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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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EGF stimulation does not induce cofilin dephosphorylation in MTLn3 cells. (A) Representative Western blot for p-cofilin in MTLn3 cells at 0, 30, 60, and 180 s after stimulation. (B) Plot of p-cofilin band intensities standardized over the corresponding actin bands (time is in seconds after stimulation). (C) Cofilin siRNA suppresses the levels of cofilin expression in MTLn3 cells. Representative Western blot of cofilin after cofilin RNAi transfection or after control treatment with oligofectamine (time is in hours after transfection). White lines indicate that intervening lanes have been spliced out. (D) Quantitation of anti-cofilin Western blotting analysis of lysates at different time points after transfection. Error bars are SEM of averages of at least three independent experiments.
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fig6: EGF stimulation does not induce cofilin dephosphorylation in MTLn3 cells. (A) Representative Western blot for p-cofilin in MTLn3 cells at 0, 30, 60, and 180 s after stimulation. (B) Plot of p-cofilin band intensities standardized over the corresponding actin bands (time is in seconds after stimulation). (C) Cofilin siRNA suppresses the levels of cofilin expression in MTLn3 cells. Representative Western blot of cofilin after cofilin RNAi transfection or after control treatment with oligofectamine (time is in hours after transfection). White lines indicate that intervening lanes have been spliced out. (D) Quantitation of anti-cofilin Western blotting analysis of lysates at different time points after transfection. Error bars are SEM of averages of at least three independent experiments.

Mentions: However, previous work has shown that resting carcinoma cells contain lower levels of phospho-cofilin (Zebda et al., 2000). Therefore, we determined the effect of EGF stimulation on phospho-cofilin levels in carcinoma cells and found that the level of phospho-cofilin increases after EGF stimulation (Fig. 6, A and B). This finding suggests that cofilin activity is unlikely to be regulated by dephosphorylation in MTLn3 cells, which is consistent with the hypothesis that PLC hydrolysis of PIP2 in response to EGF, independent of cofilin dephosphorylation, could be the major pathway for cofilin activation.


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)

EGF stimulation does not induce cofilin dephosphorylation in MTLn3 cells. (A) Representative Western blot for p-cofilin in MTLn3 cells at 0, 30, 60, and 180 s after stimulation. (B) Plot of p-cofilin band intensities standardized over the corresponding actin bands (time is in seconds after stimulation). (C) Cofilin siRNA suppresses the levels of cofilin expression in MTLn3 cells. Representative Western blot of cofilin after cofilin RNAi transfection or after control treatment with oligofectamine (time is in hours after transfection). White lines indicate that intervening lanes have been spliced out. (D) Quantitation of anti-cofilin Western blotting analysis of lysates at different time points after transfection. Error bars are SEM of averages of at least three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: EGF stimulation does not induce cofilin dephosphorylation in MTLn3 cells. (A) Representative Western blot for p-cofilin in MTLn3 cells at 0, 30, 60, and 180 s after stimulation. (B) Plot of p-cofilin band intensities standardized over the corresponding actin bands (time is in seconds after stimulation). (C) Cofilin siRNA suppresses the levels of cofilin expression in MTLn3 cells. Representative Western blot of cofilin after cofilin RNAi transfection or after control treatment with oligofectamine (time is in hours after transfection). White lines indicate that intervening lanes have been spliced out. (D) Quantitation of anti-cofilin Western blotting analysis of lysates at different time points after transfection. Error bars are SEM of averages of at least three independent experiments.
Mentions: However, previous work has shown that resting carcinoma cells contain lower levels of phospho-cofilin (Zebda et al., 2000). Therefore, we determined the effect of EGF stimulation on phospho-cofilin levels in carcinoma cells and found that the level of phospho-cofilin increases after EGF stimulation (Fig. 6, A and B). This finding suggests that cofilin activity is unlikely to be regulated by dephosphorylation in MTLn3 cells, which is consistent with the hypothesis that PLC hydrolysis of PIP2 in response to EGF, independent of cofilin dephosphorylation, could be the major pathway for cofilin activation.

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