Feline Lindhout

3 Centrosome-mediated microtubule remodeling during axon formation in human iPSC-derived neurons 75 Centrosome-associated localization of AIS protein Trim46 in early-stage human neurons An important hallmark of axon development is the assembly of the AIS in the proximal axon, which occurs after the developmental decline of the microtubule organizing function of centrosomes in dissociated rat hippocampal neurons (Stiess et al. 2010). Surprisingly, we observed that the AIS protein Trim46 was localized at centrosomes prior to AIS assembly (stage 1-2), as shown by co-localization of Trim46 with the centrosome markers Centrin and γ-Tubulin (Fig 1D). Similarly, centrosome-associated localization of Trim46 was observed in human HeLa cells, but not in dissociated rat hippocampal neurons or mouse IMCD3 cells (Fig S1A). Previous studies showed that Trim46 is a microtubule binding protein, however, a possible association with centrosomal proteins or structures has not been investigated (van Beuningen et al. 2015). Thus, we sought to identify which centrosome substructure coincided with Trim46. Localization experiments by confocal microscopy showed that Trim46 appeared as an oval structure which only partially overlapped with the centriolar and pericentriolar structures marked by Centrin and γ-Tubulin, respectively (Fig 1D). To gain more in-depth structural insights, we aimed to resolve centrosomal nanostructures by STED microscopy and observed that Trim46 appeared as a cloud of punctae surrounding but not overlapping with γ-Tubulin structures (Fig 1E). The γ-Tubulin structures mark the outer layer of the pericentriolar material as well as the minus-end nucleation sites of microtubules, suggesting that Trim46 associates with the starting points of centrosomal microtubules (Mennella et al. 2012). Examples of other cells also showed a consistent alignment of Trim46 puncta surrounding γ-Tubulin, which in turn surrounded Centrin puncta representing the centriolar core. Together, these data suggest that specifically in human cells, Trim46 localizes to centrosome-associated structures. Trim46 localization shifts from centrosomes to axonal microtubules during development Next, we studied the localization of Trim46 over time in developing human iPSC-derived neurons. We observed a clear decline of Trim46 staining at the pericentriolar region while the intensity of axonal Trim46 levels increased during development. More specifically, the pericentriolar Trim46 levels remained high in stage 2 neurons and were markedly decreased in stage 3 neurons, following the same trend as γ-Tubulin (Fig 1F). Consistently, we observed a similar shift of Trim46 staining from centrosomes to dense peripheral microtubules arrays in maturing human iPSC-derived glia cells, which are present at low abundance in the human iPSC-derived neuron cultures (Fig S1B). Together, the neuronal data suggest that Trim46 localization shifts from the pericentriolar region to peripheral axonal microtubule arrays. Centrinone-B treatment depletes centrioles in neuronal stem cells To study the effect of centrosome dysfunction on axon specification, we next aimed to remove centrioles in human iPSC-derived NSCs by using Centrinone-B treatment. The