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

Mouneimne G, Soon L, DesMarais V, Sidani M, Song X, Yip SC, Ghosh M, Eddy R, Backer JM, Condeelis J - J. Cell Biol. (2004)

Bottom Line: 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.Suppression of cofilin, using either small interfering RNA silencing or function-blocking antibodies, selectively inhibits the early transient.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461, USA. gmouneim@aecom.yu.edu

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.

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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.
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fig4: 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.

Mentions: To reveal the involvement of PI3K in the EGF-induced generation of free barbed ends, we used wortmannin at a final concentration of 100 nM to specifically inhibit PI3K. Cells were treated for 15 min with wortmannin or with equal volume of DMSO or were left untreated (only switched to BSA-free medium; unpublished data). After the different treatments, the cells were analyzed using the barbed end assay, and the relative levels of barbed ends were quantitated. Fig. 4 A shows that wortmannin treatment decreased the barbed end level in the nucleation zone at 3 min but not 1 min. Quantitation of the fluorescence intensity at the cell edge showed (Fig. 4 B) that wortmannin suppressed the level of barbed ends by almost sixfold (rendering it close to control levels) at 3 min. These results are consistent with previous work, in which function-blocking antibodies, raised against the P110α subunit of PI3K, when microinjected into these carcinoma cells, inhibited the generation of free barbed ends at 3 min after EGF stimulation (Hill et al., 2000). However, as shown in the current study for the first time, the number of barbed ends was not affected during the early transient as compared with controls. From these experiments, we concluded that PI3K activity is exclusively involved in the late barbed end transient.


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

Mouneimne G, Soon L, DesMarais V, Sidani M, Song X, Yip SC, Ghosh M, Eddy R, Backer JM, Condeelis J - J. Cell Biol. (2004)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172433&req=5

fig4: 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.
Mentions: To reveal the involvement of PI3K in the EGF-induced generation of free barbed ends, we used wortmannin at a final concentration of 100 nM to specifically inhibit PI3K. Cells were treated for 15 min with wortmannin or with equal volume of DMSO or were left untreated (only switched to BSA-free medium; unpublished data). After the different treatments, the cells were analyzed using the barbed end assay, and the relative levels of barbed ends were quantitated. Fig. 4 A shows that wortmannin treatment decreased the barbed end level in the nucleation zone at 3 min but not 1 min. Quantitation of the fluorescence intensity at the cell edge showed (Fig. 4 B) that wortmannin suppressed the level of barbed ends by almost sixfold (rendering it close to control levels) at 3 min. These results are consistent with previous work, in which function-blocking antibodies, raised against the P110α subunit of PI3K, when microinjected into these carcinoma cells, inhibited the generation of free barbed ends at 3 min after EGF stimulation (Hill et al., 2000). However, as shown in the current study for the first time, the number of barbed ends was not affected during the early transient as compared with controls. From these experiments, we concluded that PI3K activity is exclusively involved in the late barbed end transient.

Bottom Line: 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.Suppression of cofilin, using either small interfering RNA silencing or function-blocking antibodies, selectively inhibits the early transient.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461, USA. gmouneim@aecom.yu.edu

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.

Show MeSH
Related in: MedlinePlus