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First published online 15 May 2007
doi: 10.1242/jcs.004366

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Dynamic cofilin phosphorylation in the control of lamellipodial actin homeostasis

¹ Ludwig Institute for Cancer Research, UCL Branch, London, W1W 7BS, UK
² Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense, Denmark
³ Translational Research Laboratory, Institute of Women's Health, University College London, London, WC1E 6DH, UK

View larger version (64K): [in this window] [in a new window]   Fig. 1. P-cofilin levels change in response to alterations in actin filament organisation and PI3K signalling. (A) Representative 2D-DIGE gel images and 3D-fluorescence profiles of acidic and basic cofilin isoforms are shown for untreated (Ctrl) cells and cells treated with control dsRNA and dsRNA targeting cofilin, PTEN, PI3K p110 catalytic subunit (p110), Cdc42, Rac1 and Rac2 (Rac1/2), and Arp3. (B) The relative abundances of P-cofilin (acidic isoform) and cofilin (basic isoform) are shown for RNAi-treated cells from 2D-DIGE image analysis. Values represent the average of three measurements from biological replicates. Error bars represent the standard deviation. (C) Immunoblots show P-cofilin levels in untreated control and dsRNA-treated S2R+ cell lysates confirming 2D-DIGE data. Relative abundance is shown below the blot as a percentage of the control, and is the average calculated from densitometry measurements of blots from three independent experiments. (D) Immunoblot of P-Akt and total Akt levels in control and dsRNA-treated S2R+ cell lysates confirms the loss of PI3K, PTEN and Akt expression.

View larger version (86K): [in this window] [in a new window]   Fig. 2. The kinetics of actin remodelling and cofilin phosphorylation following an acute stimulus. (A) S2R+ cells stained for F-actin (Rhodamine-phalloidin) and nuclei (DAPI) are shown at various times after insulin stimulation (10 µg/ml). Bar, 50 µm. (B) Images from a time-lapse movie of S2R+ cells stimulated with insulin (10 µg/ml) for the times indicated. Cells were filmed in phase-contrast on a time-lapse microscope using a 100x oil-immersion lens; frames were acquired every 20 seconds, 10 minutes prior to insulin addition and for 30 minutes after addition. Images are representative of five independent experiments. Bar, 50 µm. Right panel shows a kymograph of the movie from which the presented images were taken, generated from pixel intensities along a line transecting the cell membrane (shown in first image). It shows increased protrusion dynamics after insulin stimulation. (C) F-actin immunostaining (Rhodamine-phalloidin) of serum-starved Kc167 cells stimulated with insulin (10 µg/ml) for the indicated times. Bar, 50 µm. (D) Cofilin is transiently phosphorylated in S2R+ cells in response to insulin. S2R+ cells were grown in serum-free medium overnight and stimulated with insulin at 10 µg/ml for the indicated times. Lysates were prepared and immunoblotted with antibody against P-cofilin. Blots were analysed by densitometry. Values represent the mean P-cofilin signal from two experiments after normalising with respect to -actin levels. Error bars represent the standard deviation. (E) Time course of Akt activation in insulin stimulated S2R+ cells. S2R+ cells were maintained in Schneider's serum-free medium overnight, and then stimulated with bovine insulin (10 µg/ml) for the times indicated. Immunoblotting was used to assess levels of P-Akt and total Akt.

View larger version (84K): [in this window] [in a new window]   Fig. 3. PI3K-signalling controls cortical actin organisation. (A) Actin staining of S2R+ cells after treatment with DMSO vehicle (Ctrl), LY294002 (100 µM) and wortmannin (100 nM). Cells were treated for 10 minutes and then fixed and stained with Rhodamine-phalloidin. Arrowheads indicate regions of cell retraction to leave protrusions. Bar, 50 µm. (B) Images of time-lapse movies of S2R+ cells treated with DMSO, LY294002 (100 µM) and wortmannin (100 nM). Cells were filmed in phase-contrast on a time-lapse microscope using a 100x oil-immersion lens. Frames were acquired every 10 seconds for 30 minutes with inhibitor treatment after 10 minutes of filming (designated 0' in the figure). Snapshots are shown at 0, 5 and 10 minutes. The corresponding kymographs show cells from 3 minutes before until 10 minutes after insulin stimulation and were constructed from pixel intensities taken along the lines depicted in the images on the left. Bar, 50 µm. (C) Immunoblotting of P-Akt and total Akt in untreated S2R+ cells and cells treated with insulin (for 10 minutes) with and without pre-treatment (for 10 minutes) with LY294002 (100 µM) or wortmannin (100 nM). (D) Immunoblotting of P-Akt, -actin and P-cofilin in S2R+ cells treated for 10 minutes with LY294002 (100 µM) and insulin (10 µg/ml), alone and in combination. Relative abundance is represented as a percentage of the control below the blot and is the average calculated from densitometry measurements of blots from three independent experiments.

View larger version (110K): [in this window] [in a new window]   Fig. 4. Functional analysis of cofilin during acute actin signalling. (A) Actin staining in dsRNA-treated cells stimulated with insulin. S2R+ cells were incubated with dsRNAs targeting LacZ, cofilin, LIMK, Ssh, Rac1and Rac2 (Rac1/2) and Arp3 for 5 days. On day 5, cells were treated with insulin for 10 minutes (10') or left untreated (0'). Cells were then fixed and stained for F-actin with Rhodamine-phalloidin. Bars, 50 µm. (B) Visualisation of actin dynamics in dsRNA-treated cells using GFP-labelled moesin. Snapshots of time-lapse movies just before the addition of insulin are shown on the left. Bars, 50 µm. The two lines used to generate the kymographs (right) are indicated on the cell images. Actin reorganisation was filmed for 3 minutes before and for 10 minutes after the addition of 10 µg/ml insulin, the point of insulin addition labelled as 0'. Images are representative of at least two independent experiments.

View larger version (58K): [in this window] [in a new window]   Fig. 5. PI3K-induced changes in cofilin phosphorylation are the result of actin cytoskeletal remodelling. (A) LIMK and Ssh are regulators of cofilin phosphorylation. S2R+ cells were left untreated (Ctrl) or were treated with dsRNA for knockdown of expression of LIMK, Ssh and cofilin for 5 days. Cells were lysed and P-cofilin and -actin levels assessed in the lysates by immunoblotting. (B) Immunoblotting of P-cofilin, P-Akt and -actin in lysates of dsRNA-treated cells stimulated with insulin. S2R+ cells were cultured in the presence of dsRNA targeting LacZ (control), Rac1+2 and Ssh and then treated with 10 µg/ml insulin for the indicated times prior to lysis and immunoblotting. (C) P-cofilin and -actin levels were assessed in extracts from S2R+ cells treated with jasplakinolide (1 µg/ml), latrunculin B (1 µg/ml) or cytochalasin D (2 µg/ml) for the times indicated. (D) P-cofilin, P-Akt and -actin levels are shown for S2R+ cells pre-treated for 10 minutes with latrunculin B (1 µg/ml) or jasplakinolide (1 µg/ml) followed by 20 minutes of insulin treatment (10 µg/ml) or vehicle alone.

View larger version (50K): [in this window] [in a new window]   Fig. 6. Slingshot is the major regulator of actin-dependent cofilin phosphorylation. (A) Ssh RNAi blocks jasplakinolide-induced loss of cofilin phosphorylation. Control S2R+ cells and cells treated with dsRNA targeting Ssh were treated with jasplakinolide (1 µg/ml) for the times indicated and P-cofilin and -actin levels assessed in the lysates by immunoblotting. (B) LIMK RNAi does not reverse Ssh dsRNA blockade of jasplakinolide-induced loss of cofilin phosphorylation. Cells treated with dsRNA targeting Ssh (SSH) or Ssh and LIMK together (SSH/LIMK) were treated with jasplakinolide (1 µg/ml) for the indicated times, and P-cofilin and -actin levels assessed in lysates by immunoblotting. (C) The effect of LIMK and Ssh on latrunculin B-induced cofilin-phosphorylation. Cells treated with control dsRNA and dsRNA targeting LIMK or LIMK and SSH together (LIMK/SSH) were treated with latrunculin B (1 µg/ml) for the indicated times, and P-cofilin and -actin levels in lysates were determined by immunoblotting. (D) Loss of actin expression induces cofilin phosphorylation. P-cofilin and -actin levels were determined by immunoblotting in lysates of control cells (untreated) and cells treated with dsRNAs targeting actin, cofilin and LacZ.

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