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

• Published in: Nature protocols Vol. 12; no. 8; pp. 1563 - 1575
• Main Authors: Bhatia, Tripta, Cornelius, Flemming, Ipsen, John H
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.08.2017; Nature Publishing Group
• Subjects: 14/19; 14/3; 14/34; 14/63; 631/1647/2258/1262; 631/1647/350/1056; 631/45/287/1192; 631/80/313; 82/1; Analytical Chemistry; Atomic force microscopy; Atomic structure; Biological membranes; Biological Techniques; Biophysical Phenomena; Chlorides; Collapse; Complications and side effects; Computational Biology/Bioinformatics; Confocal microscopy; Dosage and administration; High resolution; Information dissemination; Life Sciences; Lipid Bilayers - chemistry; Lipid Bilayers - metabolism; Lipid membranes; Lipids; Magnesium; Magnesium chloride; Membranes; Microarrays; Microscopy; Na+/K+-exchanging ATPase; Organic Chemistry; Patches (structures); Phosphatidylethanolamines; Proteins; protocol; Scanning; Spatial distribution; Unilamellar Liposomes - chemistry; Unilamellar Liposomes - metabolism; Vesicles
• ISSN: 1754-2189; 1750-2799
• DOI: 10.1038/nprot.2017.047

Bhatia et al . describe how to prepare giant unilamellar vesicles by using complex lipid mixtures and proteins (Na + /K + -ATPase) at physiological conditions; the protocol includes subsequent imaging of lateral nanoscale structures of the membrane. 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.