Structure and function of the N-terminal extension of the formin INF2

Figures

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  Fig. 1. The N-terminal extension of INF2 contains two α-helices. A INF2 has an extension N-terminal to the DID that is shorter than that of mDia1 and lacks a G domain. G (also known as GBDN) domain necessary for Rho binding that is adjacent to the N terminus of the DID, GBD GTPase-binding site, DAD diaphanous autoinhibitory domain, DID diaphanous inhibitory domain, FH1 and FH2 formin homology 1 and 2, respectively. B Ribbon representation of the NMR solution structure of the N-terminal extension of INF2. Backbone atoms are shown in black. K and R side chains are displayed in blue, D and E side chains in red, and all others in green

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  Fig. 2. The first of α-helix of INF2 is required for the normal distribution and content of F-actin. A Schematic of the INF2 proteins expressed. B–D INF2 KO cells or INF2 KO cells expressing mCherry alone or mCherry fusions of the indicated INF2 proteins were stained for F-actin. Nuclei were visualized with DAPI. An enlargement of the DAPI and F-actin staining corresponding to the boxed regions is shown in the rightmost panels. Scale bar, 10 μm (B). Box plots showing the intensity of F-actin staining at the perinuclear region (C) and the cytosol (D) relative to that of control INF2 KO cells (> 140 cells were analyzed for each experimental condition; three independent experiments; ns not significant; ***p < 0.001)

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  Fig. 3. CaM binds the N-terminal α-helix of INF2. A Schematic of the INF2 fragments used in the pull-down experiments. B, C Pull-down analysis of the association of GFP-CaM with GST fusions of the indicated INF2 fragments. D GST fusions of the fragment comprising amino acids 4–19 of INF2 with the indicated mutations in residues W11, L14 and L18 were used in pull-down experiments with GFP-CaM. The purified GST fusions were stained with Coomassie blue as a control for the amount of GST fusions used in (B–D). E Lysates of HEK293T cells expressing intact INF2 or INF2 with the triple W11L14L18A mutation fused to mCherry were immunoprecipitated with control or anti-Cherry antibodies and immunoblotted for exogenous INF2 with anti-Cherry antibodies, and with antibodies to endogenous CaM

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  Fig. 4. Surface plasmon resonance analysis of the interaction of the 2-19 INF2 peptide with CaM. A–D Representative equilibrium binding affinity measurements of the interaction of purified CaM from bovine brain with the control 2-19 peptide (A, left panel) or 2-19 peptides containing the L14L18A (B), W11A (C) and W11L14L18A (D) mutations in the presence of Ca2+. E Control of the binding of CaM to the 2-19 peptide in the absence of Ca2+. The equilibrium plots of CaM with the control 2-19 (A, right panel) is shown, with indication of the apparent KD resulting from the same experiment. RU resonance units, Req RU at equilibrium

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  Fig. 5. Conservation of the CaMBS of INF2. A Alignment of the sequence of the 1–19 peptide of human INF2 with that of 50 species from different groups of vertebrates. The corresponding position of the W11, L14 and L18 residues of human INF2 is indicated. B Compilation of allelic variants of the 6–19 sequence of INF2 in the general human population, with indication of the SNP reference, database and frequency. C Examples of CaM-binding proteins with a sequence similar to the 1-4-8 motif of INF2. AKAP79 A-kinase-anchoring protein 79, CX32 connexin 32, GRM5 glutamate metabotropic receptor 5, PDE1A and PDE1B Ca2+/CaM-dependent 3´,5´-cyclic nucleotide phosphodiesterases 1A and 1B, RIT1 and RIT2 Ras-related GTPases 1 and 2, TRPV4 transient receptor potential vanilloid subtype 4

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  Fig. 6. Structural model of the CaM/CaMBS complex. A Structural model of C-lobe of CaM bound to the 2-19 INF2 peptide. The position of the three hydrophobic amino acids comprising the 1-4-8 motif of the CaMBS of INF2 is indicated. B GST, alone or fused to the 2–21 peptide of INF2, was used in pull-down experiments with GFP fused to intact (WT) or to CaM with mutations that inactivate the two lobes (mutant 1234), the N-terminal lobe (mutant 12) or the C-terminal lobe (mutant 34). The purified GST fusions were stained with Coomassie blue as a control for the amount of GST protein used

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  Fig. 7. Integrity of the CaMBS of INF2 is crucial for actin homeostasis. A–C INF2 KO cells expressing mCherry fusions of the indicated INF2 mutants were stained for F-actin. Nuclei were visualized with DAPI. An enlargement of the DAPI and F-actin staining corresponding to the boxed region is shown in the rightmost panels. Scale bar, 10 μm(A). Box plots showing the intensity of F-actin staining at the perinuclear region (B) and the cytosol (C) relative to that of control INF2 KO cells (> 150 cells were analyzed for each experimental condition; three independent experiments; ns not significant; *p > 0.05; ***p < 0.001)

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  Fig. 8. The CaMBS of INF2 is required for actin polymerization and incorporation of GFP-MRTF-A into the nucleus in response to increased intracellular Ca2+ levels. A, B GCaMP6S INF2 KO cells expressing the indicated exogenous INF2 proteins were incubated with SiR-actin to visualize F-actin. Cells were treated with A23187 (0 min) and analyzed by videomicroscopy before and after treatment (A). Note that, although the staining of F-actin with SiR-Actin in live cells was poor compared to that of fluorescent phalloidin in fixed cells, SiR-Actin was useful for detecting the initial increase of the levels of F-actin after A23187 stimulation. B Kinetics of the perinuclear content of F-actin as measured by SiR-actin fluorescence relative to that of cells at t = 0 min (> 30 cells were analyzed for each experimental condition; three independent experiments). C–E GFP-MRTF-A INF2 KO cells expressing mCherry fusions of intact INF2 or the INF2 W11L14L18A mutant were treated with A23187 and analyzed by videomicroscopy before and after treatment (C). (D) Kinetics of the number of INF2 KO cells with nuclear distribution of GFP-MRTF-A relative to that of cells at t = 0 min (≥ 30 cells were analyzed for…

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  Fig. 9. Comparative schematics of the activation of mDia1 and INF2. mDia1 is activated by the binding of GTP-loaded Rho to the GBD, whereas INF2 activation takes place when Ca2+/CaM binds to the INF2 N-terminal extension