Feline Lindhout

3 Centrosome-mediated microtubule remodeling during axon formation in human iPSC-derived neurons 77 Figure 1. Centrosomes display MTOC function and Trim46 appearance during axon specification A. Schematic illustration of centrosomal MTOC function in neurodevelopmental stages 1, 2 and 3 in human iPSC-derived NSCs/neurons. B. Typical examples of stage 1 (day 0), stage 2 (day 7) and stage 3 (day 12) human iPSC-derived NSCs/neurons immunostained for γ-Tubulin and Centrin. Cells were co-immunostained with Ki67 (day 0) or Trim46 and MAP2 (day 7 and 12) to define their neuronal stages. Scale bars: 10 µm in overview, 2 µm in zooms. C. Quantifications of normalized γ-Tubulin fluorescent intensities at centrosomes in stage 1, 2 and 3 human iPSC-derived neurons. N=2, n=46-51 cells. D. Human iPSC-derived NSCs (day 0) immunostained for Trim46, γ-Tubulin and Centrin. Zoom represents centrosome structure. Scale bars: 5 µm in overview, 2 µm in zooms. E. Centrosomes of human iPSC-derived NSCs (day 0) with STED imaging of Trim46 and Centrin immunostaining, and confocal imaging of γ-Tubulin immunostaining. Merged images show different examples of centrosome structures. Scale bar: 1 µm. F. Typical examples of stage 2 and stage 3 human iPSC-derived neurons immunostained for Trim46 and MAP2. Inserts represent centrosomes. Zooms on the right represent a non-polarized neurite or a developing axon in stage 2 or stage 3 neurons, respectively. Scale bars: 20 µm in overview, 1 µm in insert, 10 µm in zoom. Data information: Data represent mean ± SEM. One-way ANOVA including post-hoc analysis with Bonferroni correction ( B , C ). *** p<0.001, ns p>0.05 efficient and robust centriole loss by Centrinone-B treatment has previously been validated in various other cell types (Wong et al. 2015). Centrinone-B is a PLK4-inhibitor that blocks centriole duplication during cell division in proliferating cells, thereby generating a mixed population of cells containing 0, 1 or 2 centrioles. We treated human iPSC-derived NSCs for 0, 2 or 5 days with Centrinone-B prior to neuronal induction and quantified the number of centrioles per cell. Centrioles were defined as puncta with overlapping staining of Pericentrin and Centrin (Fig 2A). We observed successful removal of either 1 or 2 centrioles in ~50% of the NSCs after 2 days of Centrinone-B treatment, which was not significantly enhanced after a prolonged 5 day treatment (Fig 2B). Additionally, Centrinone-B treatment increased the number of cells with a characteristic neuron-like morphology, even before inducing neuronal differentiation. This premature neuronal differentiation phenotype is a well-described hallmark of centrosome dysfunction in various in vivo or 3D in vitro systems, as it underlies microcephaly and other neurodevelopmental disorders (Lancaster et al. 2013). We found significantly more neurons upon 2 days of Centrinone-B treatment, measured as the relative number of cells that were positive for neuron differentiation markers MAP2 or -3-Tubulin, or proliferation marker Ki67 (Fig S2A-E). Together, these data show that Centrinone-B treatment results in successful depletion of centrioles in human iPSC-derived neuronal cells, and that it recapitulates neuronal developmental phenotypes that are broadly associated with centrosome defects.