Movie shows actin cytoskeleton in Nck-deficient cells rescued with Nck1 after stimulation with PDGF-B. In the presence of Nck1, PDFG-B stimulation induced, in addition to peripheral ruffles, very noticeable dorsal ruffles that coincided with areas of partial disassembly of actin bundles. Supporting Movie 3 for Rivera et al.
Nck-deficient cells (mouse embryonic fibroblast from double knockout Nck1/Nck2) were cotransfected with Nck1 (EBB containing human Nck1) 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 5 min beginning 5 min 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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