Myosin II controls tractions and is necessary for neutrophil chemotaxis. (A) dHL-60 cells were stimulated for 3 minutes by a uniform concentration of 1μM fMLP. Cells were fixed with 3.7% paraformaldehyde and stained with a specific antimyosin heavy chain IIA (MHCIIA) antibody, anti-p[Ser19]MRLC antibody, and rhodamine-conjugated phalloidin to localize filamentous actin (F-actin). The corresponding DIC image is also shown. Scale bar represents 10 μm. (B) dHL-60 cells were transfected with mCherry–myosin IIA (mChe-myoIIA) and stimulated by a micropipette containing 10μM fMLP for the indicated times. mCherry–myosin IIA fluorescence and the corresponding DIC images are shown. Arrows point to the locations of myosin IIA at the leading and trailing edges. Scale bar represents 10 μm. The video of the cell in panel B is available in supplemental data. (C) dHL-60 cells pretreated with blebbistatin (100μM, 30 minutes) were allowed to migrate toward chemoattractant-containing micropipette (fMLP, 10μM) on a FN-coated elastic polyacrylamide gel for the indicated times. Cells treated with blebbistatin migrated at 1.0 μm/minute on the elastic gel. Traction maps of the cell are shown. Pseudocolor bar representing traction force is given in Pascal (Pa). Scale bar represents 10 μm. The leading edge (within the first 2.2 μm of the cell) is marked by a white line (“Polyacrylamide gel substrates, TFM, and data analysis”). The image series shows part (5.6 seconds) of the whole migratory response. (D) Time series of traction maps from panel C (with 4 additional time points) was analyzed by a customized MATLAB program to determine the average tractions in both leading edge (front) and trailing edge (back) of the cells in a time-dependent manner. The graph shows part (∼ 9.6 seconds) of the whole migratory response. The x-axis indicates time in seconds; y-axis is in Pascal (Pa). (E) PSD plots of tractions at the leading edge (left panel) and the trailing edge (right panel) of a migratory cell pretreated with blebbistatin (100μM, 30 minutes). The whole migratory response was analyzed. The y-axis represents the power spectral density normalized to the highest peak value (1); x-axis shows the oscillation frequency (Hertz; top) or period (seconds; bottom). Cells depleted of myosin IIA exhibited similar response (not shown). Ten cells were analyzed, and a representative cell is shown. Additional plots are shown in supplemental Figure 2C. (F) Before exposure to attractant supplied by a micropipette containing 10μM fMLP, cells were not pretreated (control), were pretreated with blebbistatin (Blebbis, 100μM, 30 minutes), were infected with lentivirus-containing myosin IIA–targeting shRNAs (MyoII KD), or were pretreated with Y-27632 (30μM, 30 minutes). The 3 images in each row show the positions of individual cells (each identified with a superimposed letter) after the indicated times of exposure to fMLP. White and black arrows point to the poorly developed leading edges and long stretched tails, respectively. Cells infected with virus containing a scramble shRNA exhibited similar response to uninfected control cells. Lower doses of blebbistatin (≤ 50μM) were tested, which required a prolonged period of incubation to exert the same effects as 100μM blebbistatin (data not shown). Scale bar represents 10 μm. Videos of cells with or without treatments are available in supplemental data. (G) DIC kymographs of a dHL-60 cell untreated or treated with inhibitors or myosin IIA shRNAs migrating toward an fMLP (10μM)–containing micropipette. The left panel shows a portion of the neutrophils' leading edge under various conditions. 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. White scale bar represents 1 μm. The right panel shows the DIC kymographs. White scale bar represents 5 μm. In both panels, white arrows indicate the direction of protrusion. Cells a, d, f, and h in panel F were used for the analysis. Approximately 8 minutes of migration was recorded.