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

Bhatia, T., Cornelius, F., & Ipsen, J. H. (2017). Capturing suboptical dynamic structures in lipid bilayer patches formed from free-standing giant unilamellar vesicles. Nature Protocols, 12(8), 1563-1575. doi:10.1038/nprot.2017.047.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-002D-A916-8 Version Permalink: http://hdl.handle.net/11858/00-001M-0000-002D-A917-6
Genre: Journal Article

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Bhatia, Tripta1, Author              
Cornelius, Flemming, Author
Ipsen, John H, Author
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1Rumiana Dimova, Theorie & Bio-Systeme, Max Planck Institute of Colloids and Interfaces, Max Planck Society, ou_1863328              

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 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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 Dates: 2017
 Publication Status: Published in print
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 Identifiers: DOI: 10.1038/nprot.2017.047
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Title: Nature Protocols
  Other : Nat. Protoc.
Source Genre: Journal
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Publ. Info: London, UK : Nature Publishing Group
Pages: - Volume / Issue: 12 (8) Sequence Number: - Start / End Page: 1563 - 1575 Identifier: ISSN: 1750-2799