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Direct observation of the nanoscale dynamics of membrane lipids in a living cell

Nature volume 457, pages 1159–1162 (2009)Cite this article

Abstract

Cholesterol-mediated lipid interactions are thought to have a functional role in many membrane-associated processes such as signalling events1,2,3,4,5. Although several experiments indicate their existence, lipid nanodomains (‘rafts’) remain controversial owing to the lack of suitable detection techniques in living cells4,6,7,8,9. The controversy is reflected in their putative size of 5–200 nm, spanning the range between the extent of a protein complex and the resolution limit of optical microscopy. Here we demonstrate the ability of stimulated emission depletion (STED) far-field fluorescence nanoscopy10 to detect single diffusing (lipid) molecules in nanosized areas in the plasma membrane of living cells. Tuning of the probed area to spot sizes ∼70-fold below the diffraction barrier reveals that unlike phosphoglycerolipids, sphingolipids and glycosylphosphatidylinositol-anchored proteins are transiently (∼10–20 ms) trapped in cholesterol-mediated molecular complexes dwelling within <20-nm diameter areas. The non-invasive optical recording of molecular time traces and fluctuation data in tunable nanoscale domains is a powerful new approach to study the dynamics of biomolecules in living cells.

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Figure 1: STED microscopy time traces of single-molecule diffusion in live cell plasma membrane.
Figure 2: FCS of Atto647N-labelled phosphoethanolamine and sphingomyelin plasma membrane diffusion.
Figure 3: Molecular transit through nanoscale areas in live cell plasma membrane.

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Acknowledgements

We thank J. Jethwa, B. Rankin and M. Hilbert for critical reading, K. Willig for help with the setup, T. Lang, R. Wagner and H. Rigneault for valuable discussions, R. Machinek and H. Frauendorf for recording the NMR and mass spectra, and S. Yan for help with the synthesis.

Author Contributions C.R. and B.H. performed experiments, C.E. and C.R. analysed data, R.M. prepared samples and performed washing experiments, G.S., K.S., S.P. and V.N.B. synthesized fluorescently labelled lipids and performed chromatography, C.v.M., A.S., C.R. and C.E. realized and analysed simulated data, C.E. and S.W.H. designed experiments and wrote the paper. All authors discussed the results and commented on the manuscript.

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  1. Christian Eggeling and Christian Ringemann: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Nanobiophotonics, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany,

    Christian Eggeling, Christian Ringemann, Rebecca Medda, Svetlana Polyakova, Vladimir N. Belov, Birka Hein, Claas von Middendorff, Andreas Schönle & Stefan W. Hell

  2. LIMES Membrane Biology and Lipid Biochemistry Unit, University of Bonn, Gerhard-Domagk-Strasse 1, 53121 Bonn, Germany ,

    Günter Schwarzmann & Konrad Sandhoff

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  1. Christian Eggeling
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Correspondence to Christian Eggeling or Stefan W. Hell.

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Eggeling, C., Ringemann, C., Medda, R. et al. Direct observation of the nanoscale dynamics of membrane lipids in a living cell. Nature 457, 1159–1162 (2009). https://doi.org/10.1038/nature07596

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