Urbančič V , Butler R , Richier B , Peter M , Mason J , Livesey FJ , Holt CE , Gallop JL .

Wellcome Trust , Cancer Research UK, MR/L023784/1 Medical Research Council , MR/L023784/2 Medical Research Council , 281971 European Research Council, 203144 Wellcome Trust , 322817 European Research Council, 085314 Wellcome Trust , A24843 Cancer Research UK

	Figure 1. Filopodyan, a highly customizable pipeline for detection, tracking, and analysis of protrusions in time-lapse datasets. (A) Image of a Xenopus retinal ganglion cell growth cone with filopodia (left) is processed with a Laplacian of Gaussian (LoG) filter for edge contrast enhancement (middle) and binarized with a chosen threshold method (right). (B) Parameters for boundary mapping are customizable; colored lines show the different cell boundaries detected by using three different values of LoG σ with the Renyi entropy thresholding method. (C) Iterative erosion and dilation (ED) of the thresholded mask (from A, right) removes protrusions (left), and the difference between these two masks yields a mask with protrusions alone (center), leading to a fully segmented image (right; protrusions in cyan, growth cone body in magenta, and its boundary in yellow). (D) The cutoff threshold for segmenting protrusions is fully user customizable by adjusting the number of ED operations. The two panels illustrate the difference upon changing the ED number from 3 to 6, showing additional protrusions (white arrowhead) and shifted position of their bases (black arrowheads). (E) Automated filtering removes false hits that do not satisfy user-defined selection criteria. Detected candidate protrusions include true (black arrowheads) as well as erroneous (white arrowheads) annotations that are removed by the filter. Any false hits that escape automated filtering and incorrect tracking can be corrected manually. (F) A time series of images segmented into body (magenta), protrusions (cyan), their bases (orange), and tips (green). (G) Output tables contain the measurements at each time point (row) of protrusion shape, movement, fluorescence, and coordinates (column) for all detected protrusions (column sections). (H) Descriptive properties of filopodia extracted by Filopodyan plugin and the downstream analysis scripts include length, straightness, direction-corrected measures of tip movement and base movement, tip persistence, and the time spent extending, retracting, or stalling. Bars: (A and C) 5 µm; (B and D–F) 2 µm.
	Figure 2. Filopodyan detects and accurately segments filopodia from a variety of different cell types in vitro and in vivo. (A) Growth cone from Xenopus retinal ganglion cell axons in culture, expressing GAP-RFP (a single time point from Video 1). (B) Tracheal cells in a Drosophila embryo expressing btl-Gal4 UAS-Cherry-CAAX (a single time point from Video 2). (C) Leading edge cells during dorsal closure in a Drosophila embryo expressing en-Gal4 UAS-cd8mCherry (a single time point from Video 3). (D) Human iPSC–derived cortical neurons in culture expressing cytoplasmic mNeonGreen (a single time point from Video 4). Bars, 5 µm.
	Figure 3. Correlations between parameters of filopodial dynamics. (A) Properties of Xenopus RGC growth cone filopodia from a manually curated dataset of (n = 160 filopodia and N = 19 neurons). Distribution histograms are shown for selected parameters: maximum length reached during time-lapse, straightness at maximum length, median rate of tip extension during the time-lapse, median rate of base invasion during the time-lapse, proportion of time spent in extending state, and tip persistence; see full description in Materials and methods. Descriptive statistics for these and all other parameters are in Table S1. (B) Comparison of the filopodia phenotype between two analyses of the same dataset: the manually curated analysis and fully automated batch analysis. For each parameter, the normalized boxplot represents the difference of the batch analysis relative to the manual analysis; black and gray vertical lines represent the median and interquartile range of the manually curated dataset for each parameter. Boxes and thick vertical lines represent the interquartile ranges and medians of each parameter for the fully automated (batch) dataset. Full descriptive statistics are in Table S1. (C) Correlation matrix between parameters describing filopodium shape and movement in a dataset of Xenopus RGC growth cone filopodia (n = 160) expressing GAP-RFP and mNeonGreen. Color scaling indicates the sign of correlation (blue to red for positive to negative); circle size indicates correlation strength. (D) Positive correlation between tip persistence of a filopodium and its maximum reached length. (E) Negative correlation between the median rate of tip extension of a filopodium and its proportion of time spent stalling. (F) Correlation matrix visualizing the correlations between the initial movement of the filopodial tips and bases (in the first 20 s after their initial appearance) with other filopodial properties in subsequent time points (after initial 20 s). IQR, interquartile range.
	Figure 4. Predicted filopodium base position enables the measurement of protein recruitment before filopodium formation. (A) Measurements of fluorescence are taken at bases and tips of filopodia during formation (t0) and throughout their lifespan (tn). In addition, fluorescence at the boundary is recorded at the position closest to the site of nascent filopodium formation for a defined number of frames before initiation (t-n). (B) Example filopodium from a growth cone (shown in D) expressing mNeonGreen-ENA and GAP-RFP, illustrating the accumulation of ENA signal at the advancing lamellipodial edge before and during filopodium formation (orange outlined region in B). (C) Quantification of mNeonGreen-ENA fluorescence in the (predicted) base area (orange) and in the tip (green) of the single example filopodium from B. Base fluorescence and thresholded tip fluorescence are normalized against growth cone body fluorescence. After the moment of formation (t0) the enhanced fluorescence signal travels onwards with the moving filopodium tip while dropping at the base. (D) A still image from a time-lapse (Video 5) of a growth cone showing three filopodia during formation all exhibiting enriched levels of ENA fluorescence signal (arrowheads). (E) Normalized base fluorescence (mean and 95% confidence interval) at newly forming filopodia (n = 53, from eight growth cones) expressing mNeonGreen-ENA (cyan), showing accumulation at the site of future filopodium initiation in the time points immediately preceding its formation; this accumulation is not observed for the membrane marker GAP-RFP (red). (F) A still image from a time-lapse (Video 6) of a growth cone expressing mNeonGreen-VASP during the moment of filopodium formation (inset). (G) Normalized base fluorescence at newly forming filopodia in growth cones expressing mNeonGreen-VASP (n = 31, from seven growth cones), showing an increase in fluorescence before initiation. (H) Summary of filopodia properties in neurons expressing mNeonGreen-ENA, compared with mNeonGreen control. (I) Summary of filopodia properties in neurons expressing mNeonGreen-VASP, compared with mNeonGreen control. See Table S1 for full information. (J) Relationship between normalized mNeonGreen-ENA fluorescence at predicted base position before filopodium formation and the proportion of time the filopodium subsequently spent stalling, showing negative correlation. (K) Normalized mNeonGreen-ENA fluorescence at the tip and the proportion of time spent stalling, not recapitulating the negative correlation seen with predicted base fluorescence shown in J. Bars: (main panels) 5 µm; (inset) 1 µm.
	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.
	Figure 6. Heterogeneity of filopodia responses to VASP accumulation within their tips. (A) A filopodium from Xenopus RGC growth cone expressing mNeonGreen-VASP, showing a positive response to VASP accumulation within its tip (surge of forward movement from 96 s, retraction concomitant with the loss of tip fluorescence from 184 s). (B) Concurrent measurements of VASP fluorescence and tip movement for the example filopodium shown in A. (Interruption at t = 160 s represents loss of focus during those time points.) (C) Positive correlation between VASP tip fluorescence and tip movement across the time-lapse for the example filopodium shown in A (at time offset 0). (D) An example filopodium expressing mNeonGreen-VASP showing stalling at elevated levels of VASP (0–80 s) and forward tip extension during a period of reduced VASP fluorescence (112–136 s). (E) Concurrent VASP fluorescence and tip movement measurements across the time-lapse for the filopodium shown in D. (F) Absence of positive correlation between VASP fluorescence and tip movement across the time-lapse for the filopodium shown in D, at time offset 0. (G) Cross-correlation between VASP fluorescence and tip movement for each filopodium (rows) as a function of time offset (columns). n = 76 filopodia. Red shows a negative correlation and blue a positive correlation. Negative offset, fluorescence leads before movement; positive offset, fluorescence lags behind movement. Blue shaded block over the dendrogram represents most strongly positively correlating (VASP-responding) filopodia, and red indicates all other filopodia. (H) The VASP-responding and other filopodia from A and D occupy adjacent positions in the same growth cone. Dashed rectangles indicate crop regions used for the kymographs in A and D. (A still image from Video 10.) (I and J) Filopodia from the VASP-responsive subcluster have a higher rate of tip extension (I) and spend more time retracting (J) than other filopodia. Tukey box plots (box: first and third quartile; upper and lower whiskers: within 1.5 interquartile range of the upper and lower quartile, respectively); p-values are provided for Mann–Whitney test after Holm correction for multiple comparisons. Full information on subcluster properties is in Table S1. Bars: (A and D) 1 µm; (H) 2 µm.

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