Journal: The Febs Journal

Article Title: Structural and functional dissection of the WH2 / DAD motif of INF2 , a formin linked to human inherited degenerative disorders

doi: 10.1111/febs.70271

Figure Lengend Snippet: Comparison of the WH2/DAD of human INF2 with the DAD of other human formins and its conservation across vertebrates. (A) Sequence alignment of the human INF2 WH2/DAD (residues 967–991) with the DAD sequences of the other DAD‐containing human formins. Uniprot accession numbers: INF2 ( Q27J81 ), DAAM1 ( Q9Y4D1 ), DAAM2 ( Q86T65 ), DIAPH1 ( O60610 ), DIAPH2 ( O60879 ), DIAPH3 ( Q9NSV4 ), FMNL1 ( O95466 ), FMNL2 ( Q96PY5 ), FMNL3 ( Q8IVF7 ), FHOD1 ( Q9Y613 ), FHOD3 ( Q2V2M9 ). Dark blue shading (hydrophobic region, H) and red shading (basic region, B) highlight identical residues or with very strictly conserved substitutions (e.g., E and D, R, and K, or L, I and V). Light blue shading indicates less strict amino acid hydrophobic substitutions (e.g., I, M, and F). Only residues conserved in at least six of the eleven DAD‐containing human formins are highlighted. The bottom line shows amino acids conserved in more than 50% of 100 representative WH2 domain sequences from different proteins and species . (B) Summary of the amino acid substitutions in the human INF2 WH2/DAD sequence across 45 vertebrate species, including 19 mammals, 9 birds, 7 reptiles, 4 amphibians, and 5 fish species (see Fig. for detailed alignments). The leucine residues (Leu976, Leu977, and Leu986) of INF2 critical for WH2 function are indicated in bold. The bottom line summarizes the conservation of the human INF2 WH2/DAD residues across vertebrates. Identical residues (bold letters) or very strictly conserved substitutions (normal letters) are highlighted in blue (H region) and red (B region).

Article Snippet: Epithelial canine MDCK II (CVCL_0424) INF2 KO cells [ ] and human epithelial HEK293T (CVCL_0063) cells from the American Type Culture Collection ( https://www.atcc.org ) were grown in minimal essential medium and Dulbecco's modified Eagle's medium, respectively, each supplemented with 5–10% (v/v) fetal bovine serum.

Techniques: Comparison, Sequencing

Journal: The Febs Journal

Article Title: Structural and functional dissection of the WH2 / DAD motif of INF2 , a formin linked to human inherited degenerative disorders

doi: 10.1111/febs.70271

Figure Lengend Snippet: Circular dichroism and nuclear magnetic resonance (NMR) analysis of the INF2 WH2/DAD peptide. (A) Circular dichroism spectra of the INF2 WH2/DAD peptide in aqueous solution (orange line) and in the 70:30 mixed solvent TFE/H 2 O (blue line) at pH 5.5 and 25 °C. Four scans were performed. (B) 2D 1 H, 1 H‐ total correlation spectroscopy spectra acquired for the INF2 WH2/DAD peptide in aqueous solution (orange contours) and in 30% TFE (blue contours) at pH 5.5 and 25 °C. (C) Bar plots showing the Δ δ Hα values as a function of the amino acid position in aqueous solution (orange bars) and in 30% TFE (blue bars) at pH 5.5 and 25 °C. ( n = 1; averaged error in chemical shift measurements is ±0.01 ppm). Residues 1–25 of the peptide correspond to residues 967–991 of INF2. ppm, parts per million; RC, random coil value interval; TFE, 2,2,2‐trifluoroethanol. [Correction added on 2 October 2025 after first online publication: Panel 2A has been corrected.]

Article Snippet: Epithelial canine MDCK II (CVCL_0424) INF2 KO cells [ ] and human epithelial HEK293T (CVCL_0063) cells from the American Type Culture Collection ( https://www.atcc.org ) were grown in minimal essential medium and Dulbecco's modified Eagle's medium, respectively, each supplemented with 5–10% (v/v) fetal bovine serum.

Techniques: Circular Dichroism, Nuclear Magnetic Resonance, Solvent, Spectroscopy

Journal: The Febs Journal

Article Title: Structural and functional dissection of the WH2 / DAD motif of INF2 , a formin linked to human inherited degenerative disorders

doi: 10.1111/febs.70271

Figure Lengend Snippet: Structure of the INF2 WH2/DAD peptide revealed by NMR. (A) Overlay of the backbone atoms (black) from the 20 lowest target function conformers of the structure in aqueous solution (top panel) and in 30% TFE (bottom panel). (B) Overlay of the backbone atoms of the ensembles in aqueous solution (blue) and 30% TFE (black). Conformers in (A, B) were visualized with MOLMOL. (C) Ribbon representation of the first conformer of the ensemble obtained in 30% TFE. (D, E) Superimposition of the NMR structure of the INF2 WH2/DAD on the crystallographic structure of the WASP WH2 domain (PDB 2VCP) when bound to G‐Actin (D) and the mDia1 DAD (PDB 2F31) when bound to the diaphanous inhibitory domain (DID) of mDia1 (E). In (A) and (C), hydrophobic sidechains are shown in green, polar sidechains in cyan, positively charged sidechains in blue, and negatively charged sidechains in red. Statistical data for the structural ensembles in (A) and (B) are presented in Table . The lowest target function conformer is shown in (C–E) using chimerax . The hydrophobic (H) and basic (B) regions are indicated. DAD, diaphanous autoregulatory domain; TFE, 2,2,2‐trifluoroethanol; WH2, WASP homology 2.

Article Snippet: Epithelial canine MDCK II (CVCL_0424) INF2 KO cells [ ] and human epithelial HEK293T (CVCL_0063) cells from the American Type Culture Collection ( https://www.atcc.org ) were grown in minimal essential medium and Dulbecco's modified Eagle's medium, respectively, each supplemented with 5–10% (v/v) fetal bovine serum.

Techniques:

Journal: The Febs Journal

Article Title: Structural and functional dissection of the WH2 / DAD motif of INF2 , a formin linked to human inherited degenerative disorders

doi: 10.1111/febs.70271

Figure Lengend Snippet: The hydrophobic and the basic regions of the WH2/DAD of INF2 are both essential for Actin binding. (A) Sequences of the INF2 WH2/DAD fragments used in the pull‐down analysis. (B) Pull‐down experiment using GST fused to the indicated WH2/DAD sequences of INF2 and DAD of mDia1 and extracts from GFP‐Actin‐expressing cells. The analysis was performed using antibodies against GFP and Actin. (C) Identification of different classes of consensus CaM‐binding site motifs in the INF2 WH2/DAD. (D) Pull‐down experiment using GST fused to the indicated INF2 sequences and extracts from GFP‐CaM‐expressing cells. Analysis was performed using antibodies against GFP and CaM. Purified GST fusions were stained with Coomassie blue to control for the amount of GST protein used (bottom panels in B and D). Note the presence of endogenous Actin in the GST‐DAD pull‐downs in both panels (B) and (D). (E) Pull‐down experiment using GST fused to the indicated INF2 sequences and purified CaM. Analysis was performed using antibodies against CaM. In the Coomassie blue‐stained gels, filled arrowheads indicate endogenous Actin (B, D), while empty arrowheads denote exogenous GFP‐Actin (B), GFP‐CaM (D), and purified CaM (E). Three independent experiments were performed in (B, D, E). A representative experiment is shown. CaM, calmodulin; DAD, diaphanous autoregulatory domain; GFP, green fluorescence protein; GST, glutathione S‐transferase.

Article Snippet: Epithelial canine MDCK II (CVCL_0424) INF2 KO cells [ ] and human epithelial HEK293T (CVCL_0063) cells from the American Type Culture Collection ( https://www.atcc.org ) were grown in minimal essential medium and Dulbecco's modified Eagle's medium, respectively, each supplemented with 5–10% (v/v) fetal bovine serum.

Techniques: Binding Assay, Expressing, Purification, Staining, Control, Fluorescence

Journal: The Febs Journal

Article Title: Structural and functional dissection of the WH2 / DAD motif of INF2 , a formin linked to human inherited degenerative disorders

doi: 10.1111/febs.70271

Figure Lengend Snippet: The H region, but not the B region, of the WH2/DAD is essential for Actin polymerization by INF2. (A) Schematic representation of the INF2 deletion mutants expressed in this study. (B–D) INF2 KO MDCK cells expressing, or not expressing, Cherry fusions with the indicated INF2 proteins, were stained to visualize F‐Actin. Nuclei were counterstained with DAPI. The contrast in the Cherry channel was increased as indicated to visualize the cells expressing INF2 ΔDAD and INF2 DAD‐ΔH. An enlarged view of the perinuclear region (boxed) from representative cells of each condition is shown in the rightmost panels, displaying DAPI (top) and F‐Actin (bottom) fluorescence images. Scale bars, 15 μ m (B). (C) The graph shows the relative intensity of F‐Actin staining in the perinuclear region compared with control INF2 KO cells, measured across three equatorial planes. Statistical significance was assessed using the Mann–Whitney–Wilcoxon test. (D) Percentage of INF2 KO cells with mild (single micronucleus, a multilobed nucleus, or two normal nuclei) or severe (multiple micronuclei) nuclear phenotypes. Statistical significance was determined using a two‐tailed, unpaired Student's t ‐test. > 150 cells (C) and > 300 cells (D) were analyzed for each experimental condition; three independent experiments; ns, not significant; * P < 0.01; *** P < 0.001. DAD, diaphanous autoregulatory domain; DAPI, 4′,6‐diamidino‐2‐phenylindole dihydrochloride; DID, diaphanous inhibitory domain; FH, formin homology; WH2, WASP homology 2; wt, wild type.

Article Snippet: Epithelial canine MDCK II (CVCL_0424) INF2 KO cells [ ] and human epithelial HEK293T (CVCL_0063) cells from the American Type Culture Collection ( https://www.atcc.org ) were grown in minimal essential medium and Dulbecco's modified Eagle's medium, respectively, each supplemented with 5–10% (v/v) fetal bovine serum.

Techniques: Expressing, Staining, Fluorescence, Control, MANN-WHITNEY, Two Tailed Test

Journal: The Febs Journal

Article Title: Structural and functional dissection of the WH2 / DAD motif of INF2 , a formin linked to human inherited degenerative disorders

doi: 10.1111/febs.70271

Figure Lengend Snippet: Expression of natural INF2 WH2/DAD variants does not affect nuclear morphology. (A) Compilation of allelic variants in the WH2/DAD of INF2 found in the general human population. Variants annotated in the ClinVar database are highlighted in green (See Table ). Invariant residues are highlighted in blue (hydrophobic region, H) and red (basic region, B). (B) Percentage of MDCK cells expressing the indicated INF2 WH2/DAD variants displaying mild (single micronucleus, a multilobed nucleus, or two normal nuclei) or severe (multiple micronuclei) nuclear phenotypes. The pathogenic INF2 R218Q DID variant was included as a control. Statistical significance was determined using a two‐tailed, unpaired Student's t ‐test. > 300 cells were analyzed for each experimental condition; three independent experiments; n.s., not significant; ***, P < 0.001. wt, wild type.

Article Snippet: Epithelial canine MDCK II (CVCL_0424) INF2 KO cells [ ] and human epithelial HEK293T (CVCL_0063) cells from the American Type Culture Collection ( https://www.atcc.org ) were grown in minimal essential medium and Dulbecco's modified Eagle's medium, respectively, each supplemented with 5–10% (v/v) fetal bovine serum.

Techniques: Expressing, Variant Assay, Control, Two Tailed Test