Nanoscale organization of nicotinic acetylcholine receptors revealed by stimulated emission depletion microscopy

Acetylcholine receptor (AChR) supramolecular aggregates that have hitherto only been accessible to examination by electron microscopy were imaged with stimulated emission depletion (STED) fluorescence microscopy, providing resolution beyond limits of diffraction of classical wide-field or confocal microscopes. We examined a Chinese hamster ovary cell liner CHO-K1/A5, that stably expresses adult murine AChR. Whereas confocal microscopy displays AChR clusters as diffraction-limited dots of ∼200 nm diameter, STED microscopy yields nanoclusters with a peak size distribution of ∼55 nm. Utilizing this resolution, we show that cholesterol depletion by acute (30 min, 37 °C) exposure to methyl-β-cyclodextrin alters the short and long range organization of AChR nanoclusters on the cell surface. In the short range, AChRs form larger nanoclusters, possibly related to the alteration of cholesterol-dependent protein–protein associations. Ripley’s K-test on STED images reveals changes in nanocluster distribution on larger scales (0.5–3.5 μm), which possibly are related to the abolition of cytoskeletal physical barriers preventing the lateral diffusion of AChR nanoclusters.