Figure 5.
EVL regulates DF morphogenesis and motility through membrane-targeted actin polymerization. (A) Schematic of protein domains of wild-type EVL and EVL mutants used in this study. EVH1 (blue) = Enabled/VASP homology-1, P (yellow) = profilin binding region, G (orange) = G-actin binding region, F (red) = F-actin binding region, CC (gray) = coiled-coil domain, PRD = proline-rich region, EVH2 = Enabled/VASP homology-2. (B) Western blot of protein lysates from D11 cortical neurons derived from EVL knockout (KO) mice expressing mEmerald-EVL or various mutants of EVL as indicated. (C) Segment of dendrite from live primary cortical neurons derived from EVL knockout mice at indicated day in vitro 11 (D11) expressing mRuby2-LifeAct and mEmerald alone, or mEmerald-tagged wild-type EVL or mutants of EVL as indicated. From left to right, Panel 1: Merge of LifeAct and mEmerald-tagged proteins. Scale bar = 10 µm. Panel 2: mEmerald fluorescence intensity presented with an inverted LUT. Panel 3: Kymograph of DF position indicated by arrowhead (5 s interval, 5 min duration). Vertical scale bar = 1 µm, horizontal scale bar = 1 min, dashed line indicates dendrite. Panel 4: Maximum intensity projection of temporally color-coded binary mask outline (5 s interval, 5 min duration). (D) Scatterplot of average speed of DF tips, calculated as the average absolute tip displacement between successive timepoints. Gray shaded region indicates average speed less than 0.0128 µm/s (non-motile DFs). Median ± IQR. Mixed-effects model; n = 248–397 total DF from 3–6 neurons per biological replicate, N = 3–5 biological replicates. (E) Scatterplot of average length reached during the duration of imaging for indicated conditions. Median ± IQR. Mixed-effects model; n = 248–397 total DF from 3–6 neurons per biological replicate, N = 3–5 biological replicates. (F) Scatterplot of median protrusion rates of DFs for indicated conditions. Median ± IQR. Mixed-effects model; n = 248–397 total DF from 3–6 neurons per biological replicate, N = 3–5 biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s. is not significant. See also Video 5. Source data are available for this figure: SourceData F5.

EVL regulates DF morphogenesis and motility through membrane-targeted actin polymerization. (A) Schematic of protein domains of wild-type EVL and EVL mutants used in this study. EVH1 (blue) = Enabled/VASP homology-1, P (yellow) = profilin binding region, G (orange) = G-actin binding region, F (red) = F-actin binding region, CC (gray) = coiled-coil domain, PRD = proline-rich region, EVH2 = Enabled/VASP homology-2. (B) Western blot of protein lysates from D11 cortical neurons derived from EVL knockout (KO) mice expressing mEmerald-EVL or various mutants of EVL as indicated. (C) Segment of dendrite from live primary cortical neurons derived from EVL knockout mice at indicated day in vitro 11 (D11) expressing mRuby2-LifeAct and mEmerald alone, or mEmerald-tagged wild-type EVL or mutants of EVL as indicated. From left to right, Panel 1: Merge of LifeAct and mEmerald-tagged proteins. Scale bar = 10 µm. Panel 2: mEmerald fluorescence intensity presented with an inverted LUT. Panel 3: Kymograph of DF position indicated by arrowhead (5 s interval, 5 min duration). Vertical scale bar = 1 µm, horizontal scale bar = 1 min, dashed line indicates dendrite. Panel 4: Maximum intensity projection of temporally color-coded binary mask outline (5 s interval, 5 min duration). (D) Scatterplot of average speed of DF tips, calculated as the average absolute tip displacement between successive timepoints. Gray shaded region indicates average speed less than 0.0128 µm/s (non-motile DFs). Median ± IQR. Mixed-effects model; n = 248–397 total DF from 3–6 neurons per biological replicate, N = 3–5 biological replicates. (E) Scatterplot of average length reached during the duration of imaging for indicated conditions. Median ± IQR. Mixed-effects model; n = 248–397 total DF from 3–6 neurons per biological replicate, N = 3–5 biological replicates. (F) Scatterplot of median protrusion rates of DFs for indicated conditions. Median ± IQR. Mixed-effects model; n = 248–397 total DF from 3–6 neurons per biological replicate, N = 3–5 biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s. is not significant. See also Video 5. Source data are available for this figure: SourceData F5.

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