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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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WAVE2 is recruited to the leading edge of extending lamellipods after stimulation with EGF. (A) Cells were stimulated with EGF and stained with the C-14 anti-WAVE2 antibody. Bar, 10 μm. (B) Quantification of recruitment of WAVE2 in the leading edge. Mean fluorescent intensity of C-14 staining was measured at the leading edge and plotted versus time of EGF stimulation. (C) Ratio of WAVE2 fluorescent intensity at the leading edge to total cellular WAVE2 fluorescent intensity. Error bars are ±SEM of a total of 26 cells from three independent experiments.
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fig2: WAVE2 is recruited to the leading edge of extending lamellipods after stimulation with EGF. (A) Cells were stimulated with EGF and stained with the C-14 anti-WAVE2 antibody. Bar, 10 μm. (B) Quantification of recruitment of WAVE2 in the leading edge. Mean fluorescent intensity of C-14 staining was measured at the leading edge and plotted versus time of EGF stimulation. (C) Ratio of WAVE2 fluorescent intensity at the leading edge to total cellular WAVE2 fluorescent intensity. Error bars are ±SEM of a total of 26 cells from three independent experiments.

Mentions: Changes in the localization of WAVE2 after EGF stimulation were determined using immunofluorescence and compared with that previously determined for N-WASP (Sukumvanich et al., 2004). In unstimulated cells the distribution of these proteins is diffuse throughout the cytoplasm. After EGF stimulation these proteins are both recruited to the leading edge (Fig. 2 A). N-WASP (Sukumvanich et al., 2004) and WAVE2 (Fig. 2, B and C) have similar recruitment kinetics during EGF-stimulated protrusion. Recruitment of both proteins to the leading edge shows a peak at 60 s after EGF stimulation, followed by a gradual decrease.


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)

WAVE2 is recruited to the leading edge of extending lamellipods after stimulation with EGF. (A) Cells were stimulated with EGF and stained with the C-14 anti-WAVE2 antibody. Bar, 10 μm. (B) Quantification of recruitment of WAVE2 in the leading edge. Mean fluorescent intensity of C-14 staining was measured at the leading edge and plotted versus time of EGF stimulation. (C) Ratio of WAVE2 fluorescent intensity at the leading edge to total cellular WAVE2 fluorescent intensity. Error bars are ±SEM of a total of 26 cells from three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2290849&req=5

fig2: WAVE2 is recruited to the leading edge of extending lamellipods after stimulation with EGF. (A) Cells were stimulated with EGF and stained with the C-14 anti-WAVE2 antibody. Bar, 10 μm. (B) Quantification of recruitment of WAVE2 in the leading edge. Mean fluorescent intensity of C-14 staining was measured at the leading edge and plotted versus time of EGF stimulation. (C) Ratio of WAVE2 fluorescent intensity at the leading edge to total cellular WAVE2 fluorescent intensity. Error bars are ±SEM of a total of 26 cells from three independent experiments.
Mentions: Changes in the localization of WAVE2 after EGF stimulation were determined using immunofluorescence and compared with that previously determined for N-WASP (Sukumvanich et al., 2004). In unstimulated cells the distribution of these proteins is diffuse throughout the cytoplasm. After EGF stimulation these proteins are both recruited to the leading edge (Fig. 2 A). N-WASP (Sukumvanich et al., 2004) and WAVE2 (Fig. 2, B and C) have similar recruitment kinetics during EGF-stimulated protrusion. Recruitment of both proteins to the leading edge shows a peak at 60 s after EGF stimulation, followed by a gradual decrease.

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