Skip to main content
SpringerLink
Log in

Plasma membranes are asymmetric in lipid unsaturation, packing and protein shape

  • Article
  • Published:

From Nature Chemical Biology

View current issue Submit your manuscript

An Author Correction to this article was published on 15 May 2020

This article has been updated

Abstract

A fundamental feature of cellular plasma membranes (PMs) is an asymmetric lipid distribution between the bilayer leaflets. However, neither the detailed, comprehensive compositions of individual PM leaflets nor how these contribute to structural membrane asymmetries have been defined. We report the distinct lipidomes and biophysical properties of both monolayers in living mammalian PMs. Phospholipid unsaturation is dramatically asymmetric, with the cytoplasmic leaflet being approximately twofold more unsaturated than the exoplasmic leaflet. Atomistic simulations and spectroscopy of leaflet-selective fluorescent probes reveal that the outer PM leaflet is more packed and less diffusive than the inner leaflet, with this biophysical asymmetry maintained in the endocytic system. The structural asymmetry of the PM is reflected in the asymmetric structures of protein transmembrane domains. These structural asymmetries are conserved throughout Eukaryota, suggesting fundamental cellular design principles.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1: Lipidomic asymmetry of erythrocyte plasma membranes.
Fig. 2: Atomistic simulation of the biophysical asymmetry of erythrocyte plasma membrane.
Fig. 3: Biophysical asymmetry of the plasma membrane.
Fig. 4: Asymmetry of membrane packing through the endocytic pathway.
Fig. 5: Structural asymmetry in plasma membrane protein transmembrane domains is related to subcellular localization.

Similar content being viewed by others

Data availability

All data generated or analyzed during this study are included in this published article (and its Supplementary Information files) or are available from the corresponding author on reasonable request.

Change history

References

  1. Devaux, P. F. Static and dynamic lipid asymmetry in cell membranes. Biochemistry 30, 1163–1173 (1991).

    CAS  PubMed  Google Scholar 

  2. Op den Kamp, J. A. Lipid asymmetry in membranes. Annu. Rev. Biochem. 48, 47–71 (1979).

    CAS  PubMed  Google Scholar 

  3. Verkleij, A. J. et al. The asymmetric distribution of phospholipids in the human red cell membrane. A combined study using phospholipases and freeze-etch electron microscopy. Biochim. Biophys. Acta 323, 178–193 (1973).

    CAS  PubMed  Google Scholar 

  4. Schick, P. K., Kurica, K. B. & Chacko, G. K. Location of phosphatidylethanolamine and phosphatidylserine in the human platelet plasma membrane. J. Clin. Investig. 57, 1221–1226 (1976).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Sandra, A. & Pagano, R. E. Phospholipid asymmetry in LM cell plasma membrane derivatives: polar head group and acyl chain distributions. Biochemistry 17, 332–338 (1978).

    CAS  PubMed  Google Scholar 

  6. Bollen, I. C. & Higgins, J. A. Phospholipid asymmetry in rough- and smooth-endoplasmic-reticulum membranes of untreated and phenobarbital-treated rat liver. Biochem. J. 189, 475–480 (1980).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Lin, Q. & London, E. The influence of natural lipid asymmetry upon the conformation of a membrane-inserted protein (perfringolysin O). J. Biol. Chem. 289, 5467–5478 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Doktorova, M. et al. Preparation of asymmetric phospholipid vesicles for use as cell membrane models. Nat. Protoc. 13, 2086–2101 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Enoki, T. A. & Feigenson, G. W. Asymmetric bilayers by hemifusion: method and leaflet behaviors. Biophys. J. 117, 1037–1050 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Chiantia, S. & London, E. Acyl chain length and saturation modulate interleaflet coupling in asymmetric bilayers: effects on dynamics and structural order. Biophys. J. 103, 2311–2319 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Heberle, F. A. et al. Subnanometer structure of an asymmetric model membrane: interleaflet coupling influences domain properties. Langmuir 32, 5195–5200 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Cheng, H. T. & Megha, London,E. Preparation and properties of asymmetric vesicles that mimic cell membranes: effect upon lipid raft formation and transmembrane helix orientation. J. Biol. Chem. 284, 6079–6092 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Doktorova, M. et al. Gramicidin increases lipid flip-flop in symmetric and asymmetric lipid vesicles. Biophys. J. 116, 860–873 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Sharpe, H. J., Stevens, T. J. & Munro, S. A comprehensive comparison of transmembrane domains reveals organelle-specific properties. Cell 142, 158–169 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Morrot, G. et al. Asymmetric lateral mobility of phospholipids in the human erythrocyte membrane. Proc. Natl Acad. Sci. USA 83, 6863–6867 (1986).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. el Hage Chahine, J. M., Cribier, S. & Devaux, P. F. Phospholipid transmembrane domains and lateral diffusion in fibroblasts. Proc. Natl Acad. Sci. USA 90, 447–451 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Tanaka, K. I. & Ohnishi, S. Heterogeneity in the fluidity of intact erythrocyte membrane and its homogenization upon hemolysis. Biochim. Biophys. Acta 426, 218–231 (1976).

    CAS  PubMed  Google Scholar 

  18. Gupta, A., Korte, T., Herrmann, A. & Wohland, T. Plasma membrane asymmetry of lipid organization: fluorescence lifetime microscopy and correlation spectroscopy analysis. J. Lipid Res. 61, 252–266 (2020).

    CAS  PubMed  Google Scholar 

  19. Schroeder, F. Differences in fluidity between bilayer halves of tumour cell plasma membranes. Nature 276, 528–530 (1978).

    CAS  PubMed  Google Scholar 

  20. Schachter, D., Abbott, R. E., Cogan, U. & Flamm, M. Lipid fluidity of the individual hemileaflets of human erythrocyte membranes. Ann. N. Y. Acad. Sci. 414, 19–28 (1983).

    CAS  PubMed  Google Scholar 

  21. Rimon, G., Meyerstein, N. & Henis, Y. I. Lateral mobility of phospholipids in the external and internal leaflets of normal and hereditary spherocytic human erythrocytes. Biochim. Biophys. Acta 775, 283–290 (1984).

    CAS  PubMed  Google Scholar 

  22. Harayama, T. et al. Lysophospholipid acyltransferases mediate phosphatidylcholine diversification to achieve the physical properties required in vivo. Cell Metab. 20, 295–305 (2014).

    CAS  PubMed  Google Scholar 

  23. Yeung, T. et al. Membrane phosphatidylserine regulates surface charge and protein localization. Science 319, 210–213 (2008).

    CAS  PubMed  Google Scholar 

  24. Levental, I., Janmey, P. A. & Cebers, A. Electrostatic contribution to the surface pressure of charged monolayers containing polyphosphoinositides. Biophys. J. 95, 1199–1205 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Sodt, A. J., Venable, R. M., Lyman, E. & Pastor, R. W. Nonadditive compositional curvature energetics of lipid bilayers. Phys. Rev. Lett. 117, 138104 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Ingolfsson, H. I. et al. Lipid organization of the plasma membrane. J. Am. Chem. Soc. 136, 14554–14559 (2014).

    CAS  PubMed  Google Scholar 

  27. Cui, H., Lyman, E. & Voth, G. A. Mechanism of membrane curvature sensing by amphipathic helix containing proteins. Biophys. J. 100, 1271–1279 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Jin, L. et al. Characterization and application of a new optical probe for membrane lipid domains. Biophys. J. 90, 2563–2575 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Owen, D. M. et al. Fluorescence lifetime imaging provides enhanced contrast when imaging the phase-sensitive dye di-4-ANEPPDHQ in model membranes and live cells. Biophys. J. 90, L80–L82 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Sezgin, E., Sadowski, T. & Simons, K. Measuring lipid packing of model and cellular membranes with environment sensitive probes. Langmuir 30, 8160–8166 (2014).

    CAS  PubMed  Google Scholar 

  31. Wesseling, M. C. et al. Novel insights in the regulation of phosphatidylserine exposure in human red blood cells. Cell Physiol. Biochem. 39, 1941–1954 (2016).

    CAS  PubMed  Google Scholar 

  32. Diaz-Rohrer, B. B., Levental, K. R., Simons, K. & Levental, I. Membrane raft association is a determinant of plasma membrane localization. Proc. Natl Acad. Sci. USA 111, 8500–8505 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Lorent, J. H. et al. Structural determinants and functional consequences of protein affinity for membrane rafts. Nat. Commun. 8, 1219 (2017).

    PubMed  PubMed Central  Google Scholar 

  34. Kobayashi, T. & Menon, A. K. Transbilayer lipid asymmetry. Curr. Biol. 28, R386–R391 (2018).

    CAS  PubMed  Google Scholar 

  35. Yamakawa, T. & Nagai, Y. Glycolipids at the cell surface and their biological functions. Trends Biochem. Sci. 3, 128–131 (1978).

    CAS  Google Scholar 

  36. Levental, I., Cebers, A. & Janmey, P. A. Combined electrostatics and hydrogen bonding determine intermolecular interactions between polyphosphoinositides. J. Am. Chem. Soc. 130, 9025–9030 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Steck, T. L. & Lange, Y. Transverse distribution of plasma membrane bilayer cholesterol: picking sides. Traffic 19, 750–760 (2018).

    CAS  PubMed  Google Scholar 

  38. Courtney, K. C. et al. C24 sphingolipids govern the transbilayer asymmetry of cholesterol and lateral organization of model and live-cell plasma membranes. Cell Rep. 24, 1037–1049 (2018).

    CAS  PubMed  Google Scholar 

  39. Liu, S. L. et al. Orthogonal lipid sensors identify transbilayer asymmetry of plasma membrane cholesterol. Nat. Chem. Biol. 13, 268–274 (2017).

    CAS  PubMed  Google Scholar 

  40. Steck, T. L. & Lange, Y. How slow is the transbilayer diffusion (flip-flop) of cholesterol? Biophys. J. 102, 945–946 (2012); author reply 102, 947–949.

  41. Iaea, D. B. & Maxfield, F. R. Membrane order in the plasma membrane and endocytic recycling compartment. PLoS ONE 12, e0188041 (2017).

    PubMed  PubMed Central  Google Scholar 

  42. Levental, I., Levental, K. R. & Heberle, F. A. Lipid rafts: controversies resolved, mysteries remain. Trends Cell Biol. 30, 341–353 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Lin, Q. & London, E. Ordered raft domains induced by outer leaflet sphingomyelin in cholesterol-rich asymmetric vesicles. Biophys. J. 108, 2212–2222 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Kiessling, V., Crane, J. M. & Tamm, L. K. Transbilayer effects of raft-like lipid domains in asymmetric planar bilayers measured by single molecule tracking. Biophys. J. 91, 3313–3326 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Veatch, S. L. & Keller, S. L. Miscibility phase diagrams of giant vesicles containing sphingomyelin. Phys. Rev. Lett. 94, 148101 (2005).

    PubMed  Google Scholar 

  46. Kusumi, A. et al. Dynamic organizing principles of the plasma membrane that regulate signal transduction: commemorating the fortieth anniversary of Singer and Nicolson’s fluid-mosaic model. Annu. Rev. Cell Dev. Biol. 28, 215–250 (2012).

    CAS  PubMed  Google Scholar 

  47. Honigmann, A. et al. A lipid bound actin meshwork organizes liquid phase separation in model membranes. eLife 3, e01671 (2014).

    PubMed  PubMed Central  Google Scholar 

  48. Levental, K. R. et al. Homeostatic remodeling of mammalian membranes in response to dietary lipids is essential for cellular fitness. Nat. Commun. 11, 1339 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Hussain, N. F., Siegel, A. P., Ge, Y., Jordan, R. & Naumann, C. A. Bilayer asymmetry influences integrin sequestering in raft-mimicking lipid mixtures. Biophys. J. 104, 2212–2221 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Frewein, M., Kollmitzer, B., Heftberger, P. & Pabst, G. Lateral pressure-mediated protein partitioning into liquid-ordered/liquid-disordered domains. Soft Matter 12, 3189–3195 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. McIntyre, J. C. & Sleight, R. G. Fluorescence assay for phospholipid membrane asymmetry. Biochemistry 30, 11819–11827 (1991).

    CAS  PubMed  Google Scholar 

  52. Levental, K. R. et al. Omega-3 polyunsaturated fatty acids direct differentiation of the membrane phenotype in mesenchymal stem cells to potentiate osteogenesis. Sci. Adv. 3, eaao1193 (2017).

    PubMed  PubMed Central  Google Scholar 

  53. Surma, M. A. et al. An automated shotgun lipidomics platform for high throughput, comprehensive and quantitative analysis of blood plasma intact lipids. Eur. J. Lipid Sci. Technol. 117, 1540–1549 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Wu, E. L. et al. CHARMM-GUI Membrane Builder toward realistic biological membrane simulations. J. Comput. Chem. 35, 1997–2004 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Venable, RichardM. et al. CHARMM all-atom additive force field for sphingomyelin: elucidation of hydrogen bonding and of positive curvature. Biophys. J. 107, 134–145 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Shan, Y., Klepeis, J. L., Eastwood, M. P., Dror, R. O. & Shaw, D. E. Gaussian split Ewald: a fast Ewald mesh method for molecular simulation. J. Chem. Phys. 122, 054101 (2005).

    Google Scholar 

  57. Camley, B. A., Lerner, M. G., Pastor, R. W. & Brown, F. L. H. Strong influence of periodic boundary conditions on lateral diffusion in lipid bilayer membranes. J. Chem. Phys. 143, 243113 (2015).

    PubMed  PubMed Central  Google Scholar 

  58. Li, Q., Wang, X., Ma, S., Zhang, Y. & Han, X. Electroformation of giant unilamellar vesicles in saline solution. Colloids Surf. B Biointerfaces 147, 368–375 (2016).

    CAS  PubMed  Google Scholar 

  59. Steinkuhler, J., De Tillieux, P., Knorr, R. L., Lipowsky, R. & Dimova, R. Charged giant unilamellar vesicles prepared by electroformation exhibit nanotubes and transbilayer lipid asymmetry. Sci. Rep. 8, 11838 (2018).

    PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

All fluorescence microscopy was performed at the Center for Advanced Microscopy, Department of Integrative Biology & Pharmacology at McGovern Medical School, UTHealth. We thank N. Waxham for his generous sharing of the microinjection system. We acknowledge K. Simons, T. Steck, Y. Lange and G. Feigenson for their critical feedback on this manuscript. Funding for this work was provided by the NIH/National Institute of General Medical Sciences (GM114282, GM124072, GM120351 and GM134949), the Volkswagen Foundation (grant no. 93091) and the Human Frontiers Science Program (RGP0059/2019). E.S. is funded by Newton-Katip Ҫelebi Institutional Links grant no. 352333122. Anton2 computer time was provided by the National Resource for Biomedical Supercomputing (NRBSC), the Pittsburgh Supercomputing Center (PSC) and the Biomedical Technology Research Center for Multiscale Modeling of Biological Systems through grant no. P41GM103712-S1 from the National Institutes of Health.

Author information

Author notes

  1. E. Sezgin

    Present address: SciLifeLab, Karolinska Institute, Stockholm, Sweden

Authors and Affiliations

  1. McGovern Medical School, Department of Integrative Biology and Pharmacology, University of Texas Health Science Center at Houston, Houston, TX, USA

    J. H. Lorent, K. R. Levental, L. Ganesan, M. Doktorova & I. Levental

  2. Biological Sciences Department, Columbia University, New York, NY, USA

    G. Rivera-Longsworth

  3. John Radcliffe Hospital, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK

    E. Sezgin

  4. Department of Physics and Astronomy and Department of Chemistry and Biochemistry, University of Delaware, Newark, DE, USA

    E. Lyman

Authors
  1. J. H. Lorent
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. K. R. Levental
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. L. Ganesan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. G. Rivera-Longsworth
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. E. Sezgin
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. M. Doktorova
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. E. Lyman
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. I. Levental
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.H.L., I.L., E.L. and K.R.L. designed the study. J.H.L., K.R.L., L.G., G.R.-L., M.D. and E.S. performed experiments. E.L. performed and analyzed the molecular dynamics simulations. J.H.L. carried out the bioinformatics analysis. J.H.L., K.R.L. and I.L. analyzed the experimental results and wrote the paper.

Corresponding author

Correspondence to I. Levental.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Tables 1 and 2 and Supplementary Figs. 1–15.

Reporting Summary

Supplementary Dataset

Asymmetric lipidomes.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Lorent, J.H., Levental, K.R., Ganesan, L. et al. Plasma membranes are asymmetric in lipid unsaturation, packing and protein shape. Nat Chem Biol 16, 644–652 (2020). https://doi.org/10.1038/s41589-020-0529-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41589-020-0529-6

  • Springer Nature America, Inc.

This article is cited by

Search

Navigation