The plasma membrane (PM) is a lipid bilayer whose two leaflets differ in headgroup composition and lipid unsaturation, and this asymmetry shapes the lateral organization of each leaflet, including the formation of ordered nanoscale lipid domains. Coarse-grained molecular dynamics simulations by Yamada and Shinoda (2025, published 2026) showed that an asymmetric PM-composition bilayer keeps ordered domains nanoscopic in the inner leaflet while a fully scrambled (symmetric) counterpart develops large anti-registered domains resembling the microscale phase separation seen in cell-derived giant plasma membrane vesicles (GPMVs). The authors explicitly leave unresolved: the precise extent of lipid scrambling in GPMVs, which is needed to relate GPMV experiments to the intact PM; and the identity of the dominant factors governing interleaflet coupling (domain registration versus anti-registration) in complex asymmetric membranes. No published study has resolved either thread: scrambling in GPMVs has been quantified only for phosphatidylserine exposure and is vesiculation-protocol dependent, and interleaflet-coupling mechanisms have been probed mainly in symmetric or simple model bilayers, with thermodynamic frameworks for asymmetric membranes covering ternary mixtures rather than PM-like complexity. The problem therefore remains open: a lipid-class-resolved measurement of GPMV scrambling and a quantitative, predictive account of which coupling mechanisms dominate in complex asymmetric membranes are both still missing.

Background

The plasma membrane (PM) of eukaryotic cells is a lipid bilayer whose two leaflets have different compositions: the outer (exoplasmic) leaflet is enriched in sphingolipids, while the inner (cytoplasmic) leaflet contains most of the charged and amino phospholipids, and lipidomic work has further shown that inner-leaflet phospholipids are roughly twice as unsaturated as outer-leaflet ones (Lorent et al. 2020). A leaflet is one of the two monolayers that make up the bilayer. Within each leaflet, lipids can laterally segregate into coexisting liquid-ordered (Lo) and liquid-disordered (Ld) phases; the Lo phase is a tightly packed, cholesterol- and sphingolipid-rich state that underlies the lipid-raft hypothesis of membrane functional organization. Giant plasma membrane vesicles (GPMVs) are tens-of-micrometers vesicles blebbed from the surface of live cells by chemical treatment; because they retain the PM lipid inventory but can be imaged like model membranes, they are the standard experimental platform for PM phase behavior, and they routinely show micron-scale Lo/Ld phase separation near room temperature. However, the vesiculation procedure activates lipid scrambling — the bidirectional, scramblase-mediated translocation of lipids between leaflets, requiring the calcium-activated scramblase TMEM16F (Han et al. 2019) — so GPMVs lose the strict lipid asymmetry of the intact PM, and how much scrambling actually occurs is only partially known (annexin-based assays detect only phosphatidylserine exposure, and the degree depends on the vesiculation chemistry; Kakuda et al. 2022). The two leaflets of a bilayer also interact mechanically and thermodynamically across the midplane, a phenomenon called interleaflet coupling. One of its manifestations is domain registration, in which an ordered domain in one leaflet sits directly opposite an ordered domain in the other leaflet, versus anti-registration, in which ordered regions face disordered ones. Candidate mechanisms for this coupling include hydrophobic (thickness) mismatch between domains of different order, lipid interdigitation (the penetration of acyl chains from one leaflet into the other), membrane curvature and elastic stress, and rapid cholesterol flip-flop (exchange of cholesterol between leaflets), each supported by theory or simulations in simple membrane models (Putzel et al. 2011; Perlmutter and Sachs 2011; Thallmair et al. 2018; Seo et al. 2020; reviewed by Fujimoto and Parmryd 2016). Asymmetry adds a further coupling channel: differential stress, the state in which one leaflet wants more area than the other, generating compression in one leaflet and tension in the other; theory shows cholesterol distributes between leaflets in response to both composition and this stress (Hossein and Deserno 2020; Varma and Deserno 2022). Coarse-grained molecular dynamics (CG-MD), a simulation technique that groups atoms into interaction sites to reach microsecond-and-larger scales, let Yamada and Shinoda (2025; published in Biophysical Journal, 2026) simulate an asymmetric PM-composition bilayer built from erythrocyte lipidomics: the outer leaflet is uniformly ordered, the inner leaflet separates into roughly 10 nm dynamic ordered/disordered domains, and differential stress from a cholesterol bias toward the outer leaflet reinforces this asymmetry, whereas an artificially fully scrambled version of the same membrane develops much larger, anti-registered domains resembling GPMVs. Their paper states that the precise extent of lipid scrambling in GPMVs is unknown (blocking direct comparison of GPMV and intact-PM phase behavior) and that identifying the dominant interleaflet-coupling factors in complex membranes like the PM remains a challenging open problem.

Problem Statement

Establish, quantitatively and mechanistically, how transbilayer lipid asymmetry controls interleaflet coupling and lateral domain organization in plasma-membrane-like complex membranes. The problem has two linked parts that jointly constitute this one objective; completing both parts is the full resolution standard. (1) Determine the extent of lipid scrambling in giant plasma membrane vesicles — the fraction of each lipid class translocated between leaflets, for the standard vesiculation protocols — so that GPMV phase behavior (domain sizes, transition temperatures) can be related to that of the intact asymmetric PM, whose ordered domains are nanoscopic whereas GPMV domains are micron-scale. (2) Identify the dominant factors that govern interleaflet coupling in complex asymmetric membranes — that is, which of the candidate mechanisms (hydrophobic/thickness mismatch, lipid interdigitation, membrane curvature and elastic stress, cholesterol flip-flop, differential stress) principally determine whether ordered domains in the two leaflets register or anti-register in bilayers of PM-like composition, ranked by their relative contribution.

References

  1. Teppei Yamada and Wataru Shinoda, 'Asymmetry and Heterogeneity in the Plasma Membrane', bioRxiv preprint (2025), DOI 10.1101/2025.03.03.641122, https://doi.org/10.1101/2025.03.03.641122
  2. Teppei Yamada and Wataru Shinoda, 'Asymmetry and heterogeneity in the plasma membrane', Biophysical Journal 125(2):387-395 (2026), DOI 10.1016/j.bpj.2025.06.026, https://doi.org/10.1016/j.bpj.2025.06.026
  3. J. H. Lorent, K. R. Levental, L. Ganesan, G. Rivera-Longsworth, E. Sezgin, M. Doktorova, E. Lyman, I. Levental, 'Plasma membranes are asymmetric in lipid unsaturation, packing and protein shape', Nature Chemical Biology 16:644-652 (2020), DOI 10.1038/s41589-020-0529-6, https://doi.org/10.1038/s41589-020-0529-6
  4. Tina W. Han, Wenlei Ye, Neville P. Bethel, Mario Zubia, Andrew Kim, Kathy H. Li, Alma L. Burlingame, Michael Grabe, Yuh Nung Jan, Lily Y. Jan, 'Chemically induced vesiculation as a platform for studying TMEM16F activity', Proceedings of the National Academy of Sciences 116(4):1309-1318 (2019), DOI 10.1073/pnas.1817498116, https://doi.org/10.1073/pnas.1817498116
  5. Shinako Kakuda, Pavana Suresh, Guangtao Li, Erwin London, 'Loss of plasma membrane lipid asymmetry can induce ordered domain (raft) formation', Journal of Lipid Research 63(1):100155 (2022), DOI 10.1016/j.jlr.2021.100155, https://doi.org/10.1016/j.jlr.2021.100155
  6. Johnna Wellman St. Clair, Shinako Kakuda, Erwin London, 'Induction of Ordered Lipid Raft Domain Formation by Loss of Lipid Asymmetry', Biophysical Journal 119(3):483-492 (2020), DOI 10.1016/j.bpj.2020.06.030, https://doi.org/10.1016/j.bpj.2020.06.030
  7. G. Garbès Putzel, Mark J. Uline, Igal Szleifer, M. Schick, 'Interleaflet Coupling and Domain Registry in Phase-Separated Lipid Bilayers', Biophysical Journal 100(4):996-1004 (2011), DOI 10.1016/j.bpj.2011.01.021, https://doi.org/10.1016/j.bpj.2011.01.021
  8. Jason D. Perlmutter and Jonathan N. Sachs, 'Interleaflet Interaction and Asymmetry in Phase Separated Lipid Bilayers: Molecular Dynamics Simulations', Journal of the American Chemical Society 133(17):6563-6577 (2011), DOI 10.1021/ja106626r, https://doi.org/10.1021/ja106626r
  9. Sebastian Thallmair, Helgi I. Ingólfsson, Siewert J. Marrink, 'Cholesterol Flip-Flop Impacts Domain Registration in Plasma Membrane Models', The Journal of Physical Chemistry Letters 9(18):5527-5533 (2018), DOI 10.1021/acs.jpclett.8b01877, https://doi.org/10.1021/acs.jpclett.8b01877
  10. Sangjae Seo, Michio Murata, Wataru Shinoda, 'Pivotal Role of Interdigitation in Interleaflet Interactions: Implications from Molecular Dynamics Simulations', The Journal of Physical Chemistry Letters 11(13):5171-5176 (2020), DOI 10.1021/acs.jpclett.0c01317, https://doi.org/10.1021/acs.jpclett.0c01317
  11. Toyoshi Fujimoto and Ingela Parmryd, 'Interleaflet Coupling, Pinning, and Leaflet Asymmetry—Major Players in Plasma Membrane Nanodomain Formation', Frontiers in Cell and Developmental Biology 4:155 (2016), DOI 10.3389/fcell.2016.00155, https://doi.org/10.3389/fcell.2016.00155
  12. Amirali Hossein and Markus Deserno, 'Spontaneous Curvature, Differential Stress, and Bending Modulus of Asymmetric Lipid Membranes', Biophysical Journal 118(3):624-642 (2020), DOI 10.1016/j.bpj.2019.11.3398, https://doi.org/10.1016/j.bpj.2019.11.3398
  13. Malavika Varma and Markus Deserno, 'Distribution of cholesterol in asymmetric membranes driven by composition and differential stress', Biophysical Journal 121(20):4001-4018 (2022), DOI 10.1016/j.bpj.2022.07.032, https://doi.org/10.1016/j.bpj.2022.07.032
  14. Malavika Varma and Markus Deserno, 'The interplay of composition and mechanics in the thermodynamics of asymmetric ternary lipid membranes', Faraday Discussions 259:200-233 (2025), DOI 10.1039/d4fd00196f, https://doi.org/10.1039/d4fd00196f
  15. Milka Doktorova, Jessica L. Symons, Xiaoxuan Zhang, Hong-Yin Wang, Jan Schlegel, Joseph H. Lorent, Frederick A. Heberle, Erdinc Sezgin, Edward Lyman, Kandice R. Levental, Ilya Levental, 'Cell membranes sustain phospholipid imbalance via cholesterol asymmetry', Cell 188(10):2586-2602.e24 (2025), DOI 10.1016/j.cell.2025.02.034, https://doi.org/10.1016/j.cell.2025.02.034
  16. Yvonne Lange and Theodore L. Steck, 'Critique of a Radically New Model for Plasma Membrane Bilayer Organization', BioEssays 48(2):e70114 (2026), DOI 10.1002/bies.70114, https://doi.org/10.1002/bies.70114
  17. Thais A. Enoki and Frederick A. Heberle, 'Experimentally determined leaflet-leaflet phase diagram of an asymmetric lipid bilayer', PNAS 120(46):e2308723120 (2023), DOI 10.1073/pnas.2308723120, https://doi.org/10.1073/pnas.2308723120
  18. Gil-Suk Yang, Alice Wagenknecht-Wiesner, Boyu Yin, Pavana Suresh, Erwin London, Barbara A. Baird, Nirmalya Bag, 'Lipid-driven interleaflet coupling of plasma membrane order regulates FcεRI signaling in mast cells', Biophysical Journal 123(15):2256-2270 (2024), DOI 10.1016/j.bpj.2023.07.027, https://doi.org/10.1016/j.bpj.2023.07.027
  19. Jinyu Chen, Yu Cao, D. Peter Tieleman, Qing Liang, 'Cooperative Regulation of Membrane Domain Registration by Lipid Headgroup Properties and Cholesterol Dynamics', bioRxiv preprint (2026), DOI 10.64898/2026.01.16.699701, https://doi.org/10.64898/2026.01.16.699701
  20. Akshara Sharma, Aniruddha Seal, Sahithya S. Iyer, Anand Srivastava, 'Enthalpic and entropic contributions to interleaflet coupling drive domain registration and antiregistration in biological membrane', Physical Review E 105:044408 (2022), DOI 10.1103/physreve.105.044408, https://doi.org/10.1103/PhysRevE.105.044408
  21. Milka Doktorova, Jessica L. Symons, Ilya Levental, 'Structural and functional consequences of reversible lipid asymmetry in living membranes', Nature Chemical Biology 16:1321-1330 (2020), DOI 10.1038/s41589-020-00688-0, https://doi.org/10.1038/s41589-020-00688-0
  22. Frederick A. Heberle and Milka Doktorova, 'Exploring the sensitivities of experimental techniques to various types of membrane asymmetry using atomistic simulations', Faraday Discussions 259:300-320 (2025), DOI 10.1039/d4fd00200h, https://doi.org/10.1039/d4fd00200h
  23. Emily H. Chaisson, Frederick A. Heberle, Milka Doktorova, 'Quantifying Acyl Chain Interdigitation in Simulated Bilayers via Direct Transbilayer Interactions', Journal of Chemical Information and Modeling 65(8):3879-3885 (2025), DOI 10.1021/acs.jcim.4c02287, https://doi.org/10.1021/acs.jcim.4c02287
  24. Paulina Piller, Enrico F. Semeraro, Gerald N. Rechberger, Sandro Keller, Georg Pabst, 'Allosteric modulation of integral protein activity by differential stress in asymmetric membranes', PNAS Nexus 2(5):pgad126 (2023), DOI 10.1093/pnasnexus/pgad126, https://doi.org/10.1093/pnasnexus/pgad126