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WASP family members and formin proteins coordinate regulation of cell protrusions in carcinoma cells.

Sarmiento C, Wang W, Dovas A, Yamaguchi H, Sidani M, El-Sibai M, Desmarais V, Holman HA, Kitchen S, Backer JM, Alberts A, Condeelis J - J. Cell Biol. (2008)

Bottom Line: We found that WAVE2 knockdown (KD) suppresses lamellipod formation and increases filopod formation, whereas N-WASP KD has no effect.This suggests that another actin nucleation activity is at work in carcinoma cells in response to EGF.Increased RhoA activity, which stimulates mDia1 nucleation, was observed in the N-WASP/WAVE2 KD cells and was shown to be required for the N-WASP/WAVE2 KD phenotype.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Yeshiva University, Bronx, NY 10461, USA. csarmien@aecom.yu.edu

ABSTRACT
We examined the role of the actin nucleation promoters neural Wiskott-Aldrich syndrome protein (N-WASP) and WAVE2 in cell protrusion in response to epidermal growth factor (EGF), a key regulator in carcinoma cell invasion. We found that WAVE2 knockdown (KD) suppresses lamellipod formation and increases filopod formation, whereas N-WASP KD has no effect. However, simultaneous KD of both proteins results in the formation of large jagged protrusions with lamellar properties and increased filopod formation. This suggests that another actin nucleation activity is at work in carcinoma cells in response to EGF. A mammalian Diaphanous-related formin, mDia1, localizes at the jagged protrusions in double KD cells. Constitutively active mDia1 recapitulated the phenotype, whereas inhibition of mDia1 blocked the formation of these protrusions. Increased RhoA activity, which stimulates mDia1 nucleation, was observed in the N-WASP/WAVE2 KD cells and was shown to be required for the N-WASP/WAVE2 KD phenotype. These data show that coordinate regulation between the WASP family and mDia proteins controls the balance between lamellar and lamellipodial protrusion activity.

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mDia1 localization and activity. (A) Localization of mDia1 by antibody staining in N-WASP/WAVE2 double KD cells and control cells. Cells were treated with Scrambled or N-WASP/WAVE2 siRNA and stained with anti-mDia1 antibody (green) at 3 min after EGF stimulation. Rhodamine phalloidin was used to stain for actin (red). Insets show actin protrusion with filopods in N-WASP/WAVE2 KD cells. Bar, 10 μm. (B) Constitutively active mDia1 activation gives similar phenotype to N-WASP/WAVE2 double KD cells. Images of cells transfected with control GFP empty vector (top, red) or GFP-ΔGBD-mDia1 (bottom, red), a DA mDia1, for 6 h. Images were taken of cells fixed at 0, 1, and 3 min after EGF stimulation. Rhodamine phalloidin was used to stain for actin (green). Insets (indicated by dashed boxes) show active mDia1 localized at tips of filopods. Bar, 10 μm. (C) Quantification of filopod formation. P-values are compared with control GFP vector cells. (D) Total F-actin fluorescence intensity (percent) was measured using the mean value of phalloidin staining of unstimulated cells transfected with GFP or DA-mDia1 vector. P-values are in comparison to control. Error bars indicate ±SEM of a total of 19 cells from three independent experiments.
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fig8: mDia1 localization and activity. (A) Localization of mDia1 by antibody staining in N-WASP/WAVE2 double KD cells and control cells. Cells were treated with Scrambled or N-WASP/WAVE2 siRNA and stained with anti-mDia1 antibody (green) at 3 min after EGF stimulation. Rhodamine phalloidin was used to stain for actin (red). Insets show actin protrusion with filopods in N-WASP/WAVE2 KD cells. Bar, 10 μm. (B) Constitutively active mDia1 activation gives similar phenotype to N-WASP/WAVE2 double KD cells. Images of cells transfected with control GFP empty vector (top, red) or GFP-ΔGBD-mDia1 (bottom, red), a DA mDia1, for 6 h. Images were taken of cells fixed at 0, 1, and 3 min after EGF stimulation. Rhodamine phalloidin was used to stain for actin (green). Insets (indicated by dashed boxes) show active mDia1 localized at tips of filopods. Bar, 10 μm. (C) Quantification of filopod formation. P-values are compared with control GFP vector cells. (D) Total F-actin fluorescence intensity (percent) was measured using the mean value of phalloidin staining of unstimulated cells transfected with GFP or DA-mDia1 vector. P-values are in comparison to control. Error bars indicate ±SEM of a total of 19 cells from three independent experiments.

Mentions: The localization of endogenous mDia1 was determined by staining with antibodies specific for mDia1. mDia1 was localized throughout the leading edge of control cells. In N-WASP/WAVE2 KD cells, mDia1 was localized at the leading edge of the jagged protrusions and in filopods (Fig. 8 A). mDia1's localization is consistent with its role in the formation of filopods and jagged protrusions in N-WASP/WAVE2 KD cells.


WASP family members and formin proteins coordinate regulation of cell protrusions in carcinoma cells.

Sarmiento C, Wang W, Dovas A, Yamaguchi H, Sidani M, El-Sibai M, Desmarais V, Holman HA, Kitchen S, Backer JM, Alberts A, Condeelis J - J. Cell Biol. (2008)

mDia1 localization and activity. (A) Localization of mDia1 by antibody staining in N-WASP/WAVE2 double KD cells and control cells. Cells were treated with Scrambled or N-WASP/WAVE2 siRNA and stained with anti-mDia1 antibody (green) at 3 min after EGF stimulation. Rhodamine phalloidin was used to stain for actin (red). Insets show actin protrusion with filopods in N-WASP/WAVE2 KD cells. Bar, 10 μm. (B) Constitutively active mDia1 activation gives similar phenotype to N-WASP/WAVE2 double KD cells. Images of cells transfected with control GFP empty vector (top, red) or GFP-ΔGBD-mDia1 (bottom, red), a DA mDia1, for 6 h. Images were taken of cells fixed at 0, 1, and 3 min after EGF stimulation. Rhodamine phalloidin was used to stain for actin (green). Insets (indicated by dashed boxes) show active mDia1 localized at tips of filopods. Bar, 10 μm. (C) Quantification of filopod formation. P-values are compared with control GFP vector cells. (D) Total F-actin fluorescence intensity (percent) was measured using the mean value of phalloidin staining of unstimulated cells transfected with GFP or DA-mDia1 vector. P-values are in comparison to control. Error bars indicate ±SEM of a total of 19 cells from three independent experiments.
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Related In: Results  -  Collection

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fig8: mDia1 localization and activity. (A) Localization of mDia1 by antibody staining in N-WASP/WAVE2 double KD cells and control cells. Cells were treated with Scrambled or N-WASP/WAVE2 siRNA and stained with anti-mDia1 antibody (green) at 3 min after EGF stimulation. Rhodamine phalloidin was used to stain for actin (red). Insets show actin protrusion with filopods in N-WASP/WAVE2 KD cells. Bar, 10 μm. (B) Constitutively active mDia1 activation gives similar phenotype to N-WASP/WAVE2 double KD cells. Images of cells transfected with control GFP empty vector (top, red) or GFP-ΔGBD-mDia1 (bottom, red), a DA mDia1, for 6 h. Images were taken of cells fixed at 0, 1, and 3 min after EGF stimulation. Rhodamine phalloidin was used to stain for actin (green). Insets (indicated by dashed boxes) show active mDia1 localized at tips of filopods. Bar, 10 μm. (C) Quantification of filopod formation. P-values are compared with control GFP vector cells. (D) Total F-actin fluorescence intensity (percent) was measured using the mean value of phalloidin staining of unstimulated cells transfected with GFP or DA-mDia1 vector. P-values are in comparison to control. Error bars indicate ±SEM of a total of 19 cells from three independent experiments.
Mentions: The localization of endogenous mDia1 was determined by staining with antibodies specific for mDia1. mDia1 was localized throughout the leading edge of control cells. In N-WASP/WAVE2 KD cells, mDia1 was localized at the leading edge of the jagged protrusions and in filopods (Fig. 8 A). mDia1's localization is consistent with its role in the formation of filopods and jagged protrusions in N-WASP/WAVE2 KD cells.

Bottom Line: We found that WAVE2 knockdown (KD) suppresses lamellipod formation and increases filopod formation, whereas N-WASP KD has no effect.This suggests that another actin nucleation activity is at work in carcinoma cells in response to EGF.Increased RhoA activity, which stimulates mDia1 nucleation, was observed in the N-WASP/WAVE2 KD cells and was shown to be required for the N-WASP/WAVE2 KD phenotype.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Yeshiva University, Bronx, NY 10461, USA. csarmien@aecom.yu.edu

ABSTRACT
We examined the role of the actin nucleation promoters neural Wiskott-Aldrich syndrome protein (N-WASP) and WAVE2 in cell protrusion in response to epidermal growth factor (EGF), a key regulator in carcinoma cell invasion. We found that WAVE2 knockdown (KD) suppresses lamellipod formation and increases filopod formation, whereas N-WASP KD has no effect. However, simultaneous KD of both proteins results in the formation of large jagged protrusions with lamellar properties and increased filopod formation. This suggests that another actin nucleation activity is at work in carcinoma cells in response to EGF. A mammalian Diaphanous-related formin, mDia1, localizes at the jagged protrusions in double KD cells. Constitutively active mDia1 recapitulated the phenotype, whereas inhibition of mDia1 blocked the formation of these protrusions. Increased RhoA activity, which stimulates mDia1 nucleation, was observed in the N-WASP/WAVE2 KD cells and was shown to be required for the N-WASP/WAVE2 KD phenotype. These data show that coordinate regulation between the WASP family and mDia proteins controls the balance between lamellar and lamellipodial protrusion activity.

Show MeSH
Related in: MedlinePlus