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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 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.
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fig5: 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.

Mentions: To further study the contribution of PLC to the EGF-induced actin polymerization and cell motility, we used a fluorescence time-lapse microscopy technique for visualizing changes in F-actin content in living cells (Lorenz et al., 2004a,b). MTLn3 cells stably expressing GFP-β-actin were used in these experiments. This microscopy technique allowed us to quantitate both changes in F-actin levels at the leading edge and lamellipod extension in response to EGF by time-lapse, of which Fig. 5 A shows representative still pictures. This assay was used to measure the changes in F-actin levels at the leading edge (Fig. 5 B), but not free barbed ends. Inhibition of PLC activity abolished the early EGF-induced increase of F-actin (Fig. 5 B; and Fig. 5 D, i). However, F-actin levels started increasing by 200 s after EGF addition, indicating that later actin polymerization was not affected. On the contrary, inhibition of PI3K suppressed the late increase in F-actin and did not affect early F-actin levels (Fig. 5 D, ii). Similarly, analysis of lamellipodium extension revealed that inhibition of PLC halted membrane protrusion until the F-actin levels increased at 250 s after stimulation, at which lamellipod extension occurred (Fig. 5 C; and Fig. 5 E, i), and that PI3K inhibition suppressed protrusion significantly in addition to suppressing the late increase in F-actin levels (Fig. 5 D, ii; and Fig. 5 E, ii). These results revealed that PLC inhibition only delays the EGF-induced lamellipodium extension, whereas full lamellipod extension requires PI3K 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 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.
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Related In: Results  -  Collection

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fig5: 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.
Mentions: To further study the contribution of PLC to the EGF-induced actin polymerization and cell motility, we used a fluorescence time-lapse microscopy technique for visualizing changes in F-actin content in living cells (Lorenz et al., 2004a,b). MTLn3 cells stably expressing GFP-β-actin were used in these experiments. This microscopy technique allowed us to quantitate both changes in F-actin levels at the leading edge and lamellipod extension in response to EGF by time-lapse, of which Fig. 5 A shows representative still pictures. This assay was used to measure the changes in F-actin levels at the leading edge (Fig. 5 B), but not free barbed ends. Inhibition of PLC activity abolished the early EGF-induced increase of F-actin (Fig. 5 B; and Fig. 5 D, i). However, F-actin levels started increasing by 200 s after EGF addition, indicating that later actin polymerization was not affected. On the contrary, inhibition of PI3K suppressed the late increase in F-actin and did not affect early F-actin levels (Fig. 5 D, ii). Similarly, analysis of lamellipodium extension revealed that inhibition of PLC halted membrane protrusion until the F-actin levels increased at 250 s after stimulation, at which lamellipod extension occurred (Fig. 5 C; and Fig. 5 E, i), and that PI3K inhibition suppressed protrusion significantly in addition to suppressing the late increase in F-actin levels (Fig. 5 D, ii; and Fig. 5 E, ii). These results revealed that PLC inhibition only delays the EGF-induced lamellipodium extension, whereas full lamellipod extension requires PI3K 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