Movie shows actin cytoskeleton in Nck-deficient cells after stimulation with PDGF-B. In the absence of Nck proteins, prominent actin bundles and the almost complete absence of membrane ruffles even after PDGF-B stimulation reflect a very stable actin cytoskeleton. Supporting Movie 1 for Rivera et al.
Nck-deficient cells (mouse embryonic fibroblast from double knockout Nck1/Nck2) were cotransfected with an empty vector (EBB) and actin-GFP to visualize cytoskeletal dynamics, serum-starved for 24 h, and then stimulated with 30 ng/ml PDGF-B. Confocal images (five optical section per time point) were obtained every 12 seconds for 10 min beginning immediately after PDGF-B stimulation using a Nikon TE2000 inverted microscope with a 60 × 1.4 NA oil-immersion objective. The confocal system was equipped with a PerkinElmer UltraView spinning microlens and an ORCA–ER Firewire CCD camera. The display rate of the movie is five frames per second.
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