Reactive Oxygen Species Regulate Protrusion Efficiency by Controlling Actin Dynamics
Figure 8
H2O2 regulates the contractile machinery of the lamella.
(A) Kymographs and F-actin flow maps computed from quantitative FSM analysis of time-lapse movies of control, blebbistatin (50 µM) and blebbistatin (50 µM)+H2O2 500 µM-treated cells. White lines on kymographs indicate speckle translocation used to calculate flow velocities. Flow rates are color coded, ranging from slow flow in dark blue to fast flow in red. Flow maps have been averaged over 30 frames, i.e., 5 min. (B and C) Average F-actin flow rates measured at the leading edge (B) and 5 µm from the leading edge (C) of control, blebbistatin, H2O2 and blebbistatin + H2O2-treated cells. n ≥7 cells from at least four independent experiments. Five kymographs/cell were analyzed for each condition. Error bars represent s.e.m. ***, p<0.001 compared to control and blebbistatin (B). **, p<0.001 compared to control and ***, p<0.001 compared to control and H2O2 (C). (D) Average lamellipodium width of control, blebbistatin and blebbistatin + H2O2-treated cells. n≥7 cells from at least four independent experiments. Five kymographs/cell were analyzed for each condition. Error bars represent s.e.m. ***, p<0.001 compared to control and blebbistatin. (E) Immunolocalization of phosphorylated MLC (pMLC, green) and F-actin phalloidin staining (red) in starved PtK1 cells treated with H2O2 500 µM for the indicated times. The scale bar is 10 µm. Red lines highlight the leading edge of the cells. (F and G) Fluorescence intensity of pMLC (F) and F-actin (G) in cells treated with 500 µM H2O2, measured from the cell edge (0 µm) into the cell center (10 µm). (H) pMLC/F-actin fluorescence intensity ratio in cells treated with 500 µM H2O2, measured from the cell edge (0 µm) into the cell center (10 µm). In (F-H), the data shown represent one experiment and are averaged from at least 13 cells for each condition. The experiment was repeated three times with similar results (Figure S2G).