MENA and EVL overexpression enhance DF motility. (A) Live primary mouse cortical neurons at day in vitro 11 (D11) expressing mRuby2-LifeAct and EGFP (left column), EGFP-MENA (middle column), or EGFP-EVL (right column). Row 1: Full cell image. Row 2: Indicated segment of dendrite. Rows 3 and 4: Individual fluorescent channels presented with intensity-coded LUTs. Row 5: 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; n = 368–389 total DF from 4–6 neurons per biological replicate, N = 3 biological replicates. (C) Scatterplot of average length reached during the duration of imaging. Median ± IQR. Mixed-effects model; n = 368–389 total DF from 4–6 neurons per biological replicate, N = 3 biological replicates. (D) 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 = 368–389 total DF from 4–6 neurons per biological replicate, N = 3 biological replicates. (E) 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 = 368–389 total DF from 4–6 neurons per biological replicate, N = 3 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. S3 and Video 2.