PDK1 and MRCKα colocalize in nascent lamellipodia upon EGF stimulation. (A) MCF10A cells, transfected with LifeAct-mTurquoise, mCherry-MRCKα, and GFP-PDK1_WT, were observed with TIRF microscopy set at 90 nm thickness, stimulated with 5 ng/ml EGF, and monitored every 20 s. Images show a representative cell before stimulation (0 s) and after stimulation (100 s, 200 s, and 600 s). The colocalization channel between mCherry-MRCKα and GFP-PDK1_WT was calculated as described in the Materials and methods section. Lamellipodia (LP) and lamella (LM) were identified on the basis of LifeAct-mTurquoise distribution. Enlargements (taken from the boxed regions above) were obtained by bicubic 4 × 4 interpolation of the original images. Bars: (top rows) 10 µm; (enlarged panels) 3 µm. (B) MCF10A cells were infected with a lentivirus coding for myc-PDK1 and, after EGF deprivation, stimulated with 5 ng/ml EGF. PLA of PDK1 and MRCKα was performed at different time points using, as detecting antibodies, anti–c-Myc produced in mouse and anti-MRCKα produced in rabbit. Fluorescent PLA spots close to the adherent surface were observed with TIRF microscopy set at 90 nm thickness. Cellular shape was identified by Phalloidin-488 staining. Bars, 10 µm.