Figure 2. Myosin regulatory light chain (RLC) phosphorylation state directs adhesion maturation in 3D.
U2OS cells were co-transfected to express GFP-paxillin and over-express RLC phospho-mutants (-AA, -AD, and -DD). (a) Paxillin images of cells cultured on fibronectin-adsorbed glass for 2 h. RLC-AA transfected cells had numerous protrusions with lammellipodia characterized by nascent adhesions and focal complexes (arrows, see inset). RLC-AD transfected cells had more mature focal adhesions and fewer protrusive zones. RLC-DD transfected cells had large, robust adhesions bordering non-protrusive areas of the cell body. (b) Paxillin in cells cultured in 2 mg/ml bovine collagen matrices. RLC-AA: cells had few visible adhesions; those detected were very small. RLC-AD: cells had overall larger adhesions and a greater range of adhesion sizes. RLC-DD: cells had a population of very long adhesions that localized to the lateral edges of the pseudopodia and more proximal to the cell body. (c) Quantification of mean adhesion length for cells cultured in 3D (2D quantification in Fig. S1a, b). Data from cells expressing wild-type RLC (RLC-WT) are shown for comparison. All populations are significantly different except AD-WT and DD-WT (Rank-sum test; from left to right, n = 18, 20, 21 and 26 cells). (d) Histogram of adhesion lengths from cells in panel c. The histograms are normalized based on area under the curve. Overexpression of the RLC mutants results primarily in changes in the population of the longest adhesions. Bar, 10 μm (for insets, 3 μm).