Time-lapse microscopy of a spreading control Cho-K1 cell expressing RLC-D,D–GFP (a phospho-mimetic mutant of the RLC) on fibronectin. The arrow points to the site of initial accumulation of RLC-D,D–GFP. Note the protrusion opposite the concentration of RLC-D,D, which results in cell polarization and definition of the rear. Images were captured in TIRF mode using an inverted microscope (IX70; Olympus) coupled to a CCD camera (Retiga Exi; Qimaging). Live samples were acquired at 37°C using CCM1 medium. This movie corresponds to Fig. 1 C. and Video 1 from J Cell Biol 2011 193:381-396.