(A) Schematic depicting the MT growth and shrinkage cycle. MTs grow until they undergo a catastrophe, which initiates MT shrinkage, and they start growing again after a rescue event. During growth (but not during shrinkage), EB1 localizes to MT tips. (B) First frame of a live cell imaging movie of axonal EB1-GFP dynamics. Bright dots represent individual EB1-GFP puncta, which label growing MT plus-ends. (C) Maximum intensity projection of a 200-s-long movie depicting EB1-GFP dynamics in a Drosophila melanogaster axon. EB1-GFP density is increased towards the tip. (D) Schematic showing how EB1-GFP live imaging movies were visualized and analysed using kymographs. The growing tips of plus-end-out MTs (blue) and minus-end-out MTs (red) were fluorescently labelled with EB1-GFP (green tear drop shaped ‘comets’). The same axon is shown at three different time points; MTs grow at their plus-end, where EB1-GFP is located. The axonal intensity profiles of all time points are plotted underneath each other, resulting in a space-time grid called ‘kymograph’. Connecting puncta between consecutive kymograph lines with blue/red lines yields the overall displacement dg for individual MT growth events. Note that the red minus-end-out MT stops growing in the second frame and shrinks in the third frame (E) Kymograph of an axon 24 hr post plating showing EB1-GFP dynamics analysed with KymoButler (Jakobs et al., 2019). Lines with a positive slope (blue, left to right upwards) are MTs growing with their plus-end towards the axon tip, lines with a negative slope (red, left to right downwards) are MTs growing away from the tip. Horizontal bars indicate the growth lengths (dg) for individual MT growth cycles. (F) Kymographs of axonal processes expressing EB1-GFP analysed with KymoButler 4 hr post plating (G) MT orientation as a function of axon length. Longer axons exhibit a more pronounced plus-end-out MT orientation (p<10–5, Kruskal-Wallis test, **p<0.01, ***p<0.001 for pairwise comparisons, Dunn-Sidak post hoc test). (H) MT orientation at 4 and 24 hiv (hours in vitro). MT orientation increased with time (p<10–4, Wilcoxon rank sum test). (I) EB1-GFP comet density as a function of the distance from the axon tip. Most MT polymerization occurred near the advancing axon tip. (N Axons = 353, 20 biological replicates from 20 different experiment days, p<10–20, Kruskal-Wallis test, p<10–7 for pairwise comparisons between bins 1–2, 3, and 4, Dunn-Sidak post hoc test). Shown are median±95% confidence interval. (J) Added length per MT growth cycle dg as a function of distance from the axon tip. MTs grew longer in the vicinity of the axon tip (p<10–20, Kruskal-Wallis test, p<10–7 for pairwise comparisons of either bin 1 or 2 with any other bin, Dunn-Sidak post hoc test). (K) dg for plus-end-out (blue) and minus-end-out (red) MTs grouped for growth in the distalmost 10 µm of the axon tip, and further away than 10 µm from the axon tip. Each dot represents the average of one axon in the respective region, grey lines indicate median values. With dg 2.11 [2.04, 2.16] µm/cycle (bootstrapped median [95% confidence interval]), plus-end-out MTs near the axon tip grew significantly longer than minus-end-out MTs (dg = 1.39 [1.27, 1.50] µm/cycle) and MTs located further away from the tip (dg = 1.53 [1.47, 1.59] µm/cycle, plus-end-out, dg = 1.16 [1.03, 1.33] µm/cycle, minus-end-out) (N=346 (plus-end-out close to tip), 343 (plus-end-out away from tip), 177 (minus-end-out close to tip), 194 (minus-end-out away from tip) axons), 20 biological replicates; p<10–30, Kruskal-Wallis test followed by Dunn-Sidak post hoc test; ***p<10–4. Scale bars: 3 µm.