Figure S4.
Tip enrichment of EVL precedes DF protrusion. (A) Scatterplots of tip fluorescence and tip motility z-scores at individual timepoints across entire population of analyzed DFs from D11 neurons expressing EGFP-MENA or EGFP-EVL. Poor correlation of tip fluorescence and motility observed by Spearman’s correlation test across total population of DFs. Individual timepoints presented, n = 368–389 total DF from 4–6 neurons per biological replicate, N = 3 biological replicates. (B) Heatmaps of cross-correlation function (CCF) of normalized tip fluorescence intensity and tip motility per individual DF (rows) as a function of time offset (columns), of DFs from D11 neurons of indicated conditions. Positive cross-correlations indicated in color, negative cross-correlations indicated in gray. High cross-correlation occurring with negative offset values indicates that fluorescence enrichment precedes motility; high cross-correlation with positive offset values indicates fluorescence enrichment follows motility. Color shaded region denotes top-correlating subcluster (TCS) of DFs within each condition. This subpopulation was used to compare metrics for DFs in which fluorescence intensity and motility are highly correlated (TCS), versus DFs in which this correlation is weak or negatively correlated (non-TCS). Histogram (overlaid on color code scale bar) displays DF population distribution across cross-correlation values. (C) Scatterplots of tip fluorescence and tip motility z-scores at individual timepoints for top-correlating subcluster (color) overlaid on non-top-correlating subcluster (gray) DFs of indicated conditions. Strong correlation observed by Pearson’s correlation test for TCS compared to non-TCS of DFs. Individual timepoints presented, 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. (D) Line plots of cross-correlation values of normalized tip fluorescence intensity and tip motility as a function of time offset for observed top-correlating subcluster (color lines) and the TCS from randomized datasets (gray lines). Randomization was performed by shuffling eight-timepoint blocks of motility data with respect to tip fluorescence per DF (block bootstrap). N = 1,000 randomized datasets. (E) Frequency histogram displaying distribution of TCS cross-correlation values for randomized subcluster datasets (gray bars) compared to observed TCS cross-correlation values (color dashed line). Frequency of randomized mean peak cross-correlation value exceeding observed mean peak: EGFP-MENA 114/1,000 (bootstrap P = 0.114); EGFP-EVL 3/1,000 (bootstrap P = 003). See also Fig. 3.

Tip enrichment of EVL precedes DF protrusion. (A) Scatterplots of tip fluorescence and tip motility z-scores at individual timepoints across entire population of analyzed DFs from D11 neurons expressing EGFP-MENA or EGFP-EVL. Poor correlation of tip fluorescence and motility observed by Spearman’s correlation test across total population of DFs. Individual timepoints presented, n = 368–389 total DF from 4–6 neurons per biological replicate, N = 3 biological replicates. (B) Heatmaps of cross-correlation function (CCF) of normalized tip fluorescence intensity and tip motility per individual DF (rows) as a function of time offset (columns), of DFs from D11 neurons of indicated conditions. Positive cross-correlations indicated in color, negative cross-correlations indicated in gray. High cross-correlation occurring with negative offset values indicates that fluorescence enrichment precedes motility; high cross-correlation with positive offset values indicates fluorescence enrichment follows motility. Color shaded region denotes top-correlating subcluster (TCS) of DFs within each condition. This subpopulation was used to compare metrics for DFs in which fluorescence intensity and motility are highly correlated (TCS), versus DFs in which this correlation is weak or negatively correlated (non-TCS). Histogram (overlaid on color code scale bar) displays DF population distribution across cross-correlation values. (C) Scatterplots of tip fluorescence and tip motility z-scores at individual timepoints for top-correlating subcluster (color) overlaid on non-top-correlating subcluster (gray) DFs of indicated conditions. Strong correlation observed by Pearson’s correlation test for TCS compared to non-TCS of DFs. Individual timepoints presented, 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. (D) Line plots of cross-correlation values of normalized tip fluorescence intensity and tip motility as a function of time offset for observed top-correlating subcluster (color lines) and the TCS from randomized datasets (gray lines). Randomization was performed by shuffling eight-timepoint blocks of motility data with respect to tip fluorescence per DF (block bootstrap). N = 1,000 randomized datasets. (E) Frequency histogram displaying distribution of TCS cross-correlation values for randomized subcluster datasets (gray bars) compared to observed TCS cross-correlation values (color dashed line). Frequency of randomized mean peak cross-correlation value exceeding observed mean peak: EGFP-MENA 114/1,000 (bootstrap P = 0.114); EGFP-EVL 3/1,000 (bootstrap P = 003). See also Fig. 3.

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