To study the molecular mechanism by which nonmuscle myosin II (MII) regulates protrusion and adhesion dynamics in migrating cells, NIH3T3 cells were transfected with myc-tagged myosin light chain (MLC, green). After 20' exposure to PDGF, which induces myosin-inactivation, stress fiber disassembly, and stimulates fibroblast motility, the cells were fixed and co-stained for betaPIX (a Rac1/Cdc42-specific GEF highly implicated in cell motility, red) and filamentous actin (blue). These findings help elucidate a functional link between MII and Rac1/Cdc42 GTPases, which may regulate protrusion/adhesion dynamics in migrating cells. This image is the original data file from Fig. 7C, J. Cell Biol. 2010. Vol. 190(4):663–674
Cells were cultured in DME (Invitrogen) supplemented with 10% fetal bovine serum and 100 U/ml penicillin/streptomycin (Invitrogen) at 37°C in a humidified 5% CO2 incubator. For transfections, cells in 60-mm-diameter dishes or on fibronectin-coated coverslips were incu- bated with a mixture of DNA and LipofectAMINE 2000 (Invitrogen) according to the manufacturer’s instructions. In some experimental conditions, 16 h after replating onto fibronectin-coated coverslips, cells were treated with 50 ng/ml PDGF for 20 min. Cells were fixed 24–48 h after transfection using 3.7% paraformaldehyde in PBS for 15 min, permeabilized using 0.2% Triton X-100 in PBS for 2 min, blocked with 2% BSA in PBS, and co-stained for MLC (green), betaPIX (red), and actin (blue). Images were captured by Zeiss LSM 710 confocal microscope with Plan-Apochromat 63X objective.
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