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First published online July 13, 2004
doi: 10.1242/10.1242/jcs.01211

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Synergistic interaction between the Arp2/3 complex and cofilin drives stimulated lamellipod extension

¹ Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA
² Analytical Imaging Facility, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA
³ Division of Cell Biology, Institute of Ophthalmology, University College London, 11-43 Bath Street, London EC1V 9EL, UK

View larger version (31K): [in a new window]   Fig. 3. Inhibition of Arp 2/3 or cofilin function causes a reduction in barbed ends. Cells were time-lapsed after addition of EGF and the fluorescent edge intensity (GFP-ßactin) was measured every 10 seconds and barbed ends calculated (see Materials and Methods). (A) Fold GFP-actin edge intensity increase after addition of EGF for nonimmune IgG-injected cells (n=15); (B) for anti-p34-injected cells (n=18); (C) for anti-cofilin-injected cells (n=17) (error bars: ±s.e.m.). (D) The relative increase in barbed ends at the leading edge after stimulation. Barbed ends were calculated by determining the slopes of the initial fluorescence intensity increase (10-100 seconds after EGF stimulation) as a measure of the rate of GFP-ßactin incorporation (Lorenz et al., 2004). The first bar shows barbed ends present after injection of anti-p34, i.e. essentially the cofilin contribution to barbed ends. The second bar indicates barbed ends present after injection of anti-cofilin, i.e. the Arp2/3 contribution to barbed ends. The last bar shows barbed ends present in control IgG-injected cells (full length of the bar). The overlay (dark area on the bar) corresponds to the calculated sum of cofilin and Arp2/3 contributions as displayed in the first two bars. To estimate error bars for D, slopes were determined for straight lines that were calculated using the data in A-C±s.e.m.

View larger version (39K): [in a new window]   Fig. 1. Cofilin recruitment to the leading edge of the lamellipod in the initial phase after EGF stimulation is initiated faster than Arp2/3 recruitment. (A) CFP-Arp3 and YFP-cofilin distribution in the same cell before and after EGF stimulation. Arrowheads indicate accumulation of GFP-Arp3 at the leading edge. Bar, 10 µm. (B-D) Cells transfected with GFP-cofilin or GFP-Arp3 were time-lapsed after the addition of EGF, followed by measurement of the fluorescent edge intensity and cell area throughout the time-course. The area was plotted as fold area increase over time (B and C, open circles). The fluorescent edge intensity in a 0.7 µm depth from the edge of the membrane was plotted as mean fold fluorescent intensity increase over time (B: filled squares, GFP-Arp3, n=7; C: filled triangles, GFP-cofilin, n=9). Error bars indicate the standard error of the mean (s.e.m.). (D) To calculate the initial rates of intensity increase for Arp3 and cofilin, data points for the intensity plots in B and C from 20 to 60 seconds were fitted to straight lines and the slopes of these lines were taken as the initial rates of increase. To estimate errors, the slope calculations were conducted with the data from B and C plus or minus s.e.m.

View larger version (81K): [in a new window]   Fig. 2. Both cofilin and Arp2/3 are required for lamellipod extension. GFP-ßactin-expressing MTLn3 cells were either not injected (A-D), or microinjected with control nonimmune rabbit IgG (E-H), with anti-p34 function-blocking antibody (I-L), or with anti-cofilin function-blocking antibody (M-P, injected cell indicated with arrow). Cells were stimulated with EGF and time-lapsed to follow lamellipod extension and accumulation of F-actin at the leading edge. A, E, I and M show cells before the addition of EGF; B, F, J and N 90 seconds after EGF; and C, G, K and O 180 seconds after EGF. D, H, L and P show the difference in cell area before and after EGF (cell area before EGF: green; after EGF: red; overlap of areas: yellow). Some small protruding areas are indicated with arrowheads in K and O. Bar in A, 10 µm. (Q) Quantitation of cell area during lamellipod extension. Cell areas were measured every 10 seconds during time-lapse. Q shows the fold-increase in area after EGF stimulation of control nonimmune IgG-injected cells (n=15), anti-p34 (n=18) and anti-cofilin (n=17) antibody injected cells. (R) Dose-response for anti-p34 microinjected cells. Cells were co-injected with antibody and Texas Red-labeled dextran. Texas Red fluorescence was quantified as an indicator of relative amounts of antibody injected per cell, and measurements of cells were combined into four groups according to the fluorescent intensity of the cell (shown as normalized fluorescence intensity of Texas Red dextran, arbitrary units, a.u., below each column). The dose-response for each group is shown as average fold area increase 200 seconds after the addition of EGF, at which time cells have maximally extended. Measurements for uninjected cells are shown in the first column for comparison. (S) Dose-response for anti-cofilin injected cells. Error bars indicate ±s.e.m. In each case, both for R and S, there is no statistic difference between the last three columns, whereas the difference comparing the last three columns to the first two columns is statistically significant.

View larger version (125K): [in a new window]   Fig. 4. Blocking Arp2/3 function disrupts the actin network structure at the leading edge. Cells were microinjected with antibodies (E-H, anti-p34; I-L, anti-cofilin) or left untreated (A-D). After stimulation for 1 or 3 minutes with EGF, the samples were processed for replica electron microscopy. Low-magnification images are shown in A, C, E, G, I and K (Bar, 4 µm). High-magnification images (B,D,F,H,J,L) show typical leading edge areas or closest structure when leading edges were absent (Bar, 0.1 µm). Arrowheads indicate small protrusions, as opposed to broad lamellipods.

View larger version (11K): [in a new window]   Fig. 5. The filament incidence angles at the membrane are altered in anti-p34 injected cells. The value of the incidence angle of free end filaments at the leading edge 1 minute after stimulation was measured. Values plotted are mean angle value per cell (filled circle), median (triangle) and mean (square). (See Table 1 for statistics.)

View larger version (18K): [in a new window]   Fig. 6. The filament length distribution is altered in anti-cofilin treated cells. The length of filaments with free ends at the leading edge 1 minute after EGF stimulation was measured. The frequency of filaments in each category was calculated for a total of 177 filaments for the IgG control (19 cells), 173 filaments for anti-p34 (17 cells) and 218 filaments for anti-cofilin samples (20 cells). Corresponding means and standard error of the mean are provided in Table 2.

View larger version (22K): [in a new window]   Fig. 7. A model for stimulated protrusion. Upon EGF stimulation, cofilin severing initiates a burst in free barbed ends. Polymerization at these barbed ends leads to the generation of ATP-actin-rich filaments at the leading edge. These filaments promote the nucleation and branching activity of the Arp2/3 complex, leading to the formation of a dense actin network adjacent to the plasma membrane, which facilitates cell protrusion.

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