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Statistical Analysis of High-Resolution Light Microscope Images Reveals Effects of Cytoskeleton-Disrupting Drugs on the Membrane Organization of the Nicotinic Acetylcholine Receptor

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Abstract

Extensive evidence supports the notion that the cytoskeleton participates in the immobilization and membrane clustering of the nicotinic acetylcholine receptor (AChR) at the neuromuscular junction. Stimulated emission depletion fluorescence microscopy has revealed the supramolecular organization of AChR nanoclusters at the surface of CHO-K1/A5 cells with subdiffraction resolution (Kellner et al., Neuroscience 144:135–143 2007). We studied the effect of two cytoskeletal-disrupting drugs (cytochalasin D and jasplakinolide) on the nanoscale distribution of muscle-type AChR expressed in these cells by means of mathematical and statistical analysis of images obtained with the same high-resolution microscopy. AChR nanoclusters were found to be randomly distributed in both controls and cells treated with either drug for distances larger than 500 nm. Treatments altered the distribution of AChR nanoclusters according to their brightness/size. Cytochalasin D and jasplakinolide produced a statistically significant increase in the proportion of medium-size nanoclusters and a diminution of small nanoclusters, indicating higher disrupting activity on the latter. This was further corroborated by the diminution of the brightness/diameter ratio of nanoclusters (a measure of the intracluster density of AChR molecules) and by Ripley’s analysis applied to simulated patterns with intracluster aggregation of AChR molecules. The combined analytical tools bring out subtle changes in the two-dimensional organization of the AChR nanoaggregates on disruption of the cytoskeletal network and throw light on the possible link between the cytoskeleton and the distribution of the AChR at the cell surface.

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Acknowledgments

Thanks are due to Prof. S. H. Hell and his group for the use of the Leica TCS STED microscope at the facility of the Department of Nanobiophotonics, Max-Planck Institute for Biophysical Chemistry, Göttingen. Research described in this article was supported in part by PICT 01-12790 and 5-20155 from the Ministry of Science and Technology of Argentina; PIP no. 6367 from the Argentinian Scientific Research Council (CONICET); Philip Morris USA Inc., and Philip Morris International to F.J.B.

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  1. Instituto de Investigaciones Bioquímicas de Bahía Blanca and UNESCO Chair of Biophysics and Molecular Neurobiology, C.C. 857, F8000FWB, Bahía Blanca, Argentina

    Jorge J. Wenz, Virginia Borroni & Francisco J. Barrantes

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  1. Jorge J. Wenz
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  2. Virginia Borroni
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  3. Francisco J. Barrantes
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Correspondence to Jorge J. Wenz.

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Wenz, J.J., Borroni, V. & Barrantes, F.J. Statistical Analysis of High-Resolution Light Microscope Images Reveals Effects of Cytoskeleton-Disrupting Drugs on the Membrane Organization of the Nicotinic Acetylcholine Receptor. J Membrane Biol 235, 163–175 (2010). https://doi.org/10.1007/s00232-010-9261-6

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  • DOI: https://doi.org/10.1007/s00232-010-9261-6

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