MLCK inhibition/depletion impairs myosin II activity at the leading edge. (A) dHL-60 cells plated on FN-coated coverslips were stimulated for 2 minutes with 1μM fMLP, fixed with 3.7% paraformaldehyde, and stained with a specific anti-MLCK antibody (red) and AlexaFluor-488–conjugated phalloidin (green). The polarized distribution of endogenous MLCK was observed in 188 of 269 polarized cells. (B) A dHL-60 cell treated with ML-7 (25μM, 30 minutes) was exposed to a point source of 10μM fMLP for the times indicated (bottom panel). The cell fails to migrate to the micropipette and shows poorly developed pseudopod (white arrow). An untreated dHL-60 cell (top panel) with well-developed, stable pseudopod is shown. Scale bars represent 10 μm. (C) DIC kymographs (left) of a dHL-60, untreated or treated with ML-7, migrating toward an fMLP (10μM)–containing micropipette. The dotted rectangles indicate the regions of the cell used to generate the kymographs (before fMLP stimulation). The actual lengths of the rectangles are 20 μm in the direction of the arrow. Left panel: Scale bar represents 1 μm. Right panel: Scale bars represent 5 μm. Arrows indicate the direction of protrusion. Five minutes of migration was recorded. The speed of leading-edge protrusion was calculated based on the kymographs (right). The values are mean plus or minus SEM (n = 34 for control, and 32 for cells treated with ML-7). *Value for cells with ML-7 treatment differs from the corresponding control (P < .0001). (D) dHL-60 cells not pretreated or pretreated with ML-7 (25μM, 30 minutes) or Y-27632 (30μM, 30 minutes) were stimulated for 3 minutes by a uniform concentration of 1μM fMLP. Cells were fixed with 3.7% paraformaldehyde and stained with the anti-MHCIIA antibody, anti-p[Ser19]MRLC antibody, and rhodamine-conjugated phalloidin. The corresponding DIC images are also shown. Scale bar represents 10 μm. (E) The distribution of p[Ser19]MRLC in dHL-60 cells with or without ML-7 treatment was analyzed. The mean fluorescence of p[Ser19]MRLC staining at the leading edge and the trailing edge of cells was determined using ImageJ software 1.43, and the ratios between the leading edge and the trailing edge (ie, mean fluorescence intensity at the leading edge/mean fluorescence intensity at the trailing edge) are shown. Values were normalized to the ratio (100%) in control cells and are mean plus or minus SEM (n = 40 for control, and 30 for cells treated with ML-7). Student t tests compared data between experimental groups. *Results significantly different from control (P < .001). (F) Western blot of p[Ser19]MRLC. dHL-60 cells were pretreated with no inhibitors, ML-7 (25μM, 30 minutes), or Y-276322 (30μM, 30 minutes) before exposure to fMLP for 2 minutes in suspension. (Top panel) A typical blot. (Bottom panel) Quantification of blots from 4 separate experiments. Each bar represents the mean plus or minus SEM (error bars). All values were normalized to the signal (100%) detected without the inhibitors. Value for cells treated with M-7 or Y-27632 differs statistically from the control: *P < .01, **P < .001. Total MHCIIA levels were unaltered with the treatments and were used for equal loading in the different lanes. (G) The distribution of total MHCIIA in control dHL-60 cells and cells pretreated with ML-7 or Y-27632 was analyzed. The mean fluorescence of MHCIIA staining at leading and trailing edges was assessed by ImageJ software 1.43, and the ratios between leading and trailing edges are shown. Values were normalized to the ratio (100%) in control cells and are mean plus or minus SEM (n = 40 for control, 30 for cells treated with ML-7, and 25 for cells treated with Y-27632). Results significantly different from control: *P < .05, **P < .001.