Feline Lindhout

3 92 Nikon Eclipse microscope described above. We used a 355 nm passively Q-switched pulsed laser (Teem Photonics) for the photoablation, together with the S Fluor 100x 0.5-1.3 NA oil objective (Nikon). To cut several microtubules simultaneously, we moved the same laser beam along the line positioned perpendicular to several microtubules. All live-cell imaging experiments were performed in full conditioned differentiation (day 5) or maintenance (days 7 and 13) medium for human iPSC-derived neuron cultures (Axol). To keep cells at 37°C with 5% CO2 we used a stage top incubator (model INUBG2E-ZILCS, Tokai Hit). Image quantification and analysis Determining centrosomal γ-Tubulin levels : Centrosomal γ-Tubulin levels were quantified in stage 1 (day 0; Ki67-positive), stage 2 (day 7; MAP2-positive and axonal Trim46- negative) and stage 3 neurons (day 12; MAP2-positive and axonal Trim46-positive). Centrosomes were identified by co-localization of γ-Tubulin and Centrin. A region of interest was manually drawn around centrosomes defined by the Centrin signal, and the average γ-Tubulin immunofluorescence intensity within this region was measured and corrected for background intensities. The mean intensities of each neurodevelopmental stage were normalized to the mean intensity of stage 1 neurons. Measuring development of neuronal differentiation : To determine the effect of Centrinone-B treatment on neuronal differentiation over time, cells were identified using DAPI staining and scored to be positive or negative for Ki67, MAP2 or β3-Tubulin. Quantifying growth cone morphologies : Growth cone size was measured by manually drawing a region of interest based on phalloidin signal and measuring the area in µm². Growth cones were categorized as fan-like, torpedo-like or bulb-like based on shape. Live-cell imaging For microtubule dynamics experiments without laser severing, time-lapse acquisition was performed using the 491 nm 100 mW Calypse (200 ms exposure) and 561 nm 100 mW Jive (200 ms exposure) with 1 frame per second (fps) for 5 minutes, with a Plan Apo VC 60x NA 1.4 oil immersion objective (Nikon). 16-bit images were projected onto the EMCCD chip with intermediate lens 2.0X (Edmund Optics) at a magnification of 0.111 µm/ pixel at 60x, or onto the sCMOS chip with no intermediate lens at a magnification of 0.150 µm/pixel at 60x. For microtubule dynamics experiments with laser severing, Time-lapse acquisition was performed using the 491 nm 100 mW Calypse (50-200 ms exposure) and 561 nm 100 mW Jive (50-200 ms exposure) with 1 fps for 3 minutes. Selected regions were subjected to photoablation between frame 30 and 31. 16-bit images were projected onto the sCMOS chip with no intermediate lens at a magnification of 0.063 µm/pixel at 100x. For analysis of microtubule plus-end dynamics, kymographs were generated with the FIJI plugin KymoResliceWide v.0.4 (https://github.com/ekatrukha/KymoResliceWide ), and tracing microtubule growth events were manually traced.