Journal: Communications Biology

Article Title: BIN1 regulates actin-membrane interactions during IRSp53-dependent filopodia formation

doi: 10.1038/s42003-024-06168-8

Figure Lengend Snippet: a Wild-field deconvoluted images of HeLa cells transfected with GFP or GFP-tagged Amphiphysin1, BIN1 iso1 and BIN1 iso8 (in gray) and stained for F-actin (phalloidin, red). Scale bar, 10 μm. Quantification of the density of HeLa cells expressing GFP or GFP-tagged Amphiphysin1, BIN1 iso1 and BIN1 iso8. Number of cells: n = 26, 11, 25 and 18, respectively from three independent experiments. b Western-blot analysis of the endogenous expression of actin, BIN1, and Troponin T in C2C12 myoblasts in growth medium (0 h, undifferentiated) and grown in differentiation medium (DM) at 24 h and 48 h. c Western-blot analysis of the endogenous expression of actin and BIN1 on parental, CTRL shRNA (i.e., Luciferase) and Bin1 shRNA C2C12 myoblast cells. d Scanning electron microscopy images of CTRL shRNA and Bin1 shRNA C2C12 myoblasts cultured in growth factor-containing medium. Scale bar, 10 μm and 5 μm for the magnified images. e Quantification of the number of filopodia per cell at the cell periphery and at intercellular junctions measured from the SEM images. Cells: n = 25, 19 and 27, 38 for CTRL and Bin1 shRNA cells at the cell periphery and cell-cell junctions, respectively. f Snap-shots of spinning disk life cell imaging (500 ms exposure, during 60 s. Total time acquisition = 120 s) of C2C12 myoblasts co-transfected with Lifeact-mCherry (red) and GFP-BIN1 iso8 (gray) at t = 0 s. Kymograph analysis along the blue dashed line in the corresponding image at t = 0 s, highlight the recruitment and binding of BIN1 iso8 to filopodia. Scale bar, 10 μm. Scale bar in kymograph and ROI, 2 μm. Bottom, representative time-lapse snapshots from the ROI region showing the localization of BIN1 iso8 on F-actin during filopodia formation in C2C12 cells, as highlighted by the blue arrowheads. Error bars represent s.d.; ANOVA test: n.s > 0.1, * P < 0.05, **** P < 0.0001.

Article Snippet: Scale bar, 20 μm. g Snap-shots of spinning disk life cell imaging at t = 0 of C2C12 cells co-transfected with mCherry-Lifeact and either GFP (empty), GFP-BIN1 iso8, GFP-BIN1 D151N or GFP-BIN1 ΔSH3 (inverted LUT images of the actin and GFP signal).

Techniques: Transfection, Staining, Expressing, Western Blot, shRNA, Luciferase, Electron Microscopy, Cell Culture, Imaging, Binding Assay

Journal: Communications Biology

Article Title: BIN1 regulates actin-membrane interactions during IRSp53-dependent filopodia formation

doi: 10.1038/s42003-024-06168-8

Figure Lengend Snippet: a C2C12 myoblasts stained for endogenous BIN1 (C99D antibody, gray) and F-actin (phalloidin, red). Magnified images of two representative regions of interest (ROIs). Scale bar, 10 μm. b SIM images of C2C12 myoblasts expressing GFP-BIN1 (gray) stained for F-actin (phalloidin, red) and magnified images of two representative ROI in the corresponding image. Scale bar, 10 μm. c GFP pull-downs using extracts from HeLa cells expressing either GFP, or GFP-BIN1 iso8, GFP-BIN1 iso1 and GFP-Amphiphysin1. Actin was detected by western blotting. HeLa cells were used to provide an unbiased cellular context. IP = immunoprecipitate. d Domain representation (N-BAR, phosphoinositide-binding motif, PI, and SH3 domains) of BIN1 iso8 full-length. Stars highlight the D151N mutant at the N-BAR and the stop codon of the BIN1 ΔSH3 mutant. e GFP-Trap pull-downs using extracts from C2C12 myoblasts expressing either GFP alone or fused with BIN1 iso8, BIN1 ΔSH3 or BIN1 D151N. Actin was revealed by western blotting. IP = immunoprecipitate. f In vitro assay with purified actin and BIN1 iso8 or BIN1 D151N and BIN1 ΔSH3 mutants. TIRF images showing pre-polymerized F-actin filaments in the absence (-BIN1 iso8, CTRL) or in the presence of 1 µM of BIN1 iso8 and BIN1 mutants and the corresponding quantification of the BIN1 bundling activity as denoted by the actin intensity in each condition. Scale bar, 20 μm. g Snap-shots of spinning disk life cell imaging at t = 0 of C2C12 cells co-transfected with mCherry-Lifeact and either GFP (empty), GFP-BIN1 iso8, GFP-BIN1 D151N or GFP-BIN1 ΔSH3 (inverted LUT images of the actin and GFP signal). Time projection of the filopodia tracks (500 ms exposure, during 60 s. Total time acquisition = 120 s) obtained from the corresponding Lifeact images. Scale bar, 10 μm. Fire LUT color scale is 120 s. h Quantification of filopodia density; cells: n = 11, 12, 12 and 12 for GFP, BIN1 iso8, D151N and ΔSH3 mutants, respectively. i % of dynamic (filopodia that collapsed for a period ≤ 120 s, red) and static (filopodia that do not collapse for a period ≥ 120 s, gray) filopodia; Number of filopodia: n = 49, 64, 29, and 48 for GFP, BIN1 iso8, D151N and ΔSH3 mutants, respectively. Chi-square test: * P < 0.1, **** P < 0.0001. j Lifetime of dynamic filopodia (in seconds); Number of filopodia: n = 36, 20, 15, and 25 for GFP, BIN1 iso8, D151N and ΔSH3 mutants, respectively. k Quantification of filopodia density; Cells: n = 20, 19, 25, 31, 25, and 25 for CTRL shRNA and Bin1 shRNA C2C12 cells co-transfected with either GFP, GFP-BIN1 iso8, GFP-BIN1 D151N or GFP-BIN1 ΔSH3 and Lifeact-mCherry, respectively. Error bars represent s.d.; ANOVA test: n.s > 0.1, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Scale bar, 20 μm. g Snap-shots of spinning disk life cell imaging at t = 0 of C2C12 cells co-transfected with mCherry-Lifeact and either GFP (empty), GFP-BIN1 iso8, GFP-BIN1 D151N or GFP-BIN1 ΔSH3 (inverted LUT images of the actin and GFP signal).

Techniques: Staining, Expressing, Western Blot, Binding Assay, Mutagenesis, In Vitro, Purification, Activity Assay, Imaging, Transfection, shRNA

Journal: Communications Biology

Article Title: BIN1 regulates actin-membrane interactions during IRSp53-dependent filopodia formation

doi: 10.1038/s42003-024-06168-8

Figure Lengend Snippet: a GFP-Trap pull-downs from extracts of HeLa cells transfected with plasmids encoding GFP, GFP-tagged amphiphysin1, BIN1 iso1 and BIN1 iso8. Ezrin was revealed by western-blotting. b GFP-Trap pull-downs using extracts from C2C12 myoblasts expressing either GFP alone or fused with BIN1 iso8, BIN1 ΔSH3 or BIN1 D151N. Ezrin was revealed by western-blotting. c Time projected spinning disk movies (500 ms exposure, during 60 s. Total time acquisition = 120 s) of C2C12 cells co-expressing mCherry-ezrin (magenta) and GFP-BIN1 iso8 (green). d Time-lapse snapshots from the corresponding yellow ROI showing the co-localization of BIN1 and ezrin during filopodia formation (white arrows). Kymograph analysis along the blue dashed line. Scale bar, 10 μm. Scale bar in kymographs, 1 μm. e % of ezrin-positive filopodia displaying an overlapping of mCherry/GFP signal (red) or no overlapping of signals (gray) upon co-expression of GFP-BIN1 iso8, GFP-BIN1 D151N or GFP-BIN1 ΔSH3 and mCherry-ezrin. Number of filopodia: n = 384, 191 and 146, respectively. Error bars represent s.d.; Chi-square test: n.s > 0.1, **** P < 0.0001. f Quantification of filopodia density; n = 19, 17, 12, 10, and 10 for ezrin, ezrin-T567D, BIN1, ezrin + BIN1 iso8, and ezrin + BIN1 ΔSH3 mutant, respectively. n = number of cells from live cell imaging experiments. Error bars represent s.d.; ANOVA test: n.s > 0.1, * P < 0.05, *** P < 0.001, **** P < 0.0001. g Confocal images of the co-localization of recombinant BIN1-Alexa647 (magenta), ezrin or phospho-mimetic ezrin (T567D) tagged with Alexa488 (green) and TopFluor-TMR-PI(4,5)P 2 (cyan) on supported lipid bilayers containing 5% of PI(4,5)P 2 . Scale bar, 3 μm. Fold increase in the binding of recombinant ezrin and ezrin-T567D on supported lipid bilayers containing 5% PI(4,5)P 2 in the presence of BIN1 or amphiphysin1 (Amph1). h Western-blot analysis of the endogenous expression of ezrin and phosphorylated ezrin (phospho-ezrin), BIN1 and actin on cell extracts from CTRL (blue) and Bin1 shRNA (yellow) C2C12 myoblasts, and the corresponding quantification of the signal of each protein normalized by actin. i CTRL and Bin1 shRNA C2C12 myoblasts cultured in growth factor-containing medium and stained for endogenous phosphorylated ezrin (phospho-ezrin, yellow) and F-actin (phalloidin, cyan). Inset, high-magnification images showing the localization of endogenous phosphorylated ezrin. Maximum intensity projected airyscan images. Scale bar, 10 μm and 5 μm, respectively.

Article Snippet: Scale bar, 20 μm. g Snap-shots of spinning disk life cell imaging at t = 0 of C2C12 cells co-transfected with mCherry-Lifeact and either GFP (empty), GFP-BIN1 iso8, GFP-BIN1 D151N or GFP-BIN1 ΔSH3 (inverted LUT images of the actin and GFP signal).

Techniques: Transfection, Western Blot, Expressing, Mutagenesis, Live Cell Imaging, Recombinant, Binding Assay, shRNA, Cell Culture, Staining