Journal: bioRxiv

Article Title: Selective disruption of synaptic BMP signaling by a Smad mutation adjacent to the highly conserved H2 helix

doi: 10.1101/811109

Figure Lengend Snippet: (A) Western blot analysis of whole extracts from S2 cells expressing Flag-Mad and Flag-Mad-8 and treated with Dpp as indicated. Compared to control Mad, Mad-8 variant has significantly reduced pMad levels upon Dpp exposure. The pMad signals were normalized as the relative ratio pMad/Flag (A’). (B) Confocal images of S2 cells transfected with Flag-Mad variants and Tac-Tkv chimeras as indicated, spread on anti-Tac coated surfaces and labeled for pMad (red), Flag (green), actin (phalloidin-magenta), and DNA (DAPI-blue). The activated Tac-TkvA chimera induces nuclear pMad accumulation when co-transfected with Flag-Mad and, to a lesser extent, with Flag-Mad-8, as quantified in (B’). The pMad signals localize to cell surfaces only in Tac-TkvA/ Flag-Mad co-transfected cells. (C) Structure of the Type I receptor (PDM code 3TZM). The L45 loop (magenta) interacts specifically with R-Smads. The N-lobe of the receptor, including the GS box and L45, forms a docking surface for MH2 and positions the S-V-S C-tail of Mad in the catalytic pocket. (D-E) Structure of the MH2 domain of Drosophila Mad (PDM code 3DIT) shown as monomer (D) and trimer (E). The L3 loop (yellow) is engaged in exclusive interactions with either the L45 loop of the receptor or with the phosphorylated C-tail of another MH2 domain. (F) Map of MH2 Mad residues mutated in various Mad alleles. The two views of the structure are related by a 90° rotation around a vertical axis. (G) S359L in silica mutagenesis. S359 and adjacent peptide backbone form hydrogen bonds with H357 and residues on the H2 helix (purple); the S359L substitution breaks the hydrogen bonds with H357 (red asterisk) and introduces a bulky moiety, shifting the H2. (H) Lateral view of the Mad MH2 trimer showing the close proximity of the H2 helix (purple) to the charged L3 surface (colored by atoms). (I) Alignment of R-Smad sequences indicating class specific residues in the H2 region, including the S359 (yellow). Scale bars: 10 μm (B). Error bars indicate SEM. ***, p <0.0001; **, p <0.001; *, p <0.01

Article Snippet: The surface attached cells were fixed in 4% formaldehyde (Polysciences) for 15 min, then stained for pMad (anti-phospho-Smad 1/5, 41D10, 1:200, Cell Signaling), and Flag (anti-Flag, M2, 1:500, Sigma).

Techniques: Western Blot, Expressing, Control, Variant Assay, Transfection, Labeling, Mutagenesis