Feline Lindhout

3 Centrosome-mediated microtubule remodeling during axon formation in human iPSC-derived neurons 83 Figure 3. Centriole loss in NSCs is accompanied with changes in neurite growth cone morphology A. Correlative plot of changes in protein abundance between control neurons and neurons treated with Centrinone-B (day7/day3). Pearson correlation R=0.5716, p<0.001. Specific, significantly changing growth cone proteins highlighted in red. B. Protein abundance profile over time for axon-related proteins Trim46 and Tau in control neurons and neurons treated with Centrinone-B (+C). C. Protein abundance profile over time for growth cone-related proteins Basp1, Gap43 and Marcks in control neurons and neurons treated with Centrinone-B (+C). D. Representative images of fan-like growth cones at day 5 of control and Centrinone-B treated neurons in untreated, or nocodazole-treated conditions. Growth cone visualized by immunostaining for phalloidin. Scale bar = 5µm. E. Quantifications of the average area (µm²) of growth cones of control and Centrinone-B treated neurons in untreated, or nocodazole-treated conditions at different time points. N=3, n=25-83. F. Representative images of different growth cone morphological categories: fan-like, torpedo-like and bulb-like. Scale bar = 5µm. G. Quantifications of the ratios of different subtypes (fan-like, torpedo-like, bulb-like) of growth cones of control and Centrinone-B treated neurons in untreated, or nocodazole-treated conditions at different time points. H. Quantifications of the average area (µm²) of different subtypes (fan-like, torpedo-like, bulb-like) of growth cones of control and Centrinone-B treated neurons in untreated, or nocodazole-treated conditions at different time points. N=3, n=5-55. Data information: Data represent mean ± SEM. One-way ANOVA including Tukey’s post-hoc analysis ( E ) , Chi-square-test including post-hoc analysis with Bonferroni correction ( G ), One-way ANOVA including Sidak’s post-hoc analysis ( H ). *** p<0.001, ** p<0.005, ns p≥0.05 of growth cones. However, Centrinone-B as well as Nocodazole treatment did result in significantly smaller fan-like growth cones at day 5, which in control neurons are considerably larger than torpedo- and bulb-like growth cones (Fig 3H). This effect was distinct for fan- like growth cones, as the sizes of torpedo-like and bulb-like growth cones were unaffected, which suggests that the observed decrease of growth cone size is specifically due to affected fan-like growth cones (Fig 3E,H). The actin cytoskeleton is another important cytoskeletal component at growth cones, and was previously found to be controlled by microtubules as well as centrosome activity in dissociated rodent neurons (Zhao et al. 2017; Meka et al. 2019). Here, we observed no changes in local levels of F-actin at growth cones, suggesting that the reduced size of fan-like growth cones is not caused by changes in F-actin levels in growth cones (Fig S3E). Together, these results indicate that centriole depletion causes growth cone morphology defects during axon outgrowth, presumably by perturbing the underlying microtubule network.