Xenbase Image ID: 159296

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. Image published in: Urbančič V et al. (2017) © 2017 Urbančič et al. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View

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