Journal Article

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

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

2009

Overview

Nanoscale view of cell membrane lipids Cholesterol-mediated lipid interactions, such as nanodomain formation, are considered vital in a cell, but because of the lack of suitable detection techniques, their spatiotemporal range remained highly controversial. Here, Eggeling et al . use subdiffraction-resolution STED (stimulated emission depletion) fluorescence microscopy to detect the diffusion of single lipids or glycosylphosphatidylinositol (GPI)-anchored proteins on the plasma membrane of a living cell. Tuning the probing spot area up to about 70-fold below that of a confocal microscope reveals that unlike phosphoglycerolipids, sphingolipids and GPI-anchored proteins are trapped for about 10 ms in cholesterol-mediated complexes within less than 20 nm space. Optical probing in nanosized areas is a powerful new approach to study biomolecular function. Here, subdiffraction-resolution STED fluorescence microscopy is used to detect the diffusion of single lipids or GPI-anchored proteins on the plasma membrane of a living cell. Tuning the probing spot area ∼70-fold below that of a confocal microscope reveals that unlike phosphoglycerolipids, sphingolipids and GPI-anchored proteins are trapped for ∼10 ms in cholesterol-mediated complexes within <20 nm space. Cholesterol-mediated lipid interactions are thought to have a functional role in many membrane-associated processes such as signalling events 1 , 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 cells 4 , 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 nanoscopy 10 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.

• Publisher: Nature Publishing Group UK,Nature Publishing Group
• Subject: Biological and medical sciences / Cell Line / Cell Membrane - chemistry / Cell Membrane - metabolism / Cell Survival / Cells / Cellular signal transduction / Chemical synthesis / Cholesterol / Cholesterol - analysis / Cholesterol - metabolism / Diffusion / Epithelial Cells - cytology / Ethanolamines - analysis / Ethanolamines - metabolism / Fluorescence / Fundamental and applied biological sciences. Psychology / Glycosylphosphatidylinositols - metabolism / Humanities and Social Sciences / Intermolecular dynamics / Intermolecular phenomena / letter / Light microscopy / Lipids / Membrane lipids / Membrane Lipids - analysis / Membrane Lipids - metabolism / Membranes / Microscopy, Fluorescence - methods / Molecular biophysics / multidisciplinary / Nanotechnology - methods / Physiological aspects / Plasma / Proteins / Science / Science (multidisciplinary) / Sphingomyelins - analysis / Sphingomyelins - metabolism / Time Factors