Live Cell Imaging of Microtubule Dynamics

For live imaging, cells were plated on glass-bottom 35 mm dishes coated with poly-D-lysine (MatTek Corporation, Ashland, MA) and imaged in a stage-top humidified incubation chamber (Tokai Hit, Fujinomiya-shi, Japan) maintained at 30°C and 5% CO2. To visualize tubulin, 100 nM siR-Tubulin dye (Cytoskeleton, Inc., Denver, CO) was added 2 hr prior to imaging, along with 10 µM verapamil (Cytoskeleton, Inc.). Under these conditions, there was no detected defect in spindle appearance or microtubule dynamics. As described elsewhere (Elting et al., 2014 (link)), cells were imaged using a spinning disk confocal inverted microscope (Eclipse Ti-E; Nikon Instruments, Melville, NY) with a 100 × 1.45 Ph3 oil objective through a 1.5X lens, operated by MetaMorph (7.7.8.0; Molecular Devices, Sunnyvale, CA). Laser ablation (30 3-ns pulses at 20 Hz) with 551 nm light was performed using the galvo-controlled MicroPoint Laser System (Andor, Belfast, UK). For laser ablation experiments, images were acquired more slowly prior to ablation and more rapidly after ablation (typically 7 s prior and 3.5 s after ablation).