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. 2004 Jun 23;24(25):5816–5826. doi: 10.1523/JNEUROSCI.1184-04.2004

Figure 5.

Figure 5.

Disruption of the collybistin binding site on gephyrin prevents accumulation of gephyrin at postsynaptic sites. A-H, EGFP-tagged gephyrin (A, E) and the mutants EGFP-GephA4 (B, C, F, G) and EGFP-GephA5 (D, H) were transfected into cortical neurons and the cells immunostained for gephyrin (mAb7a; A-D) or GAD (mAbGAD-6; E-H). Two examples of cells are shown for EGFP-GephA4 representing comparatively low (B, F) and high (C, G) levels of expression and corresponding aggregate phenotypes. Note the perfect colocalization of transfected EGFP-gephyrin with endogenous gephyrin in yellow puncta (A). The few remaining red puncta indicate endogenous gephyrin expressed on dendrites of nontransfected neighboring cells that are therefore not colocalized with EGFP-gephyrin. EGFP-gephyrin is localized at postsynaptic sites juxtaposed to presynaptic GAD (E, arrows). Mutants EGFP-GephA4 and EGFP-GephA5 form large aggregates in the soma and dendrites, which are no longer juxtaposed to GABAergic terminals (F-H). Endogenous gephyrin has been trapped in these extrasynaptic gephyrin mutant aggregates, which therefore appear yellow (B-D). Quantitative analysis of mAb7a staining indicates a statistically significant loss of synaptic gephyrin clusters (I;*p < 0.001; Student's t test; n = 12-13 cells per construct) for the EGFP-GephA4 and EGFP-GephA5 mutants compared with EGFP-gephyrin. Scale bar, 20 μm.