Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Layer-by-layer cell membrane assembly

Abstract

Eukaryotic subcellular membrane systems, such as the nuclear envelope or endoplasmic reticulum, present a rich array of architecturally and compositionally complex supramolecular targets that are as yet inaccessible. Here we describe layer-by-layer phospholipid membrane assembly on microfluidic droplets, a route to structures with defined compositional asymmetry and lamellarity. Starting with phospholipid-stabilized water-in-oil droplets trapped in a static droplet array, lipid monolayer deposition proceeds as oil/water-phase boundaries pass over the droplets. Unilamellar vesicles assembled layer-by-layer support functional insertion both of purified and of in situ expressed membrane proteins. Synthesis and chemical probing of asymmetric unilamellar and double-bilayer vesicles demonstrate the programmability of both membrane lamellarity and lipid-leaflet composition during assembly. The immobilized vesicle arrays are a pragmatic experimental platform for biophysical studies of membranes and their associated proteins, particularly complexes that assemble and function in multilamellar contexts in vivo.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Schematic of LbL assembly.
Figure 2: Vesicle-assembly circuit schematic and imaging.
Figure 3: Membrane protein synthesis and function in LbL vesicles.
Figure 4: Chemical probing of intermediate and final products of double-bilayer assembly.
Figure 5: Quantitative LbL deposition.

Similar content being viewed by others

References

  1. van Meer, G., Voelker, D. R. & Feigenson, G. W. Membrane lipids: where they are and how they behave. Nature Rev. Mol. Cell Biol. 9, 112–124 (2008).

    Article  CAS  Google Scholar 

  2. Rosenbaum, D. M., Rasmussen, S. G. F. & Kobilka, B. K. The structure and function of G-protein-coupled receptors. Nature 459, 356–363 (2009).

    Article  CAS  Google Scholar 

  3. Peter, B. et al. BAR domains as sensors of membrane curvature: the amphiphysin BAR structure. Science 303, 495–499 (2004).

    Article  CAS  Google Scholar 

  4. Hatzakis, N. S. et al. How curved membranes recruit amphipathic helices and protein anchoring motifs. Nature Chem. Biol. 5, 835–841 (2009).

    Article  CAS  Google Scholar 

  5. Liu, A. P. & Fletcher, D. A. Biology under construction: in vitro reconstitution of cellular function. Nature Rev. Mol. Cell Biol. 10, 644–650 (2009).

    Article  CAS  Google Scholar 

  6. Bangham, A. D., Standish, M. M. & Watkins, J. C. Diffusion of univalent ions across lamellae of swollen phospholipids. J. Mol. Biol. 13, 238–252 (1965).

    Article  CAS  Google Scholar 

  7. Jahn, A., Vreeland, W. N., Gaitan, M. & Locascio, L. E. Controlled vesicle self-assembly in microfluidic channels with hydrodynamic focusing. J. Am. Chem. Soc. 126, 2674–2675 (2004).

    Article  CAS  Google Scholar 

  8. Shum, H. C., Lee, D., Yoon, I., Kodger, T. & Weitz, D. A. Double emulsion templated monodisperse phospholipid vesicles. Langmuir 24, 7651–7653 (2008).

    Article  CAS  Google Scholar 

  9. Stachowiak, J. C. et al. Unilamellar vesicle formation and encapsulation by microfluidic jetting. Proc. Natl Acad. Sci. USA 105, 4697–4702 (2008).

    Article  CAS  Google Scholar 

  10. Ota, S., Yoshizawa, S. & Takeuchi, S. Microfluidic formation of monodisperse, cell-sized, and unilamellar vesicles. Angew. Chem. Int. Ed. 48, 6533–6537 (2009).

    Article  CAS  Google Scholar 

  11. Richmond, D. L. et al. Forming giant vesicles with controlled membrane composition, asymmetry, and contents. Proc. Natl Acad. Sci. USA 108, 9431–9436 (2011).

    Article  CAS  Google Scholar 

  12. Hu, P. C., Li, S. & Malmstadt, N. Microfluidic fabrication of asymmetric giant lipid vesicles. ACS Appl. Mater. Interfaces 3, 1434–1440 (2011).

    Article  CAS  Google Scholar 

  13. Pautot, S., Frisken, B. J. & Weitz, D. A. Production of unilamellar vesicles using an inverted emulsion. Langmuir 19, 2870–2879 (2003).

    Article  CAS  Google Scholar 

  14. Pautot, S., Frisken, B. & Weitz, D. Engineering asymmetric vesicles. Proc. Natl Acad. Sci. USA 100, 10718–10721 (2003).

    Article  CAS  Google Scholar 

  15. Noireaux, V. & Libchaber, A. A vesicle bioreactor as a step toward an artificial cell assembly. Proc. Natl Acad. Sci. USA 101, 17669–17674 (2004).

    Article  CAS  Google Scholar 

  16. Matosevic, S. & Paegel, B. M. Stepwise synthesis of giant unilamellar vesicles on a microfluidic assembly line. J. Am. Chem. Soc. 133, 2798–2800 (2011).

    Article  CAS  Google Scholar 

  17. Blodgett, K. Films built by depositing successive monomolecular layers on a solid surface. J. Am. Chem. Soc. 57, 1007–1022 (1935).

    Article  CAS  Google Scholar 

  18. Hase, M., Yamada, A., Hamada, T. & Yoshikawa, K. Transport of a cell-sized phospholipid micro-container across water/oil interface. Chem. Phys. Lett. 426, 441–444 (2006).

    Article  CAS  Google Scholar 

  19. Huebner, A. et al. Static microdroplet arrays: a microfluidic device for droplet trapping, incubation and release for enzymatic and cell-based assays. Lab Chip 9, 692–698 (2009).

    Article  CAS  Google Scholar 

  20. Anna, S., Bontoux, N. & Stone, H. Formation of dispersions using ‘flow focusing’ in microchannels. Appl. Phys. Lett. 82, 364–366 (2003).

    Article  CAS  Google Scholar 

  21. Baret, J-C., Kleinschmidt, F., Harrak El, A., & Griffiths, A. D. Kinetic aspects of emulsion stabilization by surfactants: a microfluidic analysis. Langmuir 25, 6088–6093 (2009).

    Article  CAS  Google Scholar 

  22. Song, L. et al. Structure of staphylococcal alpha-hemolysin, a heptameric transmembrane pore. Science 274, 1859–1866 (1996).

    Article  CAS  Google Scholar 

  23. Shimizu, Y. et al. Cell-free translation reconstituted with purified components. Nature Biotechnol. 19, 751–755 (2001).

    Article  CAS  Google Scholar 

  24. Tawfik, D. S. & Griffiths, A. Man-made cell-like compartments for molecular evolution. Nature Biotechnol. 16, 652–656 (1998).

    Article  CAS  Google Scholar 

  25. Andes-Koback, M. & Keating, C. D. Complete budding and asymmetric division of primitive model cells to produce daughter vesicles with different interior and membrane compositions. J. Am. Chem. Soc. 133, 9545–9555 (2011).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  27. Morris, S. J., Bradley, D. & Blumenthal, R. The use of cobalt ions as a collisional quencher to probe surface charge and stability of fluorescently labeled bilayer vesicles. Biochim. Biophys. Acta 818, 365–372 (1985).

    Article  CAS  Google Scholar 

  28. Heider, E. C., Barhoum, M., Edwards, K., Gericke, K-H. & Harris, J. M. Structural characterization of individual vesicles using fluorescence microscopy. Anal. Chem. 83, 4909–4915 (2011).

    Article  CAS  Google Scholar 

  29. Kantak, C., Beyer, S., Yobas, L., Bansal, T. & Trau, D. A. ‘microfluidic pinball’ for on-chip generation of layer-by-layer polyelectrolyte microcapsules. Lab Chip 11, 1030–1035 (2011).

    Article  CAS  Google Scholar 

  30. Antonin, W., Ellenberg, J. & Dultz, E. Nuclear pore complex assembly through the cell cycle: regulation and membrane organization. FEBS Lett. 582, 2004–2016 (2008).

    Article  CAS  Google Scholar 

  31. Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nature Chem. Biol. 9, 671–675 (2012).

    CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by a National Institutes of Health Pathway to Independence Career Development Award (GM083155) and a National Science Foundation CAREER Award (1255250) to B.M.P.

Author information

Authors and Affiliations

Authors

Contributions

B.M.P. and S.M. conceived and designed the experiments, analysed the resulting data and co-authored the paper. S.M. executed all experimental work.

Corresponding author

Correspondence to Brian M. Paegel.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary information

Supplementary information (PDF 756 kb)

Supplementary movie

Supplementary movie (MP4 23121 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Matosevic, S., Paegel, B. Layer-by-layer cell membrane assembly. Nature Chem 5, 958–963 (2013). https://doi.org/10.1038/nchem.1765

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchem.1765

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing