Feline Lindhout

3 Centrosome-mediated microtubule remodeling during axon formation in human iPSC-derived neurons 81 Centriole depletion results in reduced expression of growth cone proteins The centriole-depleted NSCs develop into neurons with structural and functional perturbations in axon development. Next, we aimed to quantify effects of centriole depletion during axon specification with unbiased profiling. Therefore, we performed mass spectrometry based quantitative proteomics analysis on days 1, 3, and 7, which roughly corresponds with developmental stage 1, onset of stage 2, and onset of stage 3, respectively. We compared the proteome dynamics during early neurodevelopment of replicates of Centrinone-B treated and control neurons (Fig S3A, Table 1). Centrinone-B treatment did not markedly alter the relative protein expression over time (Fig 3A). The protein expression profile of control neurons showed a developmental shift, which corresponds to the transitions from stage 1 to stage 2, and from stage 2 to stage 3 (Fig S3B). Protein expression of centriole-depleted neurons largely follows the same trend (Fig S3C). In both populations, proteins considered specific for NSCs are downregulated at days 3 and 7 (e.g. Ki67, Nestin, Otx, Notch1), whereas neuronal proteins are upregulated (e.g. Stathmin1, Map2, Doublecortin, Tubß3) (Fig S3D). The onset of stage 3 marks axon specification and indeed we observed strong upregulation of the axonal proteins Trim46 and Tau at day 7, which was not affected by Centrinone-B treatment (Fig 3B). Expression of the growth cone proteins Basp1, Gap43, and Marcks was increased at day 3 in controls as well as Centrinone-B treated neurons. This upregulation was even stronger at day 7 in control neurons, but was markedly suppressed in the centriole-depleted neurons (Fig 3C, Fig S3D). Together, the quantitative proteome analysis shows that treatment with Centrinone-B does not dramatically alter the protein expression profile during early stages of neurodevelopment. However, these results indicate a specific effect on growth cone proteins upon loss of centrioles. Centriole loss affects neurite growth cone morphology Next, we examined if the reduced expression of growth cone proteins resulted in defects of axonal growth cone morphology. We found that in control neurons the size of growth cones is relatively large early in development and decreases over time (Fig 3D,E). Growth cones of centriole-depleted neurons remained smaller at day 5, and their size decreased even further later in development. As microtubules are essential components to shape growth cones, we investigated whether manipulation of the microtubule cytoskeleton mimicked the effect of centriole depletion on growth cone size (Dent, Gupton, and Gertler 2011). Indeed, treatment with Nocodazole, a microtubule destabilizer, also resulted in smaller growth cones already at day 5 (Fig 3D,E). Interestingly, Nocodazole treatment did not show an additional effect on growth cone size in centriole depleted neurons, which suggests similar underlying mechanisms. To study the effect of centriole depletion on growth cones more specifically, we categorized three subtypes: fan-like, torpedo-like, and bulb-like (Fig 3F) (van der Vaart et al. 2013). We found a majority of fan-like growth cones at day 5, which shifted to more torpedo-like and bulb-like growth cones as axons matured (Fig 3G). Neither Centrinone-B nor Nocodazole treatment altered the relative abundance of these types