Eggeling, C. et al. Direct observation of the nanoscale dynamics of membrane lipids in a living cell. Nature 457, 1159-1162

Department of Nanobiophotonics, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.
Nature (Impact Factor: 41.46). 12/2008; 457(7233):1159-62. DOI: 10.1038/nature07596
Source: PubMed


Cholesterol-mediated lipid interactions are thought to have a functional role in many membrane-associated processes such as signalling events. Although several experiments indicate their existence, lipid nanodomains ('rafts') remain controversial owing to the lack of suitable detection techniques in living cells. 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 nanoscopy to detect single diffusing (lipid) molecules in nanosized areas in the plasma membrane of living cells. Tuning of the probed area to spot sizes approximately 70-fold below the diffraction barrier reveals that unlike phosphoglycerolipids, sphingolipids and glycosylphosphatidylinositol-anchored proteins are transiently ( approximately 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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    • "Interestingly, syntaxin-1 molecules are only transiently associated with their nanoclusters and rapidly exchange with freely diffusing syntaxin-1 molecules . Live cell STED imaging also revealed the transient, nanoscale organization of sphingolipids and glycosylphosphatidylinositol (GPI)anchored proteins within a 20 nm cholesterol rich membrane region [53]. These studies support the notion that the PM organization is controlled by dynamic molecular interactions of associated and dissociated states. "
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    • "In fact, the size of the observed domains varies between 4 nm (lipid shell size [6]) to several micrometers in model systems [10]. The size of LRs makes them ungrateful subjects for in vivo studies, although there are several experimental evidences of local clustering of protein and lipid reporters of various size [6], observed also directly in the nanoscale in living cells [11]. Other evidences for nanoscale clustering were gathered with new microscopic techniques, as reviewed in the recent work by Simons and Gerl [12]. "
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    ABSTRACT: Lipid rafts (LRs) are dynamic, sterol- and sphingolipid-enriched nanodomains involved in the regulation of cellular functions and signal transduction, that upon stimuli, via (e.g. association of raft proteins and lipids), may cluster into domains of submicron or micron scale. Up to date, however, lipid raft clusters were observed only under artificially promoted conditions and their formation in vivo has not been confirmed. Using non-destructive approach involving Raman and Atomic Force Microscopy imaging we demonstrated the presence of clustered lipid rafts in endothelium of the aorta of the db/db mice that represent a reliable murine model of type 2 diabetes. The raft clusters in the aorta of diabetic mice were shown to occupy a considerably larger (about 10-fold) area of endothelial cells surface as compared to the control. Observation of pathology-promoted LRs confirms that the cellular increase of lipid content results in clustering of LRs. Clustering of LRs leads to the formation of assemblies with diameters up to 3 micrometers and increased lipid character. This massive clustering of lipid rafts in diabetes may trigger a signaling cascade leading to vascular inflammation.
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    • "Technical progress facilitates today the not so long ago impossible analysis of the subtle changes in lipid composition and of the topographical distribution of individual lipid species in cellular compartments. Probes have been developed to label lipid molecules such as new generation fluorescent tags (Eggeling et al., 2009) or modified toxins with specific lipid binding abilities such as the theta-toxin or lysenin, which bind cholesterol or sphingomyelin, respectively (Abe et al., 2012). These probes together with advanced microscopy techniques that achieve sub-diffraction optical resolution (i.e., near-field scanning optical microscopy (NSOM), photoactivated localization microscopy (PALM) stochastic optical reconstruction microscopy (STORM) or stimulated depletion (STED) fluorescent microscopy) allow the direct observation of the nanoscale dynamics of membrane lipids in a living cell (Eggeling et al., 2009; van Zanten et al., 2010; Castro et al., 2013). "
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