Feline Lindhout

Quantitative mapping of transcriptome and proteome dynamics during polarization of human iPSC-derived neurons 2 45 Figure 4. Axonal microtubule cytoskeleton is reorganized in a distal-to-proximal fashion during human iPSC-derived neuronal development. A. Schematic illustration of stage 2 (day 5), 3a (day 7) and 3b (day 14) human iPSC-derived neurons. Different locations of the neurons that are imaged have been outlined and annotated. B. Stills from a spinning-disk time-lapse recording of specified neurites transfected with MARCKS- tagRFP_IRES_GFP-MACF18 at specific time points. The top panel is a still of a typical example neurite in MARCKS-tagRFP. The other panels show moving GFP-MT+TIP comets (GFP-MACF18) pointing in either an anterograde direction (green arrowheads) or retrograde direction (blue arrowheads). P indicates the proximal direction and D the distal direction of the neurite. Scale bars, 5 µm. C. Kymographs and schematic representations of time-lapse recordings shown in B. Scale bars: 5 µm. D. Quantifications of the ratios of comets pointing in anterograde (green) or retrograde (blue) direction. N=3, n=8-23 cells. E. Quantifications of the number of comets per minute pointing in the anterograde direction. N=3, n=8-23 cells. F. Quantifications of the number of comets per minute pointing in the retrograde direction. N=3, n=8- 23 cells. G. Quantifications of the growth speed of comets. N=3, n=9-4723 traces in 8-23 cells. H. Quantifications of the distance of run length of comets. N=3, n=9-4723 traces in 8-23 cells. I. Schematic representation of microtubule laser-severing (LS) experiments. J. Kymographs and schematic representations of time-lapse recordings of LS experiments shown in S4B. Red arrowheads and dotted lines indicate when LS is performed. Scale bars: 5 µm. K. Quantifications of the ratios of comets pointing in anterograde (green) or retrograde (blue) direction, 10 µm before and after the LS position. N=3 , n=20-30 cells. L. Quantifications of the number of comets per minute pointing in the anterograde direction, 10 µm before and after the LS position. N=3 , n=20-30 cells. M. Quantifications of the number of comets per minute pointing in the retrograde direction, 10 µm before and after the LS position. N=3 , n=20-30 cells. Used tests: One-way ANOVA including Tukey’s post-hoc analysis ( E-H , L , M ); *** p<0.001, ** p<0.005, * p<0.05, ns p≥0.05; error bars are ± SEM days. However, on day 7, neurons fired APs with smaller after-hyperpolarization (data not shown), possibly reflecting a developmental increase in potassium channels (Song et al. 2013). Other intrinsic properties, like resting membrane potential, input resistance, AP firing threshold and maximum sodium current, remained sTable during this developmental period. Together, these results indicate a developmental maturation of specific AP properties, which coincides with the timing of the developmental transition from stage 3a to stage 3b neurons.