Phospholipase C and cofilin are required for carcinoma cell directionality in response to EGF stimulation

Abstract

The epidermal growth factor (EGF)-induced increase in free barbed ends, resulting in actin polymerization at the leading edge of the lamellipodium in carcinoma cells, occurs as two transients: an early one at 1 min and a late one at 3 min. Our results reveal that phospholipase (PLC) is required for triggering the early barbed end transient. Phosphoinositide-3 kinase selectively regulates the late barbed end transient. Inhibition of PLC inhibits cofilin activity in cells during the early transient, delays the initiation of protrusions, and inhibits the ability of cells to sense a gradient of EGF. Suppression of cofilin, using either small interfering RNA silencing or function-blocking antibodies, selectively inhibits the early transient. Therefore, our results demonstrate that the early PLC and cofilin-dependent barbed end transient is required for the initiation of protrusions and is involved in setting the direction of cell movement in response to EGF.

Figure 1.

The Barbed end assay, in MTLn3 cells, shows that EGF stimulation results in early and late barbed end transients at the leading edge. (A) Representative images of EGF-stimulated cells (0, 60, and 180 s) with the barbed end staining at the leading edge. Bar, 10 μm. (B) The relative number of barbed ends (arbitrary units of fluorescence intensity) from 1.1 μm outside the cells (the membrane is at 0 μm) to 1.1 μm inside the cell periphery. (C) The relative number of barbed ends in the zone between 0 and 0.22 μm inside the cell edge (B, shaded area) versus time after addition of EGF. Error bars are SEM of ∼60 cells, pooled from at least three independent experiments.

Figure 2.

PLCγ activity peaks at 60 s after EGF stimulation in MTLn3 cells. (A) The plot of p(Y)-PLCγ levels, standardized over total IgG levels (from the same blot), versus time after EGF addition. Error bars are SEM of averages of three independent experiments. (B) Representative Western blot of PLCγ [pY783]. (C) Effect of the PLC inhibitor (U73122) as compared with control (the inactive isoform, U73343).

Figure 3.

PLC inhibition selectively suppresses the generation of free barbed ends during the early transient and not during the late transient without affecting the total levels of F-actin. (A) Representative images of the barbed end assay of cells treated with the inactive isoform (control) or the PLC inhibitor at 0, 60, and 180 s after stimulation. Bar, 10 μm. (B) Plot of the relative number of barbed ends at 0–0.22 μm inside the cell versus time after stimulation in control (open circles) and PLC-inhibited (closed circles) MTLn3 cells. (C) Total F-actin levels in control and PLC-inhibited cells as measured by rhodamine-phalloidin staining. Error bars in both graphs are SEM of ∼60 cells, pooled from at least three independent experiments.

Figure 4.

PI3K inhibition selectively suppresses the generation of free barbed ends during the late transient. (A) Representative images of the barbed end assay of control cells (DMSO) and of PI3K-inhibited cells (wortmannin) at 0, 60, and 180 s after stimulation. Bar, 10 μm. (B) Plot of the relative number of barbed ends (arbitrary units of fluorescence intensity) at 0–0.22 μm inside the cell edge in control (closed bar) and in PI3K-inhibited (open bar) cells. Error bars are SEM of ∼50 cells, pooled from at least three independent experiments.

Figure 5.

PLC inhibition suppresses EGF-induced actin polymerization at the leading edge and delays lamellipodium extension. Live-cell fluorescent microscopy of PLC-inhibited GFP-actin MTLn3 cells shows a delay in the onset of actin polymerization and membrane protrusion in response to EGF. (A) Still images (at 0, 90, and 360 s after stimulation) of two representative cells treated with the inactive (control) or the active isoform of the PLC inhibitor. Bar, 10 μm. (B) The average fold increase (over 0 s) in GFP fluorescence intensity, corresponding to F-actin, at the cell edge in control (closed circles) and in PLC-inhibited cells (open circles). (C) The average fold increase (over 0 s) in membrane protrusion (Area) of the same cells (time is in seconds after stimulation). The fold change (over 0 s) in F-actin at the cell edge (D) and in cell area (E) at 2 and 5 min in cells treated with U73343 (i, white bars), U73122 (i, gray bars), DMSO control (ii, white bars), and wortmannin (ii, gray bars) is shown. The error bars are SEM values of the averages of 15 cells, in each group, pooled from three independent experiments.

Figure 6.

EGF stimulation does not induce cofilin dephosphorylation in MTLn3 cells. (A) Representative Western blot for p-cofilin in MTLn3 cells at 0, 30, 60, and 180 s after stimulation. (B) Plot of p-cofilin band intensities standardized over the corresponding actin bands (time is in seconds after stimulation). (C) Cofilin siRNA suppresses the levels of cofilin expression in MTLn3 cells. Representative Western blot of cofilin after cofilin RNAi transfection or after control treatment with oligofectamine (time is in hours after transfection). White lines indicate that intervening lanes have been spliced out. (D) Quantitation of anti-cofilin Western blotting analysis of lysates at different time points after transfection. Error bars are SEM of averages of at least three independent experiments.

Figure 7.

Suppression of cofilin expression, or blocking cofilin function, selectively inhibits the early (but not the late) barbed end transient. (A) Representative images of the barbed end assay (performed at 36 h after transfection) of control (oligofectamine) and of cofilin siRNA-transfected cells at 0, 60, and 180 s after stimulation. (C) Representative images of the barbed end assay of IgG and of cofilin function-blocking antibody-injected cells at 0, 60, and 180 s after stimulation (arrow indicates the limits of the cell edge as traced in phase contrast). Bars, 10 μm. (B and D) Relative number of barbed ends (closed bars represent control cells and open bars cofilin knockdown cells in B and cofilin Ab-injected cells in D) at 0–0.22 μm inside the cell edge versus time after stimulation. Error bars are SEM of ∼50 cells, pooled from at least three independent experiments.

Figure 8.

PLC inhibition (but not PI3K inhibition) selectively suppresses cofilin activity at 1 min after stimulation. (A) Cofilin activity in DMSO- (black bars), U73343- (gray bars), and U73122 (white bars)-treated cells at 0, 60, and 180 s after stimulation. (B) Cofilin activity in DMSO- (black bars) and wortmannin (white bars)-treated cells at 0, 60, and 180 s after stimulation. Cofilin activity was standardized over total protein content. Errors bars are SEM of the averages of three independent experiments.

Figure 9.

PLC activity is required for directional protrusion in response to an EGF source. (A) The response of a control (U73343) cell (top) and of a PLC-inhibited (U73122) cell (bottom) to an EGF microneedle (the white triangle represents the position of the tip of the needle, and the white arrowhead indicates the direction of protrusion). (B) The quantitation of membrane protrusion at: (a) the front of cells (corresponding to the protrusion along the front axis formed between the cell centroid and the tip of the pipette), closed diamonds represent control and open diamonds PLC-inhibited cells; (b) the side of cells (corresponding to the axis that forms a 90° angle with the front axis), closed triangles represent control and open triangles PLC-inhibited cells; (c) the back of cells (corresponding to the axis that forms a 180° angle with the front axis), closed circles represent control and open circles PLC-inhibited cells. Error bars are SEM values of the averages of 15 cells, in each group, pooled from at least three independent experiments.