Fig. 4.

The negative effect of GEF-H1 over-expression on spine length is eliminated by an inhibitor of RhoA or ROCK. (A) Hippocampal neurons (13 DIV) were transfected with mCherry and GFP (Cont) or mCherry and GEF-H1 tagged with GFP (GEF), and then stained with antibodies against GFP and GluR1 at 16 DIV. (B) Hippocampal neurons were transfected the same way as above, treated with a RhoA inhibitor C3 transferase (C3T, 1.0 μg/ml) overnight (14–16 h) at 15 DIV, and stained as above. (C) Hippocampal neurons were transfected the same way as above, treated with a ROCK inhibitor (Y27632, 100 μM) for 3 days, and stained as above. Only GFP or GFP-GEF and mCherry images are shown here for comparison (A–C). (Scale bars, 10 μm.) (D–F) The effect of drug or GEF-H1 on the density and length of spines were analyzed quantitatively as explained in Fig. 2. Compared with untreated neurons (Cont), neurons treated with C3T (C3T) or Y27632 (Y27) showed significant increases in spine density. Spine density did not changed by GEF-H1 over-expression (GEF +). (E) Compared with untransfected neurons (first bar from the left), neurons over-expressing GEF-H1 (second bar) showed significant decrease in spine length. The change of spine length by GEF-H1 over-expression is eliminated by treatment of C3T (fourth bar) or Y27632 (sixth bar). The drug treatments only (third or fifth bar) significantly increased spine length. The label of bars is the same as in (D). (F) The change of spine length by GEF-H1 over-expression was eliminated by the drug treatments. See Table S1 for values and statistical analysis (D and E). (G) Cortical neurons were treated with C3T or Y27632 as above. At 16 DIV, neurons were harvested and solubilized proteins were applied to RhoA or Rac1 assay. C3T treatment significantly decreased and increased the activity of RhoA and Rac1, respectively. See Table S2 for values and statistical analysis.