Figure 7
mTOR activation increases gephyrin clustering.
(a,b) ca-mTOR induces S6K phosphorylation. Lysates of HeLa cells transfected with plasmid vectors expressing EGFP, wt-mTOR, or ca-mTOR were analyzed by Western blotting to quantify total S6K protein (70 kDa isoform) or phosphor-S6K. Stimulation with fetal calf serum (FCS) served as a control. S6K proteins were detected by a pan-S6K antibody (S6K) or a phospho-S6K antibody (pS6K; pThr389), respectively (upper bands correspond to the 85 kDa isoform of S6K). (b) Densitometric analysis of signals obtained after western blotting as exemplified in (a). Bar charts represent ratios of pS6K to pan-S6K signals as percentage of the EGFP-transfected control. n = 6; *p < 0.05. ANOVA, Fisher-PLSD; error bars, S.E.M. (c) ca-mTOR increases gephyrin cluster densities. Either hippocampal neurons (c,d); gephyrin staining in c,d is shown in c’,d’) or HeLa cells (e,f) were transfected with plasmid vectors for wild type mTOR (WT) or ca-mTOR (CA). Neurons were cotransfected with pEGFP-N1 for the identification of mTOR transfected cells whereas HeLa cells were additionally cotransfected with EGFP-gephyrin and collybistin expression plasmids to allow for surface translocation of expressed EGFP-gephyrin. Cluster densities of immunocytochemically labeled endogenous gephyrin on dendritic segments of EGFP-positive neurons (n = 40) or of EGFP-gephyrin (n = 13) on the surface of HeLa cells were determined. Values are indicated as percentage of wt-mTOR transfected cells. ***p < 0.0001; Kruskal-Wallis-Test, followed by Dwass-Steel-Test; error bars, S.E.M. (d) Gephyrin cluster size was determined in primary neurons (cluster volumes) or in HeLa cells (surface cluster areas) after transfection of expression vectors for wt-mTOR or ca-mTOR. n > 13; ***p < 0.0001. ANOVA, Fisher-PLSD; error bars, S.E.M. scale bars 30 μm.
