EGF stimulation induces a rapid loss of PIP₂ in carcinoma MTLn3 cells. (A) EGF-induced decrease in PIP₂-FRET (black, average 23 experiments) and increase in cell area (red) were measured in MTLn3 cells overexpressing the EGF-receptor (Xue et al., 2006) as described in Materials and methods and Fig. S1 (available at http://www.jcb.org/cgi/content/full/jcb.200706206/DC1). The middle panel shows PIP₂-antibodies staining and the quantification before and after EGF (n > 25). (B) MTLn3-EGFR cells were treated with the PLC inhibitor U73122 (n = 16) or the nonactive drug U73343 (n = 9), and PIP₂ changes were measured by FRET. (C) WT MTLn3 cells were treated as described in B, and PIP₂ levels were analyzed by antibody staining. (n > 25) (D) The cholesterol from MTLn3 cells was extracted by methyl-β-cyclodextrin (MβCD). The change in the PIP₂ level was monitored by FRET (n =10) and antibody staining. Bars, 5 μm. Values were statistically tested by t test. *, P < 0.05; **, P < 0.01; error bars represent SEM.

Translocation of cofilin from the PM upon EGF stimulation in living cells. (A) MTLn3 cells were transiently transfected with GFP-cofilin. GFP-cofilin was detected by Western blot using antibodies against GFP or cofilin. (B) GFP images were taken from MTLn3 cells expressing GFP-cofilin before and 1 min after EGF stimulation. The LCS glow lookup table was used for the images. (C) Schematic representation of FRET between GFP-cofilin and mCherry-CAAX. Cofilin bound to PIP₂ is in close proximity to the membrane, and FRET occurs. The distance between the PM and cofilin will increase upon the translocation of cofilin to cortical F-actin and FRET will diminish. (D) FRET (sens) was measured in nontreated (n = 19) and U73122-treated (n = 16) MTLn3 cells expressing GFP-cofilin and mCherry-CAAX as detailed in the Materials and methods. Shown are FRET images before and after EGF stimulation and before and after MβCD treatment. For controls and the quantification of FRET decreases, see Fig. S4 (available at http://www.jcb.org/cgi/content/full/jcb.200706206/DC1). (E) FRET signal (black line) and area increase (red line) of MTLn3 cells were measured in time upon EGF stimulation on the confocal microscope at 25°C. Plotted is the average of seven experiments. Bars, 5 μm.

Cofilin binds actin upon PLC-mediated PIP₂ reduction. (A) Control or EGF-stimulated MTLn3 cells were fixed and labeled for cofilin (donor; Alexa 488) and F-actin (acceptor; rhodamine). Using a high laser power, a region was bleached in the acceptor fluorescence (white square box). The increase in the donor fluorescence was measured and FRET was calculated for un-treated, U73122-treated, or MβCD-treated cells (right panel; for all n > 15). (B) FRET was measured in a cell that was stimulated by local application of EGF using a pipette (asterisk). In the graph we plotted the normalized FRET values at the side of the cell close to the pipette (front) and the opposite side of the cell (back) (n = 5). Values were statistically tested by t test. *, P < 0.05; **, P < 0.01; error bars represent SEM.

Compartments involved in the cofilin activity cycle. Cofilin molecules cycle through three compartments in the cell; the cytosol, the actin, and the PM compartments. Cofilin at the PM compartment initially translocates to the F-actin compartment upon EGF mediated PIP₂ reduction. Cofilin binds and severs actin filaments resulting in filaments with free barbed ends, and cofilin–G-actin complex. The cofilin–G-actin complex cannot bind actin or PM, and therefore diffuses to the cytosol compartment. In the cytosol compartment, cofilin is phosphorylated by LIM-kinase, resulting in the release of cofilin from the cofilin–G-actin complex. Upon cofilin dephosphorylation by SSH, cofilin can reenter the PM or F-actin compartment, starting a new cycle. The cycling of cofilin through the three compartments increases free barbed ends, resulting in newly polymerized actin filaments. The ARP2/3 complex prefers to bind to these newly formed actin filaments, which amplifies the cofilin-induced actin polymerization, resulting in protrusion formation.

PM-associated cofilin translocates from the PM upon PLC-dependent PIP₂ reduction. (A) MTLn3 cells were stimulated with EGF or treated with MβCD. Shown are images of fixed cells stained for cofilin before and after stimulation or treated as indicated (n > 25). The Leica confocal software glow lookup table was used for all images. Bars, 10 μm. (B) MTLn3 cells were fixed and labeled for PIP₂ and for total cofilin or the phosphorylated form of cofilin. Images were collected before and 1 min after EGF stimulation. The spatial relationship between PIP₂ and cofilin was analyzed with Pearson's correlation coefficient analysis (all conditions n > 25). (C) Shown are the merge and the scatterplot images of the cofilin and PIP₂ channels. Scatterplots are from the first 1 μm from the cell edge and were obtained from images that were smoothed ones using ImageJ. The loss in PIP₂ signal was compensated by longer exposure times. Note that the higher the spatial relationship between PIP₂ and cofilin, the more pixels in the scatterplot align along the diagonal (n = 8). (D) The amount of cofilin in the low-density membrane fraction was analyzed by Western blot in cells stimulated with EGF or treated with MβCD. Note that in contrast to cellular cofilin (Cell Cof.), pure cofilin added to the isolation procedure does not localize in these gradient fractions (three independent experiments). The amount of PIP₂ in low-density membrane fractions of cells treated with the PLC-inhibitor U73122 or the nonactive form U73343 was analyzed by dot blot (E) and the amount of cofilin in the same fraction was determined by Western blot (F) (three independent experiments). Values were statistically tested by t test. *, P < 0.05; **, P < 0.01; error bars represent SEM.

5-ptase induced reduction of PIP₂ leads to cofilin translocation from the PM. MTLn3 cells were transfected with an mRFP-tagged 5-ptase and a PM-targeted CFP-FRB domain. Upon rapamycin addition, the 5-ptase translocated to the PM (for explanation see Fig S5 and Varnai et al. [2006]). PIP₂ levels were followed by YFP-PH(PLCδ1) (A), and the cofilin membrane localization was followed by GFP-cofilin (B). Below the images, the intensity profiles of FRB, PH, and 5-ptase fluoresence through the cell are shown. The blue lines represent pre and the red lines represent post-rapamycin (n > 15). Bars, 5 μm. (C) The formation of lamellipodia upon rapamycin (t = 0) addition was followed by confocal microscopy (see also Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200706206/DC1). Shown are the images of PM-targeted CFP-FRB domains, before (t = 0) or 21 s (t = 21) after rapamycin addition. The right image represent a merge of binary threshold images (ImageJ) of image t = 0 and t = 21. Yellow represents no change, red represents increase lamellipod formation. The bottom images show the kymograph of the formation of the lamellipod, and the area of the cell that was used for the kymograph. From the kymographs, the size of the lamellipod was measured and plotted against time (right panel) for nonstimulated cells (control, red dashed line), rapamycin-stimulated cells (rapa, red line), and for cells that were treated with U73122 and stimulated with rapamycin (U73122 + rapa, blue line) and for cells that were treated with U73122 only (U73122 control, blue dashed line). n = 12; error bars represent SEM.

Cofilin translocates from the PM to F-actin upon PLC-mediated PIP₂ reduction. (A) The membrane and actin binding of GFP-cofilin was followed by FLIP experiments on the confocal microscope at 25°C. At time 0, the cytosolic GFP-cofilin was bleached briefly (bleach region indicated in yellow) and the fluorescence at the cell periphery (Per in blue) and the cytosol (Cyt in red) were followed over time (middle graph). Upon bleaching, the Per/Cyt ratio increases, and because of exchange of molecules in the Cyt and Per, the ratio decreases to baseline. To normalize for bleach efficiencies, the initial ratio was set to 0 and the first point after bleaching was set to 1. (B) Experiments as described in A for cells expressing GFP and GFP-CAAX, and cells expressing GFP. The dark green and pink lines represent the GFP-CAAX/GFP experiments in unstimulated and EGF stimulated cells, respectively. The light green line represents the GFP experiment. Note that EGF-induced cell shape changes did not influence the GFP-CAAX/GFP FLIP, and therefore did not influence the GFP-cofilin FLIP results (for all n > 15). (C) Experiments as described in B for cells expressing GFP-cofilin. The blue and red squares represent unstimulated and EGF-stimulated (1 min) cells, respectively. The pink squares represent EGF-stimulated (1 min) U73122-treated cells. Black lines are the two component exponential decays fits with taus of 3.7 and 26 s (for both n > 15). (D) The fractions of the fast and slow taus before and after EGF (for 1 min) stimulation in U3122-treated or untreated cells (all n > 15). (E) Cofilin activity in untreated, 5 min MβCD-treated, 1 min EGF-stimulated, or EGF-stimulated and U73122-treated cells. Cofilin activity was standardized over total protein content and normalized to untreated cells. (average of three independent experiments). Bars, 5 μm. Values were statistically tested by t test. *, P < 0.05; **, P < 0.01; error bars represent SEM.