Feline Lindhout

3 Centrosome-mediated microtubule remodeling during axon formation in human iPSC-derived neurons 87 during early axon development, thereby setting-up the foundation for subsequent axon maturation and functioning. Trim46 localizes at the centrosome and AIS at different stages of neuronal development Trim46 was identified as a microtubule-associated protein localizing to the proximal part of the AIS, where it locally binds and stabilizes parallel microtubule bundles (van Beuningen et al. 2015). In this study we report the uncharacteristic localization of Trim46 near centrosomes in unpolarized neurons, which is shifted towards axons during neuronal polarization. Interestingly, a similar developmental shift was previously reported for NDEL1, a dynein regulating protein (Kuijpers et al. 2016). It remains unknown if there are additional microtubule associated proteins showing the developmental translocation fromcentrosomes to axons. Remarkably, the axonal accumulation of Trim46 was perturbed upon centriole removal, likely signifying a differentially organized axonal microtubule network. Consistent with this idea, we observed less parallel plus-end out microtubules in axons upon centriole loss. Previous in vitro reconstitution assays revealed that Trim46 binding favors bundles of parallel oriented microtubules (Freal et al. 2019). High resolution imaging showed that Trim46 appeared as small punctae surrounding the outer layer of the pericentriolar material and did not coincide with previously resolved structures of centrosomal proteins (Mennella et al. 2012). Considering that Trim46 is a microtubule-associated protein favoring parallel microtubule bundles, we speculate that Trim46 localizes to the starting points of centrosomal microtubule arrays nucleated from yTuRCs at the PCM (Freal et al. 2019). Hence, centrosomes are likely to represent the site with the highest occurrence of parallel oriented microtubules in cells with a radial microtubule network. The function of centrosome-associated Trim46 remains elusive, although it is tempting to speculate that it plays a role in stabilizing the parallel oriented microtubules, consistent with its previous reported function in axons (van Beuningen et al. 2015). Dissecting the mechanistic role of centrosomes in axon development Our data indicates that the centrosome is a critical regulator of axonal microtubule organization during early neuronal development. First, we show that centrosomes display microtubule organizing functions during the process of axon specification, and not in polarized neurons. This illustrates the potential of centrosomes, as main MTOCs, to contribute to microtubule remodeling during onset of axon development. Second, centriole loss had distinct effects on AIS assembly and function, as illustrated by the absence of microtubule binding Trim46 proteins, and more immature AP firing and reduced sodium currents. It is likely that the perturbed centrosome-mediated microtubule remodeling underlies the observed functional defects. Third, at earlier developmental stages, centriole loss affected growth cone formation, which was mimicked by drug treatments that cause microtubule destabilization. Fourth, we found that centriole loss results in a marked delay in the axon-specific uniform plus-end out microtubule reorganization. The precise mechanism