Type of presentation: Poster

LS-3-P-1626 Dynamics and organization of homomeric alpha3 glycine receptors in the plasma membrane using single particle detection

Notelaers K.1,2, Rocha S.2, Paesen R.1, Smisdom N.1, Jochen M. C.3, Rigo J.1, Hofkens J.2, Ameloot M.1
1Biomedical Research Institute, Hasselt University and School of Life Sciences, transnational University Limburg, Agoralaan building C, 3590 Diepenbeek, Belgium, 2Department of Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200F, B-3001 Heverlee, Belgium, 3RNA Editing and Hyperexcitability Disorders Helmholtz Group, Max Delbrück Center for Molecular Medicine, Robert-Rössle-Strasse 10, 13092 Berlin, Germany
kristof.notelaers@uhasselt.be

The mobility and spatial organization of membrane proteins are important factors in their interaction capabilities and physiological roles. In the case of neurotransmitter receptors, lateral diffusion and local trapping are essential in the regulation of their synaptic localization and functionality. One of these receptors is the strychnine-sensitive glycine receptor (GlyR), one of the main mediators of synaptic inhibition. Currently this research project is focusing on homomeric GlyRs consisting of alpha3 subunits. This subunit, encoded by the GLRA3 gene, exhibits two splice variants (GlyR alpha3 L and K) forming receptors with different clustering and desensitization properties. Single particle techniques are now being used to gather more information on nanoscale properties of these different receptors in the plasma membrane. This includes studying the diffusion in living cells using single particle tracking (SPT) and investigating the spatial distribution in the membrane of fixed cells using direct stochastic optical reconstruction microscopy (dSTORM) [1]. GlyR trajectories measured by SPT are analyzed for the detection of anomalous diffusion. Both confined and directed motion are revealed in the receptor populations, offering new perspectives on the regulation of GlyR alpha3 membrane trafficking [2]. The super-resolution images generated by dSTORM are analyzed using pair-correlation analysis [3]. Hereby complementary quantitative information is retrieved concerning the clustering properties of the GlyRs.

[1] K. Notelaers, N. Smisdom, S. Rocha, D. Janssen, J.C. Meier, J.M. Rigo, J. Hofkens, M. Ameloot, Ensemble and single particle fluorimetric techniques in concerted action to study the diffusion and aggregation of the glycine receptor alpha3 isoforms in the cell plasma membrane, Biochimica et biophysica acta, 1818 (2012) 3131-3140.
[2] K. Notelaers, S. Rocha, R. Paesen, N. Smisdom, B. De Clercq, J.C. Meier, J.-M. Rigo, J. Hofkens, M. Ameloot, Analysis of α3 GlyR single particle tracking in the cell membrane, Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1843 (2014) 544-553.
[3] K. Notelaers, S. Rocha, R. Paesen, N. Swinnen, J. Vangindertael, J. Meier, J.-M. Rigo, M. Ameloot, J. Hofkens, Membrane distribution of the glycine receptor α3 studied by optical super-resolution microscopy, Histochem. Cell. Biol., (2014) 1-12.

Fig. 1: Representation of the experimental approach used for studying homomeric alpha3 glycine receptors (GlyR) in the plasma membrane of HEK 293 cells. Imaging is sub-divided in live-cell diffusion measurements and fixed-cell aggregation measurements.

Fig. 2: Identification of anomalous diffusion sections in GlyR alpha3 trajectories obtained by live-cell imaging. A. Detection of confined motion (circle), based on confinement probability level (Lc). B. Detection of directed motion (arrow), based on directed motion probability level (Ld).

Fig. 3: Co-expression aggregation study of the GlyR alpha3K (A, C: red) and alpha3L (B, C: green) in fixed HEK 293 cells, showing micrographs and pair crosscorrelation analysis. The splice variants exhibit co-clustering (C, magenta inset). The crosscorrelation is fitted in order to determine the co-cluster radius (ξ^c).