Live-Cell Plasma Membrane Organization Studied with Super-Resolution STED Microscopy
Christian Eggeling, Alf Honigmann, Veronika Mueller, Stefan W. Hell, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany

Interactions of lipids and proteins in the plasma membrane of living cells such as the integration into lipid nanodomains are considered to play a functional part in a whole range of cellular processes. Unfortunately, the direct and non-invasive observation of these interactions in living cells is impeded by the resolution limit of >200nm of a conventional far-field optical microscope. We report the detection of membrane heterogeneities in nanosized areas in the plasma membrane of living cells using the superior spatial resolution of stimulated emission depletion (STED) far-field nanoscopy. By combining a (tunable) resolution of down to 30 nm with tools such as fluorescence correlation spectroscopy (FCS), we obtain new details of molecular membrane dynamics. Our first studies revealed how sphingolipids or other proteins are transiently (~ 10 ms) trapped on the nanoscale in molecular complexes, while others diffuse freely or show a kind of hopping diffusion [1-3]. Distinct differences especially with respect to the dependence on cholesterol and the underlying cytoskeleton showed up [4]. We will revisit these results and – besides novel applications of this novel STED-FCS technique - show a comparison of the plasma membrane data to STED experiments on model membranes, which highlight potential influences of the fluorescent tag and give new insights into the existence of lipid nanodomains. Our observations shed new light on the role of lipid-protein interactions and nanodomains for membrane bioactivity.

References:

[1] C. Eggeling et al. Direct observation of the nanoscale dynamics of membrane lipids in a living cell. Nature 457, 1159-1163 (2009).

[2] C. Ringemann et al. Exploring single-molecule dynamics with fluorescence nanoscopy, New J. Physics 11, 103054 (2009).

[3] S.J. Sahl et al. Fast molecular tracking maps nanoscale dynamics of plasma membrane lipids, Proc. Natl. Acad. Soc. USA 107, 6829 – 6834 (2010).

[4] V. Mueller et al. STED nanoscopy reveals molecular details of choleterol- and cytokeleton-modulated lipid interactions in living cells, Biophys. J., 101, 1651-1657 (2011).

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