Ensemble and single particle fluorimetric techniques in concerted action to study the diffusion and aggregation of the glycine receptor α3 isoforms in the cell plasma membrane

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Item Type:	Article
Title:	Ensemble and single particle fluorimetric techniques in concerted action to study the diffusion and aggregation of the glycine receptor α3 isoforms in the cell plasma membrane
Creators Name:	Notelaers, K., Smisdom, N., Rocha, S., Janssen, D., Meier, J.C., Rigo, J.M., Hofkens, J. and Ameloot, M.
Abstract:	The spatio-temporal membrane behavior of glycine receptors (GlyR) is known to be of influence on receptor homeostasis and functionality. In this work, an elaborate fluorimetric strategy was applied to study the GlyR {alpha}3K and L isoforms. Previously established differential clustering, desensitization and synaptic localization of these isoforms implies that membrane behavior is crucial in determining GlyR {alpha}3 physiology. Therefore diffusion and aggregation of homomeric {alpha}3 isoform-containing GlyRs were studied in HEK 293 cells. A unique combination of multiple diffraction-limited ensemble average methods and subdiffraction single particle techniques was used in order to achieve an integrated view of receptor properties. Static measurements of aggregation were performed with image correlation spectroscopy (ICS) and, single particle based, direct stochastic optical reconstruction microscopy (dSTORM). Receptor diffusion was measured by means of raster image correlation spectroscopy (RICS), temporal image correlation spectroscopy (TICS), fluorescence recovery after photobleaching (FRAP) and single particle tracking (SPT). The results show a significant difference in diffusion coefficient and cluster size between the isoforms. This reveals a positive correlation between desensitization and diffusion and disproves the notion that receptor aggregation is a universal mechanism for accelerated desensitization. The difference in diffusion coefficient between the clustering GlyR {alpha}3L and the non-clustering GlyR {alpha}3K cannot be explained by normal diffusion. SPT measurements indicate that the {alpha}3L receptors undergo transient trapping and directed motion, while the GlyR {alpha}3K displays mild hindered diffusion. These findings are suggestive of differential molecular interaction of the isoforms after incorporation in the membrane.
Keywords:	Glycine Receptor, alpha3 Isoforms, Nanoscopy, Single Particle, Ensemble Average, Anomalous Diffusion
Source:	Biochimica et Biophysica Acta - Biomembranes
ISSN:	0005-2736
Publisher:	Elsevier
Volume:	1818
Number:	12
Page Range:	3131-3140
Date:	December 2012
Official Publication:	https://doi.org/10.1016/j.bbamem.2012.08.010
PubMed:	View item in PubMed

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