Elucidating membrane structure and protein behavior using giant plasma membrane vesicles

Abstract

The observation of phase separation in intact plasma membranes isolated from live cells is a breakthrough for research into eukaryotic membrane lateral heterogeneity, specifically in the context of membrane rafts. These observations are made in giant plasma membrane vesicles (GPMVs), which can be isolated by chemical vesiculants from a variety of cell types and microscopically observed using basic reagents and equipment available in any cell biology laboratory. Microscopic phase separation is detectable by fluorescent labeling, followed by cooling of the membranes below their miscibility phase transition temperature. This protocol describes the methods to prepare and isolate the vesicles, equipment to observe them under temperature-controlled conditions and three examples of fluorescence analysis: (i) fluorescence spectroscopy with an environment-sensitive dye (laurdan); (ii) two-photon microscopy of the same dye; and (iii) quantitative confocal microscopy to determine component partitioning between raft and nonraft phases. GPMV preparation and isolation, including fluorescent labeling and observation, can be accomplished within 4 h.

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Figure 1: Overview of the protocol.
Figure 2: GPMV visualization.
Figure 3: C-laurdan characterization of GPMVs.
Figure 4: Temperature-controlled imaging.
Figure 5: Quantification of component partitioning.

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Acknowledgements

The work was supported by the Max Planck Society; Humboldt Foundation Postdoctoral Fellowship, Technical University of Dresden; Deutsche Forschung Gemeinschaft (DFG) 'Schwerpunktprogramm1175' grant SI459/2-1, 'Transregio 83' grant TRR83 TP02; European Science Foundation 'LIPIDPROD' grant SI459/3-1; Bundesministerium für Bildung und Forschung 'ForMaT' grant 03FO1212; US National Institutes of Health grant R21AI073409; and the Klaus Tschira Foundation.

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Contributions

E.S. and I.L. gathered the data; E.S. and H.J.K. designed the laurdan experiments; I.L. and T.B. designed GPMV isolation, microscopy and partitioning protocols; and E.S., P.S., K.S. and I.L. wrote the manuscript.

Corresponding author

Correspondence to Ilya Levental.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Video 1

GPMV formation. Shown is the initial 1 h of GPMV formation as in Step 10. (MP4 979 kb)

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Sezgin, E., Kaiser, H., Baumgart, T. et al. Elucidating membrane structure and protein behavior using giant plasma membrane vesicles. Nat Protoc 7, 1042–1051 (2012). https://doi.org/10.1038/nprot.2012.059

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