Limits...
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.

Show MeSH

Related in: MedlinePlus

PLC inhibition selectively suppresses the generation of free barbed ends during the early transient and not during the late transient without affecting the total levels of F-actin. (A) Representative images of the barbed end assay of cells treated with the inactive isoform (control) or the PLC inhibitor at 0, 60, and 180 s after stimulation. Bar, 10 μm. (B) Plot of the relative number of barbed ends at 0–0.22 μm inside the cell versus time after stimulation in control (open circles) and PLC-inhibited (closed circles) MTLn3 cells. (C) Total F-actin levels in control and PLC-inhibited cells as measured by rhodamine-phalloidin staining. Error bars in both graphs are SEM of ∼60 cells, pooled from at least three independent experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172433&req=5

fig3: PLC inhibition selectively suppresses the generation of free barbed ends during the early transient and not during the late transient without affecting the total levels of F-actin. (A) Representative images of the barbed end assay of cells treated with the inactive isoform (control) or the PLC inhibitor at 0, 60, and 180 s after stimulation. Bar, 10 μm. (B) Plot of the relative number of barbed ends at 0–0.22 μm inside the cell versus time after stimulation in control (open circles) and PLC-inhibited (closed circles) MTLn3 cells. (C) Total F-actin levels in control and PLC-inhibited cells as measured by rhodamine-phalloidin staining. Error bars in both graphs are SEM of ∼60 cells, pooled from at least three independent experiments.

Mentions: To determine the role of PLC in the EGF-induced generation of free barbed ends, we inhibited PLC activity with U73122 and measured the relative number of barbed ends after EGF addition using the barbed end assay (Fig. 3). U73343, the inactive isoform of the drug, and DMSO, the vehicle for both compounds, were applied as control treatments. Cells were treated for 10 min with U73122, U73343, or DMSO before EGF addition. Cells treated with U73122 showed a selective decrease in the barbed end edge staining during the early transient, as compared with DMSO (not depicted) and U73343 (Fig. 3 B). However, inhibition of PLC did not affect the barbed end staining during the late transient where the generation of free barbed ends still peaked at 3 min after EGF addition. These results showed that PLC inhibition selectively inhibits the generation of new barbed ends during the early barbed end transient but not the late transient. From this result, we concluded that the activation of PLC in response to EGF stimulation is a major regulator of the early transient. However, the inhibition of PLC did not have an obvious effect on the levels of total F-actin in resting cells, as were measured by rhodamine-phalloidin staining (Fig. 3 C). This result indicates that PLC inhibition is specifically suppressing actin polymerization during the early transient, and not through an indirect effect by inhibition of actin depolymerization.


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 selectively suppresses the generation of free barbed ends during the early transient and not during the late transient without affecting the total levels of F-actin. (A) Representative images of the barbed end assay of cells treated with the inactive isoform (control) or the PLC inhibitor at 0, 60, and 180 s after stimulation. Bar, 10 μm. (B) Plot of the relative number of barbed ends at 0–0.22 μm inside the cell versus time after stimulation in control (open circles) and PLC-inhibited (closed circles) MTLn3 cells. (C) Total F-actin levels in control and PLC-inhibited cells as measured by rhodamine-phalloidin staining. Error bars in both graphs 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

fig3: PLC inhibition selectively suppresses the generation of free barbed ends during the early transient and not during the late transient without affecting the total levels of F-actin. (A) Representative images of the barbed end assay of cells treated with the inactive isoform (control) or the PLC inhibitor at 0, 60, and 180 s after stimulation. Bar, 10 μm. (B) Plot of the relative number of barbed ends at 0–0.22 μm inside the cell versus time after stimulation in control (open circles) and PLC-inhibited (closed circles) MTLn3 cells. (C) Total F-actin levels in control and PLC-inhibited cells as measured by rhodamine-phalloidin staining. Error bars in both graphs are SEM of ∼60 cells, pooled from at least three independent experiments.
Mentions: To determine the role of PLC in the EGF-induced generation of free barbed ends, we inhibited PLC activity with U73122 and measured the relative number of barbed ends after EGF addition using the barbed end assay (Fig. 3). U73343, the inactive isoform of the drug, and DMSO, the vehicle for both compounds, were applied as control treatments. Cells were treated for 10 min with U73122, U73343, or DMSO before EGF addition. Cells treated with U73122 showed a selective decrease in the barbed end edge staining during the early transient, as compared with DMSO (not depicted) and U73343 (Fig. 3 B). However, inhibition of PLC did not affect the barbed end staining during the late transient where the generation of free barbed ends still peaked at 3 min after EGF addition. These results showed that PLC inhibition selectively inhibits the generation of new barbed ends during the early barbed end transient but not the late transient. From this result, we concluded that the activation of PLC in response to EGF stimulation is a major regulator of the early transient. However, the inhibition of PLC did not have an obvious effect on the levels of total F-actin in resting cells, as were measured by rhodamine-phalloidin staining (Fig. 3 C). This result indicates that PLC inhibition is specifically suppressing actin polymerization during the early transient, and not through an indirect effect by inhibition of actin depolymerization.

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