Figure 1.

Mutations in Shank3 affect its recruitment to the tips of actin filaments and actin polymerization. (a) Localization of rare non-synonymous variations or truncating SHANK3 mutations identified in autism spectrum disorder.^{6, 7} ANK, ankyrin repeats; Cbs, cortactin-binding site; Hbs, Homer-binding site; PDZ, postsynaptic density 95/discs large/zona occludens-1 homology domain; SAM, sterile alpha motif domain; SH3, Src homology 3 domain. (b) Western blot analysis of the green fluorescent protein (GFP) constructs expressed in HEK293T cells. We observed similar sizes of Shank3^WT and fusion proteins carrying point mutations (Shank3^R12C, Shank3^R300C and Shank3^Q321R). The frameshift mutation results in a truncated protein (222 kDa GFP–Shank3^WT and 169 kDa GFP–Shank3^STOP). (c) Localization of Shank3^WT in COS-7 cells transiently transfected with GFP–Shank3^WT (green) and labeled with phalloidin 647 to visualize F-actin (blue) and with anti-vinculin or anti-phospho-paxillin (red), two focal adhesion proteins. Shank3^WT is located at the membrane and in intracellular clusters that colocalize with vinculin or phospho-paxillin at the tips of actin filaments. On the right side, magnification of representative stress fibers with clusters of Shank3^WT. (d) Localization of Shank3^WT and mutated forms in COS-7 cells. COS-7 cells transiently transfected with GFP–Shank3^WT or mutated forms (green), and labeled with phalloidin 546 to visualize F-actin (red) and 4′,6-diamidino-2-phenylindole to visualize the nucleus (blue). Shank3^R12C, Shank3^R300C and Shank3^Q321R are located at the membrane and in intracellular clusters. Conversely, Shank3^STOP is restricted to around the nucleus. (e) Quantification of the accumulation of GFP constructs at the tips of actin filaments. Percentage of filaments with Shank3 clusters per cell (%). This accumulation is significantly reduced with mutated Shank3 and absent with Shank3^STOP (n=30–40 cells). Scale bars represent 15 μm. (f) Coimmunoprecipitation of Shank3 and actin in an adult rat brain. Lysate (input) was incubated with control immunoglobulin or anti-Shank3. Immunoprecipitation was performed, followed by western blotting using anti-β-actin. (g) Shank3^STOP inhibits the effect of Shank3^WT on actin polymerization. HEK293T were transiently co-transfected with red fluorescent protein–actin and GFP–C3 (control), GFP–Shank3^WT or GFP–Shank3^STOP. Representative immunoblots of G-actin (G) and F-actin (F) fractions obtained with an Actin Polymerization Assay kit. G-actin and F-actin were probed with anti-actin antibody. (h) Ratio of F-actin/G-actin was measured for each condition. The ratio is normalized to GFP–C3 and is represented as the mean±s.e.m. Shank3^WT enhances the polymerization of actin (^***P<0.001; n=6 independent experiments). Shank3^STOP inhibits the effect of Shank3 on actin polymerization.