Pak1 regulates focal adhesion strength, myosin IIA distribution, and actin dynamics to optimize cell migration

p21-activated kinases are essential for spatial and temporal coordination of cytoskeletal dynamics with cellular adhesion during cell migration

Reactive oxygen species regulate protrusion efficiency by controlling actin dynamics.

Productive protrusions allowing motile cells to sense and migrate toward a chemotactic gradient of reactive oxygen species (ROS) require a tight control of the actin cytoskeleton. However, the mechanisms of how ROS affect cell protrusion and actin dynamics are not well elucidated yet. We show here that ROS induce the formation of a persistent protrusion. In migrating epithelial cells, protrusion of the leading edge requires the precise regulation of the lamellipodium and lamella F-actin networks. Using fluorescent speckle microscopy, we showed that, upon ROS stimulation, the F-actin retrograde flow is enhanced in the lamellipodium. This event coincides with an increase of cofilin activity, free barbed ends formation, Arp2/3 recruitment, and ERK activity at the cell edge. In addition, we observed an acceleration of the F-actin flow in the lamella of ROS-stimulated cells, which correlates with an enhancement of the cell contractility. Thus, this study demonstrates that ROS modulate both the lamellipodium and the lamella networks to control protrusion efficiency

H₂O₂ regulates the contractile machinery of the lamella.

<p>(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 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041342#pone.0041342.s002" target="_blank">Figure S2G</a>).</p

H₂O₂ regulates cell migration and protrusion dynamics in PtK1 cells.

<p>(A) Representative schema of the chemotaxis experiment. Using a microinjection system, PtK1 cells were exposed (flow) or not (no flow) to a constant flow of H₂O (control) or 1.5 mM H₂O₂ mixed with rhodamine dextran (shown in red). The overlay of phase-contrast image and rhodamine dextran shows the direction of the flow. (B–E) Quantification of motility parameters of PtK1 cells exposed or not to a flow of H₂O (control) or H₂O₂, including total path length (B), the total distance traversed by cells over time; net path length (C), the net distance that the cells traversed from the first to the last frame; directionality (D), the ratio of net to total path length; and cell velocity (E). The data result from n ≥38 cells analyzed for each condition. Error bars represent s.e.m. ***, p<0.001 compared to control (flow) and H₂O₂ (no flow). (F) PtK1 cells were incubated with the H₂O₂-sensitive probe PY1-AM (5 µM for 30 min) and then treated with H₂O₂ (500 µM for 20 min). One representative image is shown before and after H₂O₂ stimulation. Red lines highlight the leading edge of the cells. The scale bar is 30 µm. In the right panel, fluorescence intensity of PY1-AM was measured in cells located at the front and the back of PtK1 islands before and after H₂O₂ addition. Fluorescence intensity was averaged from 15 cells. Error bars represent s.e.m. **, p<0.01 compared to - H₂O₂ (front) and + H₂O₂ (back). (G) After starvation, PtK1 cells were incubated for 15 min in media only and 45 min with control media (+H₂O) or media containing 500 µM H₂O₂ alone or in combination with 5 mM sodium pyruvate, a ROS scavenger. In this case, cells were pretreated with ROS scavenger for 30 min before the experiment. Phase-contrast images were taken from movies of each condition. Images of control (top row), H₂O₂- (center row) and H₂O₂+ROS scavenger-treated cells (bottom row) are shown before and after 45 min of treatment. The scale bar is 30 µm. White arrows highlight locations used to generate kymographs. Three representative kymographs of control (a, b and c), H₂O₂- (d, e and f) and H₂O₂+ROS scavenger-treated cells (g, h and i) are shown on the rightmost panels. Black lines on kymographs indicate when the cells were treated. The scale bar is indicated by black arrows corresponding to d = 5 µm and t = 15 min. (H–J) Protrusion width (H), persistence of protrusion (I) and protrusion/retraction velocities (J) resulting from the analysis of 25 control, ROS scavenger-, H₂O₂- and H₂O₂+ROS scavenger-treated cells and 125 kymographs per condition. Error bars represent s.e.m. ***, p<0.001 compared to control.</p

ERK is activated in response of H₂O₂ and contributes to H₂O₂-induced protrusion dynamics.

<p>(A) Cell lysates from starved PtK1 cells treated with 500 µM H₂O₂ alone or in combination with ROS scavenger (5 mM) for 0-15-30-45-60 min were immunoblotted with antibodies against pERK and ERK. In (B), the graph represents the averaged normalized pERK values. Data are from four and three independent experiments for H₂O₂ and H₂O₂+ROS scavenger, respectively. Error bars represent s.e.m. *, p<0.05 compared to 0 min H₂O₂ and 15 min H₂O₂+scavenger. (C) Immunolocalization of phosphorylated ERK (pERK, 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. (D and E) Fluorescence intensity of pERK (D) and F-actin (E) in cells treated with H₂O₂ 500 µM, measured from the cell edge (0 µm) into the cell center (10 µm). (F) pERK/F-actin fluorescence intensity ratio in cells treated with H₂O₂ 500 µM, measured from the cell edge (0 µm) into the cell center (10 µm). In (D)–(F), 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 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041342#pone.0041342.s002" target="_blank">Figure S2D</a>). Protrusion width (G), persistence of protrusion (H) and protrusion/retraction velocities (I) in starved Ptk1 cells incubated for 45 min with control media (+DMSO) or media containing 500 µM H₂O₂ alone or in combination with UO126, a MEK inhibitor. In (G)–(I), the data shown result from the analysis of at least 23 cells and 115 kymographs per condition. Error bars represent s.e.m. ***, p<0.001 compared to control and H₂O₂+UO126.</p

H₂O₂ increases the formation of free barbed ends and F-actin turnover.

<p>(A) Free barbed end actin incorporation (green) and F-actin phalloidin staining (red) in starved PtK1 cells treated with 500 µM H₂O₂ for the indicated times. The scale bar is 10 µm. (B and C) Fluorescence intensity of free barbed end actin incorporation (B) and F-actin (C) in cells treated with 500 µM H₂O₂, measured from the cell edge (0 µm) into the cell center (10 µm). (D) Fluorescence intensity ratio of free barbed end actin incorporation relative to F-actin in cells treated with 500 µM H₂O₂, measured from the cell edge (0 µm) into the cell center (10 µm). In (B)-(D), the data shown represent one experiment and are averaged from at least 16 cells for each condition. The experiment was repeated four times with similar results (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041342#pone.0041342.s002" target="_blank">Figure S2B</a>). (E) F-actin turnover maps computed from quantitative FSM time-lapse movies of starved PtK1 cells treated or not with 500 µM H₂O₂. Turnover maps depict F-actin polymerization (red) and depolymerization (green) rates. Maps have been averaged over 6 frames, i.e., 1 min. The scale bar is 10 µm. Boxed regions are magnified in the bottom left of each panel. n = 14 cells analyzed for control and H₂O₂ treatment.</p

Cofilin is activated upon H₂O₂ stimulation in PtK1 cells.

<p>(A) Immunolocalization of phosphorylated cofilin (P-cofilin, green) and F-actin phalloidin staining (red) in starved PtK1 cells treated with 500 µM H₂O₂ for the indicated times. The scale bar is 10 µm. Red lines highlight the leading edge of the cells. (B and C) Fluorescence intensity of P-cofilin (B) and F-actin (C) in cells treated with 500 µM H₂O₂, measured from the cell edge (0 µm) into the cell center (10 µm). (D) P-cofilin/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 (B)–(D), the data shown represent one experiment and are averaged from at least 18 cells for each condition. The experiment was repeated three times with similar results (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041342#pone.0041342.s002" target="_blank">Figure S2A</a>). (E) Cell lysates from starved PtK1 cells treated with 500 µM H₂O₂ alone or in combination with ROS scavenger (5 mM) for 0-15-30-45-60 min were immunoblotted with antibodies against P-cofilin and cofilin. In (F), the graph represents the averaged P-cofilin/cofilin values. Data are from six and three independent experiments for H₂O₂ and H₂O₂+ROS scavenger, respectively. Error bars represent s.e.m. ***, p<0.001 compared to 0 min H₂O₂ and **, p<0.01 compared to 45–60 min H₂O₂.</p

H₂O₂ modulates actin dynamics in PtK1 cells.

<p>(A) Single frames of actin fluorescent speckle time-lapse series of starved PtK1 control (top row) and treated with 500 µM H₂O₂ (bottom row). The scale bar is 10 µm. White arrows highlight the locations used to generate kymographs. (B) Kymographs of control and H₂O₂-treated cells depicted in (A). White lines indicate speckle translocation used to calculate flow velocities. (C) F-actin flow maps computed from quantitative FSM analysis of time-lapse movies of control and H₂O₂-treated cells. Flow rates are color coded, ranging from slow flow in dark blue to fast flow in red. Flow maps have been averaged over 60 frames, i.e., 10 min. (D and E) Average F-actin flow rates measured at the leading edge (D) and 5 µm from the leading edge (E) of control and H₂O₂-treated cells. n = 14 cells analyzed for control and H₂O₂ treatment. Error bars represent s.e.m. ***, p<0.001 compared to control.</p

H₂O₂ induces Arp2/3 recruitment at the leading edge of PtK1 cells.

<p>(A) Immunolocalization of p34-Arc (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. Boxed regions are magnified in the bottom left of the Merge panel. (B and C) Fluorescence intensity of p34-Arc (B) and F-actin (C) in cells treated with 500 µM H₂O₂, measured from the cell edge (0 µm) into the cell center (10 µm). (D) p34-Arc/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 (B)–(D), the data shown represent one experiment and are averaged from at least 12 cells for each condition. The experiment was repeated three times with similar results (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041342#pone.0041342.s002" target="_blank">Figure S2C</a>).</p