Figure 4
Figure 4. Localization-specific myosin activities are necessary for tractions in neutrophils. (A) dHL-60 cells depleted of MLCK were allowed to migrate toward chemoattractant-containing micropipette (fMLP, 10μM) on a FN-coated elastic polyacrylamide gel for the indicated times. Traction maps of the cell are shown. Pseudocolor bar representing tractions is given in Pascal (Pa). Scale bar represents 5 μm. The leading edge (within the first 2.2 μm of the cell) is marked by a white line. The image series shows part (5.6 seconds) of the whole migratory response. Cells treated with ML-7 exhibited similar responses. Cells treated with ML-7 or depleted of MLCK migrated at 1.1 μm/minute on the elastic gel. The video of the cell in panel A is available in supplemental data. (B) Time series of traction maps from panel A (with 5 additional time points) was analyzed by a customized MATLAB program to determine the average traction force 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). (C) PSD plots of tractions at the leading edge (left panel) and the trailing edge (right panel) of a migratory cell depleted of MLCK. 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 pretreated with ML-7 exhibited similar response (not shown). Ten cells were analyzed, and a representative cell is shown. Additional plots for ML-7 treatment and MLCK depletion are shown in supplemental Figure 9A and B. (D) dHL-60 cells pretreated with Y-27632 (30 μm, 30 minutes) were allowed to migrate toward chemoattractant-containing micropipette (fMLP, 10μM) on a FN-coated elastic polyacrylamide gel for the indicated times. Traction force 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 3 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. (E) Time series of traction maps from panel D (with 5 additional time points) was analyzed by a customized MATLAB program to determine the average traction force 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). (F) PSD plots of tractions at the leading edge (left panel) and the trailing edge (right panel) of a migratory cell pretreated with Y-27632. 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). Eight cells were analyzed, and a representative cell is shown. Additional plots are shown in supplemental Figure 9C.

Localization-specific myosin activities are necessary for tractions in neutrophils. (A) dHL-60 cells depleted of MLCK were allowed to migrate toward chemoattractant-containing micropipette (fMLP, 10μM) on a FN-coated elastic polyacrylamide gel for the indicated times. Traction maps of the cell are shown. Pseudocolor bar representing tractions is given in Pascal (Pa). Scale bar represents 5 μm. The leading edge (within the first 2.2 μm of the cell) is marked by a white line. The image series shows part (5.6 seconds) of the whole migratory response. Cells treated with ML-7 exhibited similar responses. Cells treated with ML-7 or depleted of MLCK migrated at 1.1 μm/minute on the elastic gel. The video of the cell in panel A is available in supplemental data. (B) Time series of traction maps from panel A (with 5 additional time points) was analyzed by a customized MATLAB program to determine the average traction force 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). (C) PSD plots of tractions at the leading edge (left panel) and the trailing edge (right panel) of a migratory cell depleted of MLCK. 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 pretreated with ML-7 exhibited similar response (not shown). Ten cells were analyzed, and a representative cell is shown. Additional plots for ML-7 treatment and MLCK depletion are shown in supplemental Figure 9A and B. (D) dHL-60 cells pretreated with Y-27632 (30 μm, 30 minutes) were allowed to migrate toward chemoattractant-containing micropipette (fMLP, 10μM) on a FN-coated elastic polyacrylamide gel for the indicated times. Traction force 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 3 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. (E) Time series of traction maps from panel D (with 5 additional time points) was analyzed by a customized MATLAB program to determine the average traction force 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). (F) PSD plots of tractions at the leading edge (left panel) and the trailing edge (right panel) of a migratory cell pretreated with Y-27632. 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). Eight cells were analyzed, and a representative cell is shown. Additional plots are shown in supplemental Figure 9C.

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