Figure 7.
Phosphorylation of Abi1 at the serine 88 is critical for Rac activation and spine maturation. A, HeLa cells were cotransfected with Flag-CaMKIIα, Myc-Rac1, or GFP-Abi1 as indicated. B, HeLa cells were cotransfected with Myc-Rac1 with either GFP-Abi1, GFP-Abi1S88A, or GFP-Abi1S88D. In A and B, cell lysates were subjected to GST-PBD (p21-binding domain) pull-down (PD) and immunoblotted (IB) with anti-Myc antibodies. The ratio of GTP-Rac (bound Rac to GST-PBD)/Myc-Rac was quantified and expressed as the percentage of the amount in control cells from three independent experiments in both A and B (mean ± SD, *p = 0.04, **p = 0.02, ***p = 0.007; ns, not significant; Student's t test). C, Rat hippocampal neurons were transfected with GFP, GFP-Abi1, GFP-Abi1ΔtSNARE, GFP-Abi1S88A, and GFP-Abi1S88D at 7 DIV and fixed at 14 DIV. DsRed was also coexpressed to show overall morphology of the neurons. Representative neurites for each condition are shown. Scale bars, 5 μm. D–F, Results after 1500 10 μm dendritic segments from 100 neurons were subjected to morphometric analysis. D, Number of total protrusions, spines, and filopodia per 10 μm dendritic segments in hippocampal neurons transfected as indicated in C were quantified and compared with control cells using Student's t test (mean ± SEM, *p = 0.04, **p = 0.02, ***p = 0.009, #p = 0.005, ##p = 0.004, ###p = 0.001; ns, not significant; Student's t test). E, Protrusion length was measured for neurons transfected as indicated. F, Spine widths were measured for neurons transfected as indicated. TCL, Total cell lysate.