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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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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.
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fig8: 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.

Mentions: To test the hypothesis that PLC regulates actin polymerization by regulating cofilin activity, we measured cofilin activity in MTLn3 cell lysates prepared from cells after EGF stimulation with or without inhibition of PLC. We used a modified version of a light microscope severing assay (Chan et al., 2000; Ichetovkin et al., 2000). We previously demonstrated, using this assay, that the severing activity of cofilin increases in MTLn3 cells at 1 min after EGF stimulation (Chan et al., 2000). Using this assay, EGF stimulation was observed to induce an increase in cofilin activity at 1 min, which then dropped to nonstimulated levels at 3 min (Fig. 8). This increase was completely inhibited by cofilin function-blocking antibodies (unpublished data), indicating that severing is due to cofilin activity.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

fig8: 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.
Mentions: To test the hypothesis that PLC regulates actin polymerization by regulating cofilin activity, we measured cofilin activity in MTLn3 cell lysates prepared from cells after EGF stimulation with or without inhibition of PLC. We used a modified version of a light microscope severing assay (Chan et al., 2000; Ichetovkin et al., 2000). We previously demonstrated, using this assay, that the severing activity of cofilin increases in MTLn3 cells at 1 min after EGF stimulation (Chan et al., 2000). Using this assay, EGF stimulation was observed to induce an increase in cofilin activity at 1 min, which then dropped to nonstimulated levels at 3 min (Fig. 8). This increase was completely inhibited by cofilin function-blocking antibodies (unpublished data), indicating that severing is due to cofilin activity.

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