Live-Cell Imaging of Cellular Dynamics

Cells were seeded onto poly-d-lysine–coated glass-bottom 35-mm dishes (MatTek Corp.), with media and transfection conditions as described above. Spinning disk and laser scanning confocal live-cell imaging was performed under environmentally controlled conditions, at 37°C and 5% CO₂. For experiments using the spinning disk microscope, the protocol is as described in Stehbens et al. (2014 (link)) except that the system was upgraded with a next-generation scientific CCD camera (cMyo, 293 Photometrics) with 4.5-µm pixels, allowing optimal spatial sampling using a 60× NA 1.49 objective (CFI 294 APO TIRF; Nikon). For experiments using the Olympus Fluoview 1000 laser scanning confocal microscope, a 60× oil-immersion objective with NA 1.42 was used to obtain confocal images (1,024 × 1,024 pixels). Z-stack images were acquired with a step size of 0.5–1 μm and processed using the Fiji software package.