(A) CHO.K1 cells were transfected with the indicated mutant of RLC, RLC-18D,19A (RLC-D,A), -18A,19D (RLC-A, D), or 18D,19D (RLC-D,D), coupled to GFP (green), plated for 45 min on 2 μg/ml fibronectin-coated coverslips, fixed and stained for endogenous vinculin to visualize adhesions (blue) and with phalloidin to visualize F-actin (red). Representative cells from >200 visualized are shown. Bar = 10 μm
(B–E) Representative time-lapse images of protrusions in cells expressing comparable amounts of wild type RLC (B) or the D,A (C), A,D (D), or D,D (E) mutants coupled to GFP (green), and paxillin-mOrange (magenta). Hollow arrowheads point to representative adhesions that turn over, solid arrowheads point to adhesions that mature over the course of the experiment. Bar = 5 μm (except RLC-D, D, where bar = 10 μm).
(F) Percent of adhesions undergoing maturation in cells expressing the RLC mutants. Only cells expressing comparable amounts of the RLC mutant were analyzed. For RLC-D,D, the value on top corresponds to adhesions in the rear where RLC-D,D is concentrated; the value on the bottom corresponds to adhesions forming in protrusions opposite the rear. Data are the mean ± SD of ~30 cells (n≥150 in each case) from three independent experiments.

(A) CHO.K1 cells were plated for 45 min on different doses of fibronectin. Cells were then lysed, proteins separated by SDS/PAGE, transferred to PVDF membranes and probed for RLC~P and RLC~P,P by immunoblotting. Membranes were also probed for MHCII-A (top) and then reprobed for total RLC (bottom). A representative experiment out of three performed is shown. Right, densitometric analysis of the three experiments performed in these conditions. Average fold induction ± standard deviation is calculated with respect to the normalized value for suspended cells.
(B) CHO.K1 cells were plated for the indicated time points on 1 μg/ml fibronectin-coated dishes and probed as in (A). A representative experiment out of three performed is shown. Right, densitometric analysis as in (A). Data is normalized with respect to the value at 20 min.
(C–D) CHO.K1 cells were plated for 45 min on 2 μg/ml fibronectin-coated coverslips, fixed and permeabilized. RLC~P (C) and RLC~P,P (D) were then visualized by indirect immunofluorescence. Cells were co-stained as indicated to visualize F-actin (using phalloidin) and vinculin to visualize adhesions. Dotted boxes denote protrusions, which contain RLC~P, but not RLC~P,P; arrows point to RLC~P,P-decorated bundles. Representative cells from >200 visualized are shown. Bar = 10 μm.

(A) CHO.K1 cells were transfected with the indicated RLC mutant, plated on 1 μg/ml fibronectin-coated dishes for 45 min, lysed (see M&M) and centrifuged; the supernatant is the soluble fraction (right). The pellet was solubilized with RIPA, constituting the insoluble fraction (left). Both fractions were blotted for endogenous MHCII-A and -B. β-tubulin is used as a loading control as its solubility is not affected by expression of the mutants. A representative experiment out of four is shown.
(B–C) CHO.K1 cells were co-transfected with GFP-coupled MHCII-A (B) and GFP-MHCII-B (C) and the indicated phosphomimetic mutants of RLC coupled to mCherry, plated for 45 min on 2 μg/ml fibronectin-coated coverslips.
Fluorescence recovery after photobleaching (FRAP) was then measured on actomyosin bundles, decorated with both fluorescent proteins (note that myosin IIA and IIB do not overlap in bundles). Data are the mean ± SE of 36 individual measurements per condition in four independent experiments.