Feline Lindhout

2 52 equipped with a perfect focus system (Nikon) and a spinning disk-based confocal scanner unit (CSU-X1-A1, Yokogawa). The system was also equipped with an ASI motorized stage with the piezo plate MS-2000-XYZ (ASI), Photometric Evolve Delta 512 EMCCD camera (Photometric) controlled by the MetaMorph 7.8 software (Molecular Devices), or Photometric PRIME BSI sCMOS camera (version USB 3) as upgrade of EMCCD and controlled by the MetaMorph 7.10 software (Molecular Devices). The system was equipped with Plan Apo VC 60x NA 1.4 oil-immersion objective (Nikon) and S Fluor 100x NA 0.5-1.3 oil-immersion objective (Nikon) for photoablation experiments. A 491 nm 100 mW Calypso (Cobolt) and a 561 nm 100 mW Jive (Cobolt) laser were used as the light sources. We used an ET-GFP filter set (49002, Chroma) for imaging of proteins tagged with GFP and an ET-mCherry filter set (49008, Chroma) for imaging of proteins tagged with tag-RFP. For the photoablation experiments we used an ILas system (Roper Scientific France/ PICT- IBiSA, Institut Curie, currently Gataca Systems) mounted on a Nikon Eclipse microscope described above. A 355 nm passively Q-switched pulsed laser (Teem Photonics) was used for the photoablation together with a S Fluor 100x 0.5-1.3 NA oil objective (Nikon). Image quantification and analysis Quantifying neuronal differentiation and polarization: To measure neuronal differentiation and polarization over time, cells were identified using DAPI staining and scored to be positive or negative for the indicated NSC, neuron differentiation and axon markers. Quantification of stage 2a, 2b, 3a and 3b neurons: To determine the transition of neurodevelopmental stages over time, neurons were identified using DAPI and MAP2- positive immunofluorescence, and scored for neurodevelopmental stage 2a, 2b, 3a and 3b. Stage 2a and stage 2b neurons contained unipolar neurites of similar lengths. In stage 2a neurons, all neurites were negative for Trim46. In stage 2b neurons, one or more neurites were positive for Trim46. Stage 3a and stage 3b neurons were identified by the presence of a single elongated neurite, the future axon, that was at least twice as long as the other neurites. In stage 3a neurons, Trim46 appeared as distal non-continuous stretches at distal axons. In stage 3b neurons, Trim46 showed a dense accumulation at proximal axons. Live-cell imaging For all live-cell imaging of microtubule dynamics without laser severing, time-lapse acquisition was performed using the 491 nm 100 mW Calypse (200ms exposure) and 561 nm 100 mW Jive (200ms exposure) with 1 frame per second (fps) for 5 minutes. Sixteen-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 all live-cell imaging of microtubule dynamics with laser severing, time-lapse acquisition was performed using the 491 nm 100 mW Calypse (50-200 ms exposure, depending on the expression level) and 561nm 100mW Jive (50-200ms exposure, depending on the expression level) with 1 fps for 3 minutes, and photoablation was induced after 30s. Sixteen-bit images were projected onto