Feline Lindhout

2 46 Mapping microtubule reorganization in the newly identified developmental stages The transcriptomic and proteomic profiles point to the importance of microtubule cytoskeleton remodeling during axon specification. Indeed, AIS dynamics are reported to be regulated by the underlying axonal microtubule cytoskeleton (Stepanova et al. 2003; Kleele et al. 2014; van Beuningen et al. 2015; Yau et al. 2016). Therefore, we assessed changes in the microtubule network in axons and dendrites of developing human iPSC-derived neurons by systematically analyzing plus-end dynamics and orientations of microtubules at different locations (Fig 4A). We performed two-color live-cell imaging to visualize neuronal morphology and microtubule plus-end tracking proteins (MT+TIPs), respectively (Fig 4B,C). Bidirectional MT+TIP movement, as shown by comets moving in both the anterograde and retrograde direction, was observed during stage 2 (day 5), with a preference for the anterograde direction (Fig 4D). Over time, this preference shifted towards more retrograde movement in developing dendrites, and towards unidirectional anterograde movement in developing axons (Fig 4D). These changes are consistent with differences in microtubule organization in axons and dendrites found in rodent neurons (Yau et al. 2016; Schatzle, Kapitein, and Hoogenraad 2016). In similar fashion to the distal to proximal reorganization of AIS proteins (Fig 3), the observed shift towards a unidirectional microtubule organization in axons occurred first in distal parts of the axon, followed by proximal reorganization. The observed developmental changes in anterograde and retrograde ratios are mostly explained by changes in retrograde comets, as the total number of retrograde comets was increased in dendrites and decreased in axons of (Fig 4E,F). The comet growth speed is slightly reduced during development, while comet run length increased markedly, suggesting a reduction in catastrophe events during axon and dendrite development (Fig 4G,H, S4A,B). Imaging of MT+TIPs provides information about the dynamic ends of microtubules, but the fraction of moving comets does not directly reflect the orientations of all microtubules (Yau et al. 2016). To analyze microtubule orientations of both dynamic and stable microtubules, we combined our previous live-cell imaging approach with laser-severing. Cutting microtubules with a short-pulsed laser generates new microtubule ends, which allows for analysis of newly formed MT+TIPs (Fig 4I,J, S4C,D). Following laser severing, a strong shift towards a balanced, bidirectional orientation in developing dendrites is observed (Fig 4K). The number of comets in both directions increased during development, with those moving in the retrograde direction increasing more (Fig 4L,M). This suggests the presence of a larger pool of stable, minus-end out microtubules in dendrites. In developing axons, the shift towards unidirectional, plus-end out microtubules was more notably observed following laser severing (Fig 4K). Laser severing led to a large increase in the number of anterogradely moving comets (Fig 4L), suggesting the presence of a stable pool of plus- end out microtubules. These results indicate that the microtubule remodeling in a distal to proximal fashion coincides with relocation of AIS proteins. Together, these data show that axon specification is characterized by the reorganization of the axonal microtubule network and relocation of the AIS from the distal to proximal axon (Fig S4E).