Feline Lindhout

3 86 plus-end out organization of microtubules in axons over time, in a distal-to-proximal fashion (Fig 4C-F). This shift towards a uniform orientation between day 7 and 13 corresponded to a decrease of retrogradely moving comets in most control neurons (Fig S4C-F). This developmental shift is less prevalent upon centriole loss, as shown by a decrease in neurons with uniform anterogradely moving comets (Fig 4D-F). Moreover, this was accompanied by a less distinct decrease in the number of retrograde comets over time compared to control (Fig S4C-F). The reorganization of microtubules corresponded with trends towards a decrease in the comet growth speed in control but not in Centrinone-B treated neurons, whereas comet run length was not changed over time in both conditions (Fig S4J-O, Table2,3). Imaging of MT+TIPs only provides information about the dynamic ends of microtubules, but does not account for stabilized microtubules. Thus, we next aimed to analyze the microtubule orientations of the total axonal microtubule network, including both sTable and dynamic microtubules. This was addressed by combining our approach with laser-severing to generate new microtubule ends by cutting microtubules with a short- pulsed laser, which triggers newly formed MT+TIPs (Fig 4G,H, S4Q, Movie S2) (Yau et al. 2016). Following laser severing of microtubules in axons, we observed an increase in the total number of mostly retrograde comets, which reflects the sTable pool of minus-end out microtubules (Fig S4D,F,S,U). In line with previous results, we found that the impaired shift towards uniform anterogradely moving comets in Centrinone-B treated neurons was more profound following laser-severing (Fig 4I-J, S4R-U). This is most strikingly observed at distal axon regions of day 13 neurons, the most mature axonal stage, as ~90% of the axons of control neurons showed a uniform plus-end out microtubule whereas this is ~50% in Centrinone-B treated neurons (Fig 4L). In dendrites, a shift towards a mixed microtubule network is observed in both control and Centrinone-B treated neurons (Fig S4A,G-I,P,V-X). Together, these data suggest that centriole loss is important for the unique axon-specific reorganization towards uniform plus-end out microtubules. DISCUSSION Multiple studies attempted to address the possible role of centrosomes during axon development. Early reports suggested that microtubule nucleation is important for axon formation and outgrowth in dissociated rodent neurons, and proposed that centrosomes could play an important role in axon development (Ahmad et al. 1994). Indeed, in Drosophila neurons, centrosome dysfunction affected axon formation and outgrowth (de Anda et al. 2005). In dissociated rodent neuron cultures, centrosome positioning was found to correlate with sites of newly emerging axons (Zmuda and Rivas 1998; de Anda et al. 2005). However, centrosome removal in polarized neurons did not affect further axon outgrowth in dissociated rodent neurons (Stiess et al. 2010). Nevertheless, the role of centrosomes in axon specification remained largely unexplored. This is mostly due to technical challenges that prevent investigation of the very early molecular processes that drive axon specification. Here, we used human iPSC-derived neurons to investigate centrosome function during axon specification. We uncovered that centrosomes are important for microtubule remodeling