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Figure 7


Fig. 7. Differential aggregation of EGFP-gephyrin mutant constructs. (A) Postsynaptic clusters of EGFP-geph-L1 transfected in neurons, as seen at 6+6 by double staining with the {alpha}2 subunit (red) and gephyrin (blue). (B,C) In HEK293 cells, EGFP-geph-L2B (B) was strongly expressed but also produced a diffuse fluorescence excluding the nucleus, whereas EGFP-geph-L2C (C) formed intracellular aggregates. (D,E) In neurons, EGFP-geph-L2B also produced a diffuse fluorescence, but induced a variable reduction of endogenous gephyrin clusters (open arrowheads; compare D and E). Despite the reduction of gephyrin clusters, {alpha}2 subunit clusters still were detected on transfected dendrites (open arrows). In neighboring non-transfected dendrites, gephyrin clusters associated with the {alpha}2 subunit (arrows) were not affected. (F) EGFP-geph-L2C formed numerous clusters associated with the {alpha}2 subunit (yellow). Nearly all endogenous gephyrin clusters (blue) on transfected cells contained EGFP-geph-L2C; except intracellular aggregates, all EGFP-positive clusters were also colocalized with the {alpha}2 subunit (white; F'). (G) Co-staining for the {alpha}2 subunit (red) and synapsin-1 (blue) revealed that almost all EGFP-geph-L2C-positive clusters were postsynaptic. (H,I) PSD-95 staining (red) in combination with EGFP-geph-L2C (H) and EGFP-geph-GCE (I). As expected, no colocalization was apparent between the two markers. EGFP-geph-L2C transfected cells showed an increase in gephyrin clusters relative to control. (J-L) Reduced EGFP cluster size (J; cumulative distribution analysis) but increased density (K) in 6+6 cells transfected with EGFP-geph-L2C compared with EGFP-geph-GCE. (L) Opposite changes in EGFP-positive clusters versus PSD-95-positive clusters in EGFP-geph-GCE and EGFP-geph-L2C transfected neurons (mean ± s.d.). The significant increase of EGFP-geph-L2C cluster number was accompanied by a concomitant decrease of PSD95 clusters whereas the total number of postsynaptic sites remains unchanged. Bars, 10 µm (A-E); 5 µm (F-I).





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