Figure 5.
Figure 5. Analysis of tip fluorescence and tip movement. (A) For the purpose of quantifying fluorescence in filopodia tips, the tip fitting mode of Filopodyan searches for accumulation of fluorescence signal in the immediate vicinity of initially assigned tip positions and readjusts the assigned tip position (white arrowhead) to match the position of the fluorescence signal (cyan arrowheads). (B) Filopodyan also allows connecting disconnected fragments (e.g., caused by loss of focus; arrowheads) with the reconstructed filopodium. (C) A time-lapse series of a filopodium from Xenopus RGC growth cone expressing mNeonGreen-ENA (Video 8), showing enhanced forward tip movement upon increased ENA fluorescence (at 0–20 s and 160–192 s) and absence of movement or tip retraction upon reduced ENA fluorescence (40–160 s and 192–240 s). (D) Concurrent tip fluorescence (normalized to growth cone fluorescence) and tip movement (smoothed with a five-step rolling mean) measurements for the example filopodium shown in C, showing positive correlation between fluorescence and movement. Dashed gray lines at −32.5 and +32.5 nm/s represent the thresholds for retraction and extension. (E) Positive correlation between ENA tip fluorescence and tip movement across the time-lapse for the example filopodium shown in C (with no time offset). (F) An example filopodium showing lack of correlation between tip ENA accumulation and tip movement (e.g., extending without ENA enrichment during 10–34 s). See also Video 9. (G) Concurrent tip fluorescence and tip movement measurements for the example filopodium shown in F. (H) Absence of correlation between ENA tip fluorescence and tip movement measurements across the time-lapse for the example filopodium shown in F. (I) Cross-correlation function (CCF) between normalized ENA tip fluorescence and tip movement for each filopodium (rows) for each value of time offset (columns) between fluorescence and movement, displayed as a heatmap. Blue shows a positive correlation and red a negative correlation. Negative offset, fluorescence precedes movement; positive offset, fluorescence lags behind movement. n = 46 filopodia. Blue shaded block on the dendrogram indicates the subcluster of positively correlating (ENA-responding) filopodia. Line plot shows collective CCF values for each subcluster (ENA-responding filopodia and all other filopodia); lines and shading represent means ± 95% confidence intervals. Bars, 1 µm.

Analysis of tip fluorescence and tip movement. (A) For the purpose of quantifying fluorescence in filopodia tips, the tip fitting mode of Filopodyan searches for accumulation of fluorescence signal in the immediate vicinity of initially assigned tip positions and readjusts the assigned tip position (white arrowhead) to match the position of the fluorescence signal (cyan arrowheads). (B) Filopodyan also allows connecting disconnected fragments (e.g., caused by loss of focus; arrowheads) with the reconstructed filopodium. (C) A time-lapse series of a filopodium from Xenopus RGC growth cone expressing mNeonGreen-ENA (Video 8), showing enhanced forward tip movement upon increased ENA fluorescence (at 0–20 s and 160–192 s) and absence of movement or tip retraction upon reduced ENA fluorescence (40–160 s and 192–240 s). (D) Concurrent tip fluorescence (normalized to growth cone fluorescence) and tip movement (smoothed with a five-step rolling mean) measurements for the example filopodium shown in C, showing positive correlation between fluorescence and movement. Dashed gray lines at −32.5 and +32.5 nm/s represent the thresholds for retraction and extension. (E) Positive correlation between ENA tip fluorescence and tip movement across the time-lapse for the example filopodium shown in C (with no time offset). (F) An example filopodium showing lack of correlation between tip ENA accumulation and tip movement (e.g., extending without ENA enrichment during 10–34 s). See also Video 9. (G) Concurrent tip fluorescence and tip movement measurements for the example filopodium shown in F. (H) Absence of correlation between ENA tip fluorescence and tip movement measurements across the time-lapse for the example filopodium shown in F. (I) Cross-correlation function (CCF) between normalized ENA tip fluorescence and tip movement for each filopodium (rows) for each value of time offset (columns) between fluorescence and movement, displayed as a heatmap. Blue shows a positive correlation and red a negative correlation. Negative offset, fluorescence precedes movement; positive offset, fluorescence lags behind movement. n = 46 filopodia. Blue shaded block on the dendrogram indicates the subcluster of positively correlating (ENA-responding) filopodia. Line plot shows collective CCF values for each subcluster (ENA-responding filopodia and all other filopodia); lines and shading represent means ± 95% confidence intervals. Bars, 1 µm.

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