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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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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.
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fig7: 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.

Mentions: We used the barbed end assay to examine the effect of cofilin siRNA on the EGF-induced generation of free barbed ends. Cells were transfected with cofilin siRNA, or similarly treated with oligofectamine (control), and then the assay was performed at 36 h after transfection. The knock down of cofilin selectively suppressed the early barbed end transient but did not affect the late transient (Fig. 7, A and B).


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)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172433&req=5

fig7: 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.
Mentions: We used the barbed end assay to examine the effect of cofilin siRNA on the EGF-induced generation of free barbed ends. Cells were transfected with cofilin siRNA, or similarly treated with oligofectamine (control), and then the assay was performed at 36 h after transfection. The knock down of cofilin selectively suppressed the early barbed end transient but did not affect the late transient (Fig. 7, A and B).

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