Figure 1.
EVL is the predominant Ena/VASP paralog regulating dendritic filopodia. (A) Live primary mouse cortical neurons at day in vitro 11 (D11) expressing EGFP-LifeAct and doxycycline-inducible mCherry-AP4-MITO (left column) or mCherry-FP4-MITO (right column), imaged 12 h after doxycycline induction. Top: Full cell image. Inset: mCherry-AP4/FP4-MITO expression. Middle: Indicated segment of dendrite presented with an intensity-coded LUT. Bottom: Maximum intensity projection of temporally color-coded binary mask outline, illustrating DF dynamics during imaging (5 s interval, 5 min duration). (B) 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 ± interquartile range (IQR). Mixed-effects model was used for statistical comparisons; n = 731–917 total DF from 4–7 neurons per biological replicate, N = 3 biological replicates. (C) Bar graph of percent of total DF population with average tip speeds greater than 0.0128 µm/s (motile) or less than 0.0128 µm/s (non-motile). Mann-Whitney test; n = 731–917 total DF from 4–7 neurons per biological replicate, N = 3 biological replicates. (D) Scatterplot of average length reached during the duration of imaging. Median ± IQR. Mixed-effects model; n = 731–917 total DF from 4–7 neurons per biological replicate, N = 3 biological replicates. (E) RT-qPCR of RNA samples from primary mouse cortical neuron cultures at indicated days in vitro. CTs were normalized to the average of three housekeeping genes, and relative copy numbers were generated using2-ΔCT × 106. Floating bars span the minimum and maximum data points, central line denotes mean. N = 3 biological replicates. (F) Representative western blot of protein lysates from primary neuron cultures at indicated days in vitro, probed with antibodies targeting MENA, EVL, and VASP. (G) Live D11 neurons expressing EGFP-LifeAct and pLKO-shRNA-TurboRFP targeting Enah, Evl, or non-targeting (n.t.) as indicated. Cells were transduced with shRNA lentiviral particles on D7. Top: Full cell image. Inset: TurboRFP expression identifying shRNA-positive neurons. Middle: Indicated segment of dendrite. Bottom: Maximum intensity projection of temporally color-coded binary mask outline (5 s interval, 5 min duration). (H) Scatterplot of average speed of DF tips. Gray shaded region indicates average speed <0.0128 µm/s (non-motile). Median ± IQR. Mixed-effects model; n = 630–1013 total DF from 4–5 neurons per biological replicate, N = 3–5 biological replicates. (I) Scatterplot of average length reached during duration of imaging. Median ± IQR. Mixed-effects model; n = 630–1,013 total DF from 4–5 neurons per biological replicate, N = 3–5 biological replicates. (J) Bar graph of percent time motile (percent of time per DF in which instantaneous speed was greater than 0.0128 µm/s). Median ± IQR. Kruskal-Wallis test corrected for multiple comparisons; n = 630–1,013 total DF from 4–5 neurons per biological replicate, N = 3–5 biological replicates. (K) Scatterplot of median protrusion and retraction rates of DFs (the median of values when instantaneous change in length was greater than ±0.0128 µm/s [motile]). Median ± IQR. Mixed-effects model; n = 630–1,013 total DF from 4–5 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. Scale bars = 10 µm. See also Fig. S2 and Video 1. Source data are available for this figure: SourceData F1.

EVL is the predominant Ena/VASP paralog regulating dendritic filopodia. (A) Live primary mouse cortical neurons at day in vitro 11 (D11) expressing EGFP-LifeAct and doxycycline-inducible mCherry-AP4-MITO (left column) or mCherry-FP4-MITO (right column), imaged 12 h after doxycycline induction. Top: Full cell image. Inset: mCherry-AP4/FP4-MITO expression. Middle: Indicated segment of dendrite presented with an intensity-coded LUT. Bottom: Maximum intensity projection of temporally color-coded binary mask outline, illustrating DF dynamics during imaging (5 s interval, 5 min duration). (B) 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 ± interquartile range (IQR). Mixed-effects model was used for statistical comparisons; n = 731–917 total DF from 4–7 neurons per biological replicate, N = 3 biological replicates. (C) Bar graph of percent of total DF population with average tip speeds greater than 0.0128 µm/s (motile) or less than 0.0128 µm/s (non-motile). Mann-Whitney test; n = 731–917 total DF from 4–7 neurons per biological replicate, N = 3 biological replicates. (D) Scatterplot of average length reached during the duration of imaging. Median ± IQR. Mixed-effects model; n = 731–917 total DF from 4–7 neurons per biological replicate, N = 3 biological replicates. (E) RT-qPCR of RNA samples from primary mouse cortical neuron cultures at indicated days in vitro. CTs were normalized to the average of three housekeeping genes, and relative copy numbers were generated using2-ΔCT × 106. Floating bars span the minimum and maximum data points, central line denotes mean. N = 3 biological replicates. (F) Representative western blot of protein lysates from primary neuron cultures at indicated days in vitro, probed with antibodies targeting MENA, EVL, and VASP. (G) Live D11 neurons expressing EGFP-LifeAct and pLKO-shRNA-TurboRFP targeting Enah, Evl, or non-targeting (n.t.) as indicated. Cells were transduced with shRNA lentiviral particles on D7. Top: Full cell image. Inset: TurboRFP expression identifying shRNA-positive neurons. Middle: Indicated segment of dendrite. Bottom: Maximum intensity projection of temporally color-coded binary mask outline (5 s interval, 5 min duration). (H) Scatterplot of average speed of DF tips. Gray shaded region indicates average speed <0.0128 µm/s (non-motile). Median ± IQR. Mixed-effects model; n = 630–1013 total DF from 4–5 neurons per biological replicate, N = 3–5 biological replicates. (I) Scatterplot of average length reached during duration of imaging. Median ± IQR. Mixed-effects model; n = 630–1,013 total DF from 4–5 neurons per biological replicate, N = 3–5 biological replicates. (J) Bar graph of percent time motile (percent of time per DF in which instantaneous speed was greater than 0.0128 µm/s). Median ± IQR. Kruskal-Wallis test corrected for multiple comparisons; n = 630–1,013 total DF from 4–5 neurons per biological replicate, N = 3–5 biological replicates. (K) Scatterplot of median protrusion and retraction rates of DFs (the median of values when instantaneous change in length was greater than ±0.0128 µm/s [motile]). Median ± IQR. Mixed-effects model; n = 630–1,013 total DF from 4–5 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. Scale bars = 10 µm. See also Fig. S2 and Video 1. Source data are available for this figure: SourceData F1.

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