosh S, Veatch SL (2021). Critical phenomena in plasma membrane organization and function. Annual Review of Physical Chemistry 72:51-72. DOI: 10.1146/annurev-physchem-090419-115951. https://doi.org/10.1146/annurev-physchem-090419-115951
  9. Allender DW, Schick M (2020). Recent experiments support a microemulsion origin of plasma membrane domains: dependence of domain size on physical parameters. Membranes 10(8):167. DOI: 10.3390/membranes10080167. https://doi.org/10.3390/membranes10080167
  10. Graf I, Machta BB (2022). Thermodynamic stability and critical points in multicomponent mixtures with structured interactions. Physical Review Research 4:033144. DOI: 10.1103/PhysRevResearch.4.033144. https://doi.org/10.1103/PhysRevResearch.4.033144
  11. Schick M (2018). Strongly correlated rafts in both leaves of an asymmetric bilayer. Journal of Physical Chemistry B 122(13):3251-3258. DOI: 10.1021/acs.jpcb.7b08890. https://doi.org/10.1021/acs.jpcb.7b08890
  12. Urbančič I, Schiffelers L, Jenkins E, Gong W, Santos AM, Schneider F, O'Brien-Ball C, Vuong MT, Ashman N, Sezgin E, Eggeling C (2021). Aggregation and mobility of membrane proteins interplay with local lipid order in the plasma membrane of T cells. FEBS Letters 595:2127-2146. DOI: 10.1002/1873-3468.14153. https://doi.org/10.1002/1873-3468.14153
  13. Lorizate M, Terrones O, Nieto-Garai JA, Rojo-Bartolomé I, Ciceri D, Morana O, Olazar-Intxausti J, Arboleya A, Martin A, Szynkiewicz M, Calleja-Felipe M, Bernardino de la Serna J, Contreras F-X (2021). Super-resolution microscopy using a bioorthogonal-based cholesterol probe provides unprecedented capabilities for imaging nanoscale lipid heterogeneity in living cells. Small Methods 5:2100430. DOI: 10.1002/smtd.202100430. https://doi.org/10.1002/smtd.202100430
  14. Yang G-S, Wagenknecht-Wiesner A, Yin B, Suresh P, London E, Baird BA, Bag N (2024). Lipid-driven interleaflet coupling of plasma membrane order regulates FcεRI signaling in mast cells. Biophysical Journal 123:2256-2270. DOI: 10.1016/j.bpj.2023.07.027. https://doi.org/10.1016/j.bpj.2023.07.027
  15. Bag N, London E, Holowka D, Baird B (2022). Transbilayer coupling of lipids in cells investigated by imaging fluorescence correlation spectroscopy. Journal of Physical Chemistry B 126(12):2325-2336. DOI: 10.1021/acs.jpcb.2c00117. https://doi.org/10.1021/acs.jpcb.2c00117
  16. Svobodová B, Šťastný D, Blom H, Mikhalyov I, Gretskaya N, Balleková A, Sezgin E, Hof M, Šachl R (2025). Revised diffusion law permits quantitative nanoscale characterization of membrane organization. Analytical Chemistry 97:11478-11485. DOI: 10.1021/acs.analchem.5c00021. https://doi.org/10.1021/acs.analchem.5c00021
  17. Fujiwara TK, Kusumi A (2026). Development of ultrafast single fluorescent-molecule imaging and its application to unravel plasma membrane structure and function in live cells. Biophysics and Physicobiology 23:e230018. DOI: 10.2142/biophysico.bppb-v23.0018. https://doi.org/10.2142/biophysico.bppb-v23.0018
  18. Kondratowicz M, Wu A, Grela E, Raducka-Jaszul O, Luchowski R, Błaszczak E, Wójtowicz K, Hamon Y, Gruszecki WI, Cieślik-Boczula K, Trombik T (2025). ABCA1 modifies plasma membrane organization of living cells. Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids 1870:159667. DOI: 10.1016/j.bbalip.2025.159667. https://doi.org/10.1016/j.bbalip.2025.159667
  19. Chaisson EH, Mehta D, Heberle FA (2026). Nanodomain formation in lipid bilayers I: quantifying the nanoscopic miscibility transition with FRET. Biochimica et Biophysica Acta - Biomembranes 1868:184505. DOI: 10.1016/j.bbamem.2026.184505. https://doi.org/10.1016/j.bbamem.2026.184505
  20. Mehta D, Chaisson EH, Cooper AM, Ahmed M, Waxham MN, Heberle FA (2026). Nanodomain formation in lipid bilayers II: the influence of mixed-chain saturated lipids. Biochimica et Biophysica Acta - Biomembranes 1868:184507. DOI: 10.1016/j.bbamem.2026.184507. https://doi.org/10.1016/j.bbamem.2026.184507
  21. Suresh P, London E (2022). Using cyclodextrin-induced lipid substitution to study membrane lipid and ordered membrane domain (raft) function in cells. Biochimica et Biophysica Acta - Biomembranes 1864:183774. DOI: 10.1016/j.bbamem.2021.183774. https://doi.org/10.1016/j.bbamem.2021.183774
  22. Stefanski KM, Huang H, Luu DD, Li GC, Hutchison JM, Saksena N, Fisch AJ, Hasaka TP, Bauer JA, Kenworthy AK, Van Horn WD, Sanders CR (2026). Pharmacological tools to modulate ordered membrane domains and order-dependent protein function. Communications Chemistry 9:72. DOI: 10.1038/s42004-025-01874-8. https://doi.org/10.1038/s42004-025-01874-8
  23. Anselmo S, Bonaccorso E, Gangemi C, Sancataldo G, Conti Nibali V, D'Angelo G (2025). Lipid rafts in signalling, diseases, and infections: what can be learned from fluorescence techniques? Membranes 15(1):6. DOI: 10.3390/membranes15010006. https://doi.org/10.3390/membranes15010006