Figure 4
From: Identification and functional characterization of rare SHANK2 variants in schizophrenia
Analysis of the effect of SHANK2 wild type and mutants on actin structures and polymerization. (a) YFP-SHANK2E, YFP-SHANK2-SH3 and YFP-Shank3 wild type were co-transfected together with RFP-actin into COS-7 cells. Live cell widefield epifluorescence and TIRF images are presented. SHANK2-SH3 wild type formed intracellular aggregates of different sizes and did not localize to the actin fiber tips. YFP-SHANK2E formed compact aggregates around the actin fiber tips. The same localization was observed with C-terminal SHANK2-SH3-YFP and SHANK2E-YFP constructs (data not shown). YFP-Shank3 was used as a control because it co-localizes to the actin fiber tips. Scale bars: 20 μm (main panels); 5 μm (insets). (b) YFP-SHANK2E wild type was co-transfected together with the focal adhesion marker GFP-vinculin into COS-7 cells. TIRF images are presented. SHANK2E protein accumulated around the focal adhesions but did not co-localize with vinculin. Scale bars: 20 μm (main panels); 5 μm (insets). (c) F/G-actin ratio measurements for all variants tested and SHANK2E-WT in HEK293 cells. Each condition was tested in three independent transfections with two replicates each. (d) A1731S variant and SHANK2E-WT were additionally analyzed in three independent transfections with three replicates each. The A1731S/WT results were normalized to the SHANK2 protein amounts via second western blot with anti-SHANK2 and anti-GAPDH antibodies. All values are shown as mean+standard deviation. Wild-type value is normalized to 1. *P≤0.05 (two-tailed paired t-test).
