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Cell-sized asymmetric lipid vesicles facilitate the investigation of asymmetric membranes

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Abstract

Asymmetric lipid giant vesicles have been used to model the biochemical reactions in cell membranes. However, methods for producing asymmetric giant vesicles lead to the inclusion of an organic solvent layer that affects the mechanical and physical characteristics of the membrane. Here we describe the formation of asymmetric giant vesicles that include little organic solvent, and use them to investigate the dynamic responses of lipid molecules in the vesicle membrane. We formed the giant vesicles via the inhomogeneous break-up of a lipid microtube generated by applying a jet flow to an asymmetric planar lipid bilayer. The asymmetric giant vesicles showed a lipid flip-flop behaviour in the membrane, superficially similar to the lipid flip-flop activity observed in apoptotic cells. In vitro synthesis of membrane proteins into the asymmetric giant vesicles revealed that the lipid asymmetry in bilayer membranes improves the reconstitution ratio of membrane proteins. Our asymmetric giant vesicles will be useful in elucidating lipid–lipid and lipid–membrane protein interactions involved in the regulation of cellular functions.

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Figure 1: Schematic representation of asymmetric GV formation from a planar lipid bilayer using pulsed microfluidic jet flow.
Figure 2: Formation of GVs via inhomogeneous break-up of a lipid microtube by a jet flow.
Figure 3: Characterization of GV membranes.
Figure 4: Formation and flip-flop measurement of DOPS asymmetric GVs.
Figure 5: Membrane dynamics of asymmetric GVs with the outer leaflet composed of pure DOPC and the inner leaflet composed of DOPS/DOPE/DOPC in the presence of cinnamycin.
Figure 6: Reconstituted amount and function of membrane proteins into asymmetric GVs.

Change history

  • 22 June 2016

    In the version of this Article originally published, in Fig. 1 some of the lipid heads in the bottom right panel were mistakenly coloured grey and in Fig. 5b the red arrow labelling ‘flop’ in the right panel was out of place. These errors have been corrected in all versions of the Article.

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Acknowledgements

We thank Y. Hasegawa for advising us on the simulation of the GV-formation process. We also thank M. Uchida and Y. Nozaki for technical support, K. Okumura and M. Takinoue for providing useful discussion, A. Y. Hsiao for useful comments on the manuscript and A. Sato for the artwork for Fig. 1. This work was partly supported by Grant-in-Aid for Research Activity Start-up No. 23850022 (K.K.), Challenging Exploratory Research No. 25620138 (K.K.), Young Scientists (A) No. 15H05493 (K.K.) and Scientific Research (A) No. 25246017 (S.T.) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) Japan, by Platform for Dynamic Approaches to Living System (MEXT) Japan, by the Regional Innovation Strategy Support Program (MEXT) Japan and by PRESTO (Design and Control of Cellular Functions) (K.K.) of Japan Science of Technology (JST).

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K.K. and S.T. designed the research; K.K. performed the research and analysed data; K.K., K.A. and S.T. designed the electrophysiological measurement of a single Cx43-eGFP channel current; R.K. and T.O. contributed to the device concept and fabrication and K.K. and S.T. co-wrote the paper. All the authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Shoji Takeuchi.

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The authors declare no competing financial interests.

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Kamiya, K., Kawano, R., Osaki, T. et al. Cell-sized asymmetric lipid vesicles facilitate the investigation of asymmetric membranes. Nature Chem 8, 881–889 (2016). https://doi.org/10.1038/nchem.2537

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