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First published online 27 June 2006
doi: 10.1242/jcs.03017

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Initiation of cofilin activity in response to EGF is uncoupled from cofilin phosphorylation and dephosphorylation in carcinoma cells

Department of Anatomy and Structural Biology, Albert Einstein College of Medicine of Yeshiva University, F628, 1300 Morris Park Avenue, Bronx, New York, NY 10461, USA

View larger version (23K): [in a new window]   Fig. 1. Cofilin severing is activated in the leading edge and its severing activity peaks at 60 seconds after stimulation with EGF. (A) Severing activity in cell lysates prepared at the times shown after addition of EGF was measured in an F-actin-severing assay. (Error bars indicate the standard error of the mean (s.e.m.) (B) Pre-incubation of cell lysates with an antibody that blocks cofilin-severing activity abolished the severing-activity peak at 60 seconds after stimulation with EGF (c). However, pre-incubation of nonspecific IgG (d) had no effect on the severing activity peak at 60 seconds after stimulation with EGF, demonstrating that cofilin is the severing protein activated in the cell lysates. Severing activity in the same experiment at 0 seconds (a) and 60 seconds (b) after stimulation with EGF is shown for comparison. (Error bars indicate the s.e.m.) (C) Cofilin-severing acitivity in cell lysates prepared with or without Na3VO4 at the times shown after addition of EGF was measured in an F-actin-severing assay. (D) Changes in the amounts of phosphorylated cofilin with or without the addition of Na3VO4 and NaF.

View larger version (24K): [in a new window]   Fig. 2. The majority of cofilin is dephosphorylated in resting cells. (A) Changes in the percentage of p-cofilin in MTLn3 cell lysates at 0 and 60 seconds after stimulation with EGF were analyzed by isoelectric focusing gel electrophoresis (IEF). (B) Time course of p-cofilin and p-LIMK levels in cell lysates after EGF stimulation analyzed by western blotting with anti-p-cofilin and anti-p-LIMK antibodies (a). Change of p-cofilin levels in cell lysates at resting state and 30 minutes after EGF stimulation analyzed by western blotting with anti-p-cofilin antibodies (b). (C) Western blot quantification of the change in p-cofilin and p-LIMK after EGF stimulation standardized to ß-actin. (Error bars indicate the s.e.m. based on five independent experiments. The analysis of the representative image shown in Fig. 2B falls within the error bars in Fig. 2C.)

View larger version (80K): [in a new window]   Fig. 3. Dephosphorylation of cofilin does not occur at the leading edge following stimulation with EGF. (A) MTLn3 cells were fixed at 0 and 60 seconds following stimulation with EGF and stained with antibodies against cofilin and p-cofilin. The F-actin cytoskeleton was stained with Alexa Fluor-633-labeled phalloidin. The dephosphorylated cofilin image was prepared by subtracting the scaled p-cofilin image from the total cofilin image using Image J software. (Bar, 10 µm.) (B) Immunofluorescence quantification of dephosphorylated cofilin and p-cofilin throughout the whole cytoplasm of resting and stimulated cells 60 seconds after stimulation with EGF. (Error bars indicate the s.e.m.) (C) Fluorescence intensity analysis of dephosphorylated cofilin and p-cofilin throughout the lamellipod. The lamellipod is defined as the region extending from the cell edge to 2.5 µm into the cell interior, whereas the leading edge is the region where most of the actin nucleation occurs and extends 1 µm from the membrane (Chan et al., 2000; DesMarais et al., 2002). (Error bars indicate the s.e.m.)

View larger version (61K): [in a new window]   Fig. 4. Dephosphorylated cofilin, but not p-cofilin or LIMK1, is associated with the actin cytoskeleton. (A) Representative immunofluorescence images of cofilin and p-cofilin staining in fixed MTLn3 cells compared with Triton X-100-insoluble cytoskeletons. Cofilin is observed throughout the cytoplasm and in the leading-edge compartment in both non-extracted and Triton X-100-extracted cells. However, p-cofilin is not retained in cells extracted with Triton X-100, indicating that it is not bound to the cytoskeleton. (Bar, 10 µm.) (B) Western blot analysis of the retention of cofilin, p-cofilin and LIMK1 in the Triton X-100-insoluble cytoskeleton of MTLn3 cells at 0 and 60 seconds following stimulation with EGF. Western blot was standardized to total protein.

View larger version (68K): [in a new window]   Fig. 5. Distribution of LIMK1 and phosphorylated-cofilin. (A) Representative images of LIMK1, p-LIMK1 and p-cofilin immunolocalization in MTLn3 cells fixed at 0 seconds and 60 seconds following stimulation with EGF. The two localization patterns do not significantly overlap. (Bar, 10 µm.) (B) Analysis of LIMK1 (red) and p-cofilin (green) amount from the membrane to inside of the cell 60 seconds after stimulation with EGF, showing the lack of co-enrichment in the leading edge (shaded). (Error bars indicate the s.e.m.) (C) Pearson's correlation coefficient analysis of the overlap of LIMK1 and p-cofilin channels. Both proteins show a low level of correlation throughout and particularly in the leading edge compartment (shaded) at both before () and 60 seconds after () EGF stimulation. (Error bars indicate the s.e.m.) (D) Pearson's correlation coefficient analysis of the overlap of p-LIMK (red) and p-cofilin (green) channels. Both proteins show a low level of correlation in the leading edge compartment both before and after stimulation. (Error bars indicate the s.e.m.)

View larger version (22K): [in a new window]   Fig. 6. ROCK-dependent activation of LIMK1 is required for the increase in cofilin phosphorylation following EGF stimulation. (A) MTLn3 cells were preincubated with the ROCK inhibitor Y-27632 (10 µM) for 30 minutes prior to stimulation with EGF. Cell lysates were prepared at 0 and 60 seconds following stimulation with EGF, western blotted and probed with antibodies against p-cofilin and activated p-LIMK. (B,C) Quantification of western blot for changes of LIMK1 and p-cofilin showing the inhibition of LIMK1 and cofilin phosphorylation caused by ROCK inhibitor. (Error bars indicate the s.e.m.)

View larger version (91K): [in a new window]   Fig. 7. Phosphorylation of LIMK1 and cofilin is not required for EGF-stimulated lamellipod protrusion and barbed end formation following stimulation with EGF. (A) MTLn3 cells were pre-incubated with the ROCK inhibitor Y-27632 (10 µM) for 30 minutes and fixed at 0 and 60 seconds following stimulation with EGF to inhibit the phosphorylation of both LIMK1 and cofilin. Panel shows representative images of the F-actin cytoskeleton stained with Alexa Fluor-488-labeled phalloidin in control and Y-27632-treated cells. (Bar, 10 µm) (B) The change in lamellipod protrusion area is unaffected by ROCK inhibition. MTLn3 cells were treated with or without the ROCK inhibitor, Y-27632 and fixed at 0 and 60 seconds after stimulation with EGF. The average fold change in cell area measured at 0 and 60 seconds EGF in control and Y-27632-treated cells is identical. Student's t-test showed no statistical difference in lamellar protrusion between control and Y-27632 cells. (Error bars indicate the s.e.m.) (C) Time lapse microscopy of MTLn3 cells demonstrate no change in the initial rate or final extent of lamellipod protrusion in Y-27632-treated cells () compared with control cells () following stimulation with EGF. (D) The change in the appearance of new barbed ends is unaffected by ROCK inhibition. Time lapse microscopy of ß-actin-GFP MTLn3 cells demonstrate there is no effect on the fold change in the appearance of new barbed ends in Y-27632-treated cells following stimulation with EGF compared with control cells. For panels C and D, error bars indicate the s.e.m. for nine control cells and 11 Y-27632-treated cells (n=2 separate experiments).

View larger version (45K): [in a new window]   Fig. 8. LIMK1 siRNA has no effect on EGF-stimulated lamellipod protrusion and barbed-end formation. (A) MTLn3 cells were treated with control or LIMK1-specific siRNA oligonucleotides for 36 hours prior to EGF stimulation. Cell lysates were prepared at 0 and 60 seconds following EGF stimulation, western blotted and probed with antibodies against p-cofilin and LIMK-1. (B) Quantification of western blots showing the decrease of LIMK1 expression and cofilin phosphorylation levels in control (left) and LIMK1 siRNA treated cells (right). Error bars indicate the standard error of the mean. (n=3 separate experiments). (C) The change in protrusion area following EGF stimulation of MTLn3 cells is not affected by LIMK1 siRNA treatment. Time lapse microscopy of MTLn3 cells demonstrate no change in the initial rate or final extent of lamellipod protrusion in LIMK1 siRNA-treated cells () compared to control cells () following stimulation with EGF. (D) The increase in free barbed ends following EGF stimulation of MTLn3 cells is unaffected in LIMK1 siRNA. Time lapse microscopy of ß-actin-GFP MTLn3 cells demonstrate there is no effect on the fold change in the appearance of new barbed ends in LIMK1 siRNA-treated cells following EGF stimulation compared to control cells. For panel C and D, the error bars indicate the s.e.m. (n=2 separate experiments).

View larger version (28K): [in a new window]   Fig. 9. PLC activation is not required for the increase in p-cofilin levels following EGF. (A) MTLn3 cells were preincubated with the PLC inhibitor U-73312 or the inactive control U-73343 for 10 minutes prior to stimulation with EGF. Cells were lysed at 60 seconds following stimulation with EGF and the amount of p-cofilin measured in western blots. (B) Quantification of the p-cofilin level of western blots. The error bars indicate the s.e.m. (n=2 separate experiments).