Figure 3.
Tip enrichment of EVL precedes DF protrusion. (A) Filmstrip of live primary mouse cortical neurons at day in vitro 11 (D11) expressing EGFP (top row), EGFP-MENA (middle row), or EGFP-EVL (bottom row) in a representative DF showing localization dynamics presented with an intensity-coded LUT. Line scan of normalized fluorescence intensity over time at tracked tip (right; 5 s interval, 1 min duration). Scale bar = 1 µm. (B) Scatterplot of tip fluorescence variance at DF tips normalized to local background demonstrates range of tip enrichment per DF and hence magnitude of on-off dynamics. Median ± interquartile range (IQR). Mixed-effects model; n = 368–389 total DF from 4–6 neurons per biological replicate, N = 3 biological replicates. (C) Live D11 neurons expressing mRuby2-LifeAct and EGFP (top row), EGFP-MENA (middle row), or EGFP-EVL (bottom row). Segment of dendrite (left); scale bar = 10 µm. Numbers indicate DF position analyzed by kymograph (right), highlighting protein localization dynamics during DF motility (5 s interval, 5 min duration). Vertical scale bar = 1 µm, horizontal scale bar = 1 min, dashed line indicates dendrite. (D) Line plots of cross-correlation function (CCF) of normalized tip fluorescence intensity and tip motility as a function of time offset for top-correlating subcluster (TCS; color lines) versus non-top-correlating subcluster (non-TCS; gray lines) determined in Fig. S3 B, of DFs from D11 neurons expressing EGFP, EGFP-MENA, or EGFP-EVL. Peak cross-correlation at negative offset values indicates that fluorescence enrichment precedes motility, while peak cross-correlation at positive offset values indicates fluorescence enrichment follows motility. Mean ±95% CI. Peak cross-correlation GFP = 0.43 at offset 0.413 at offset −5s; MENA = 0.475 at offset 0, 0.464 at offset +5 s; EVL =, 0.482 at offset −10 s, 0.480 at offset −5 s. Statistical significance determined by peak cross-correlation >2/√(n-|offset|). (E) Percent of area under the curve at positive and negative offset values for indicated conditions. (F) Line plots (left) and scatterplots (right) for one representative DF from the top-correlating subcluster in D11 neurons from indicated conditions. Left: Line plot of tip motility (gray, left y-axis) and normalized tip fluorescence (color, right y-axis) during imaging (5 s interval, 5 min duration). Right: Scatterplot of normalized tip fluorescence and tip motility at individual timepoints demonstrating strength of relationship by Pearson’s correlation test. (G) Scatterplot of median protrusion rates for top-correlating subcluster versus non-top-correlating subcluster DFs from indicated conditions. Protrusion rate is the median of values when instantaneous change in length was greater than +0.0128 µm/s (motile, protruding). Median ± interquartile range (IQR). Kruskal-Wallis test corrected for multiple comparisons. TCS n = 42 (MENA) and 39 (EVL); non-TCS n = 320 (MENA) and 321 (EVL) total DF from 4–6 neurons per biological replicate, N = 3 biological replicates. (H) Bar graph of percent of time in protrusion during the duration of imaging for top-correlating subcluster versus non-top-correlating subcluster DFs from indicated conditions. Percent time in protrusion is calculated as the percent of time per DF in which positive change in length between successive timepoints was greater than 0.0128 µm/s. Median ± IQR. Kruskal-Wallis test corrected for multiple comparisons. TCS n = 42 (MENA) and 39 (EVL); non-TCS n = 320 (MENA) and 321 (EVL) 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. See also Fig. S4.

Tip enrichment of EVL precedes DF protrusion. (A) Filmstrip of live primary mouse cortical neurons at day in vitro 11 (D11) expressing EGFP (top row), EGFP-MENA (middle row), or EGFP-EVL (bottom row) in a representative DF showing localization dynamics presented with an intensity-coded LUT. Line scan of normalized fluorescence intensity over time at tracked tip (right; 5 s interval, 1 min duration). Scale bar = 1 µm. (B) Scatterplot of tip fluorescence variance at DF tips normalized to local background demonstrates range of tip enrichment per DF and hence magnitude of on-off dynamics. Median ± interquartile range (IQR). Mixed-effects model; n = 368–389 total DF from 4–6 neurons per biological replicate, N = 3 biological replicates. (C) Live D11 neurons expressing mRuby2-LifeAct and EGFP (top row), EGFP-MENA (middle row), or EGFP-EVL (bottom row). Segment of dendrite (left); scale bar = 10 µm. Numbers indicate DF position analyzed by kymograph (right), highlighting protein localization dynamics during DF motility (5 s interval, 5 min duration). Vertical scale bar = 1 µm, horizontal scale bar = 1 min, dashed line indicates dendrite. (D) Line plots of cross-correlation function (CCF) of normalized tip fluorescence intensity and tip motility as a function of time offset for top-correlating subcluster (TCS; color lines) versus non-top-correlating subcluster (non-TCS; gray lines) determined in Fig. S3 B, of DFs from D11 neurons expressing EGFP, EGFP-MENA, or EGFP-EVL. Peak cross-correlation at negative offset values indicates that fluorescence enrichment precedes motility, while peak cross-correlation at positive offset values indicates fluorescence enrichment follows motility. Mean ±95% CI. Peak cross-correlation GFP = 0.43 at offset 0.413 at offset −5s; MENA = 0.475 at offset 0, 0.464 at offset +5 s; EVL =, 0.482 at offset −10 s, 0.480 at offset −5 s. Statistical significance determined by peak cross-correlation >2/√(n-|offset|). (E) Percent of area under the curve at positive and negative offset values for indicated conditions. (F) Line plots (left) and scatterplots (right) for one representative DF from the top-correlating subcluster in D11 neurons from indicated conditions. Left: Line plot of tip motility (gray, left y-axis) and normalized tip fluorescence (color, right y-axis) during imaging (5 s interval, 5 min duration). Right: Scatterplot of normalized tip fluorescence and tip motility at individual timepoints demonstrating strength of relationship by Pearson’s correlation test. (G) Scatterplot of median protrusion rates for top-correlating subcluster versus non-top-correlating subcluster DFs from indicated conditions. Protrusion rate is the median of values when instantaneous change in length was greater than +0.0128 µm/s (motile, protruding). Median ± interquartile range (IQR). Kruskal-Wallis test corrected for multiple comparisons. TCS n = 42 (MENA) and 39 (EVL); non-TCS n = 320 (MENA) and 321 (EVL) total DF from 4–6 neurons per biological replicate, N = 3 biological replicates. (H) Bar graph of percent of time in protrusion during the duration of imaging for top-correlating subcluster versus non-top-correlating subcluster DFs from indicated conditions. Percent time in protrusion is calculated as the percent of time per DF in which positive change in length between successive timepoints was greater than 0.0128 µm/s. Median ± IQR. Kruskal-Wallis test corrected for multiple comparisons. TCS n = 42 (MENA) and 39 (EVL); non-TCS n = 320 (MENA) and 321 (EVL) 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. See also Fig. S4.

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