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 phosphomimetic constitutively active MLC mutants (MLCee, 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 TRIO (a multifunctional, multidomain Rho guanine nucleotide exchange factor, 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 original data from Fig. S5 "Expression of active MLC mutants inhibits the dissociation of MII–Trio complex," in 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. The cDNA for MLC was subcloned into pCMV-myc (Takara Bio Inc.). Mutant constructs of MLC were generated using a QuikChange site-directed mutagenesis kit (Agilent Technologies) according to the manufacturer’s protocol. In this myc-tagged MLC mutant, Thr18/Ser19 were replaced by glutamic acids, and expressed in NIH3T3 cells. 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), TRIO (red), and actin (blue). Images were captured by Zeiss LSM 710 confocal microscope with Plan-Apochromat 63X objective.
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