Ti e b microscope

For TIRF microscopy, Lifeact or vinculin-transfected cells were imaged using a NIKON TI-E/B microscope, equipped with an Evolve EMCCD camera. The illumination system used was from from Roper Scientific (iLas²), and the objective was a Nikon CFI Plan APO VC oil immersion objective (60X, N.A. 1.4). The microscope was controlled with MetaMorph software (Molecular Devices). Temperature, CO₂, and humidity control was performed using a chamlide TC system (TC-A, Quorum technologies). The laser used for excitation of EGFP was a 491 nm cobolt Calypso laser, and the one for excitation of mCHerry was a 561 nm MPBC Green Visible Fiber laser. ET525/50M (Chroma) and ET605/52M (Chroma) emission filters were used for for EGFP and mCherry fluorescence collection, respectively.

For live-cell imaging, cells were seeded on 35 mm glass-bottom tissue culture dishes (Greiner Bio-one, Germany), triple transfected with ^GFPBAP31, ^FKBP-mChKIF5 DN and KIF5 motor^BFP-FRB, and allowed to express the proteins for at least 24 h before infected with SV40 (MOI 20). Cells (16 hpi) were treated with the rapa linker. In total, 23 cells were imaged for the experimental condition (cells expressing ^GFPBAP31, ^FKBP-mChKIF5 DN and KIF5 motor^FRB), whereas ten cells were imaged for the control condition (cells expressing ^GFPBAP31 and ^FKBP-mChKIF5 DN). Imaging of the cells was started at 0 h post-rapa addition for 2 h in cDMEM media without phenol red (Gibco). During imaging, cells were maintained at 5% CO₂ in a humidified chamber (Tokai Hit) regulated at 37 °C. The entire set-up was placed on a Nikon Ti-E/B microscope equipped with a × 100 1.49 numerical aperture oil-immersion objective warmed to 37 °C. Imaging was recorded using 20 mW diode lasers (488 nm) every 5 min for 2 h. ImageJ software (NIH) was used for image processing, analysis and assembly.