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Fig. 2. Expression of EGFP-tagged full-length gephyrin (EGFP-geph-GCE). Images from immunofluorescence staining with the markers indicated in each panel were visualized by confocal laser-scanning microscopy. (A) In HEK293 cells, EGFP-geph-GCE forms intracellular aggregates independently of the presence of GABA_A receptors (stained for the 1 subunit in red) at the cell surface. (B,B') In 6+2 hippocampal neurons, a clustered staining for the GABA_A receptor 2 subunit (red) was detected whereas only few endogenous (blue) and heterologous gephyrin clusters were present (B'); the gephyrin antibody stained both endogenous gephyrin and EGFP-geph-GCE. The latter formed cytosolic aggregates (arrows) and a few clusters co-localized with the 2 subunit in dendrites (open arrowheads). Open arrows point to non-specific gephyrin staining of a cell nucleus. (C) In 6+6 cells, the number of gephyrin and 2 subunit clusters increased markedly, although some 2 subunit clusters lacking gephyrin were still detectable. No endogenous gephyrin clusters devoid of EGFP were present, as shown at higher magnification in the inset (c1-c3; open arrowheads), suggesting that both proteins interact within individual clusters. (D-D'') In cells with a strong EGFP-geph-GCE expression, large aggregates devoid of 2 subunit staining were formed in dendrites (arrow). (E) In 10+2 cells, the 2 subunit (red) and gephyrin (blue) had the same appearance as at 6+6; EGFP-geph-GCE (green) was associated with 2 subunit immunoreactivity in clusters (white), but not in cytosolic aggregates (cyan), as shown at higher magnification in the inset (e1-e3). (F-F'') Labeling for synapsin 1 (red) demonstrates the selective postsynaptic localization of EGFP-geph-GCE clusters. Bars, 10 µm (A,B); 10 µm (C-E); 5 µm (F).

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