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3 Centrosome-mediated microtubule remodeling during axon formation in human iPSC-derived neurons 85 Figure 4. Centriole loss restrains microtubule remodeling during early axon development A. Schematic illustration of stage 3 human iPSC-derived neurons. Different locations of the neurons that are imaged have been outlined and annotated. B. Kymographs and schematic representations of time-lapse recordings as shown in S4 B, for different positions (proximal axon; distal axon), time points (day 7; day 13) and conditions (control; Centrinone-B). Scale bar: 5 µm. C . Quantifications of the ratios of comets pointing in anterograde (green) or retrograde (blue) direction in the proximal axon. N= 3, n= 7-11 cells. D. Quantifications of the percentage of neurons exhibiting uniform, or not uniform comet orientations in the anterograde direction in the proximal axon. N= 3, n= 7-11 cells. E. Quantifications of the ratios of comets pointing in anterograde (green) or retrograde (blue) direction in the distal axon. N= 3, n= 8-12 cells. F. Quantifications of the percentage of neurons exhibiting uniform, or not uniform comet orientations in the anterograde direction in the distal axon. N= 3, n= 8-12 cells. G . Schematic representation of microtubule laser-severing (LS) experiments. H. Kymographs and schematic representations of time-lapse recordings as shown in S4Q following LS, for different positions (proximal axon; distal axon), time points (day 7; day 13) and conditions (control; Centrinone-B). Red line and red arrowhead denote location and time of LS. Scale bars: 5 µm. I. Quantifications of the ratios of comets pointing in anterograde (green) or retrograde (blue) direction in the proximal axon following LS. N=3 , n=19-30 cells. J. Quantifications of the percentage of neurons exhibiting uniform, or not uniform comet orientations in the anterograde direction in the proximal axon following LS. N= 3, n= 19-30 cells. K . Quantifications of the ratios of comets pointing in anterograde (green) or retrograde (blue) direction in the distal axon following LS. N=3 , n=19-30 cells. L. Quantifications of the percentage of neurons exhibiting uniform, or not uniform orientations in the anterograde direction in the distal axon following LS. N= 3, n= 19-30 cells. Data information: Data represent mean ± SEM. Chi-square-test including post-hoc analysis with Bonferroni correction ( D,F,J,L ); *** p<0.001, ** p<0.005, * p<0.05, ns p≥0.05 Centriole loss perturbs microtubule reorganization in developing axons To gain more insight into the potential role of centrosomes on the organization of the axonal microtubule cytoskeleton, we studied the effect of centriole loss on the microtubule remodeling processes during development. We systematically analyzed plus-end dynamics and orientations of microtubules at different cellular compartments on day 7 and 13, which coincide with the onset of stage 3 and late stage 3, respectively, in control and Centrinone-B treated neurons (Fig 4A). We used two-color live-cell imaging to visualize neurite morphology and microtubule plus-end tracking proteins (MT+TIPs) (Fig 4B, S4B, Movie S1). In control neurons, we observed the characteristic adoption of a unidirectional,