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Capturing suboptical dynamic structures in lipid bilayer patches formed from free-standing giant unilamellar vesicles

Nature Protocols volume 12, pages 15631575 (2017) | Download Citation

Abstract

There is accumulating evidence that the small-scale lateral organization of biological membranes has a crucial role in signaling and trafficking in cells. However, it has been difficult to characterize these features with existing methods for preparing and analyzing freestanding membranes, because the dynamics occurs below the optical resolution possible with these protocols. We have developed a protocol that permits the imaging of lipid nanodomains and lateral protein organization in membranes of giant unilamellar vesicles (GUVs). Freestanding GUVs are transferred onto a mica support, and after treatment with magnesium chloride, they collapse to form planar lipid bilayer (PLB) patches. Rapid GUV collapse onto the mica preserves the lateral organization of freestanding membranes and thus makes it possible to image 'snapshots' of GUVs up to nanometer resolution by high-resolution microscopy. The method has been applied to classical lipid raft mixtures in which suboptical domain fluctuations have been imaged in both the liquid-ordered and liquid-disordered membrane phases. High-resolution scanning by atomic force microscopy (AFM) of membranes composed of binary and ternary lipid mixtures reconstituted with Na+/K+-ATPase (NKA) has revealed the spatial distribution and orientations of individual proteins, as well as details of membrane lateral structure. Immunolabeling followed by confocal microscopy can also provide information about the spatial distribution of proteins. The protocol opens up a new avenue for quantitative biophysical studies of suboptical dynamic structures in biomembranes, which are local and short-lived. Preparation of GUVs, PLB patches and their imaging takes <24 h.

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Acknowledgements

We thank DAMBIC (Danish Molecular Bioimaging Center) for access to equipment. This work was supported by the Danish Council for Independent Research–Natural Sciences (FNU; grant 95-305-23443 to J.H.I. and F.C.), the Human Frontier Science Program (HFSP; grant 95-305-73458 to J.H.I.) and the Novo Nordisk Foundation (grant DFF-418300011 to F.C.), T.B. acknowledges O.G. Mouritsen (SDU), A.C. Simonsen (SDU), P.L. Hansen (SDU), L.A. Bagatolli (SDU), J. Brewer (SDU), L. Duelund (SDU), B. Franchi (AU) and H. Kidmose (AU) for useful discussions.

Author information

Affiliations

  1. MEMPHYS Center for Biomembrane Physics and Department of Physics, Chemistry, and Pharmacy, University of Southern Denmark, Odense, Denmark.

    • Tripta Bhatia
    •  & John H Ipsen
  2. Max Planck Institute of Colloids and Interfaces, Potsdam, Germany.

    • Tripta Bhatia
  3. Department of Biomedicine, Aarhus University, Aarhus, Denmark.

    • Flemming Cornelius

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Contributions

T.B. performed the research and analyzed the data; designed the vesicle collapse method and developed it through many useful discussions with J.H.I.; and developed the SUV-mixing method together with J.H.I. F.C. prepared and characterized proteoliposomes for their functional and structural properties; prepared the primary antibodies for NKA; and discussed the protein-reconstitution protocol, antibody-labeling experiments and NKA data with T.B. J.H.I. planned the experiments with T.B. and contributed to data interpretation. T.B. and J.H.I. wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Tripta Bhatia.

Supplementary information

Videos

  1. 1.

    Time sequences showing GUV collapse after the addition of MgCl2 salt

    The starting frame (t = 0 s) shows GUVs of ternary lipid mixture DOPC-DPPC-chol (30-50-20)% sitting on mica substrate displaying macroscopic ld (bright) and lo (dark) domains. There is no MgCl2 salt present in the fluid chamber. GUV collapse starts at t 3.820 s after the addition of MgCl2 salt, and planar lipid bilayer patches are formed that retain macroscopic ld (bright) and lo (dark) domains. GUVs contain the membrane probe RhPE, which preferentially partitions into the ld phase and is imaged as a bright region in GUVs and patches. GUVs were imaged in an epifluorescence microscope equipped with an EMCCD camera. Scale bar, 10 μm. Reproduced with permission from ref. 21.

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https://doi.org/10.1038/nprot.2017.047

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