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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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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.
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fig1: 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.

Mentions: In carcinoma cells, the EGF-induced actin polymerization activity is directly correlated with the generation of free barbed filament ends. EGF induces an increase in the number of free barbed ends in the nucleation zone (corresponding to the most peripheral 0.22 μm; Fig. 1 B, shaded area) of the leading edge, and this increase has two transients, with a major peak around 1 min after stimulation and a second smaller peak at 3 min (Fig. 1 C). These results were consistent with the previously determined high temporal resolution kinetic time course of barbed ends (Chan et al., 1998). In this work, we refer to the peak of barbed ends at 1 min as the early transient and to the peak of the barbed ends at 3 min as the late 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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: 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.
Mentions: In carcinoma cells, the EGF-induced actin polymerization activity is directly correlated with the generation of free barbed filament ends. EGF induces an increase in the number of free barbed ends in the nucleation zone (corresponding to the most peripheral 0.22 μm; Fig. 1 B, shaded area) of the leading edge, and this increase has two transients, with a major peak around 1 min after stimulation and a second smaller peak at 3 min (Fig. 1 C). These results were consistent with the previously determined high temporal resolution kinetic time course of barbed ends (Chan et al., 1998). In this work, we refer to the peak of barbed ends at 1 min as the early transient and to the peak of the barbed ends at 3 min as the late 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