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Cancer-associated mutations in the protrusion-targeting region of p190RhoGAP impact tumor cell migration

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ABSTRACT

p190RhoGAP (p190A) is a negative regulator of RhoA and localizes to membrane protrusions, where its GAP activity is required for directional migration. Here, Binamé et al. identify the protrusion-localization sequence in p190A and show that cancer-associated mutations in this region affect p190A localization and function as well as tumor cell migration.

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Cortactin recruits p190A to the lamellipodium’s edge. Huh7 cells were transfected with control siRNA (siControl) or siRNA targeting cortactin (siCortactin1 and 2) and treated with 100 µM C8N6-BPA to induce lamellipodia growth. (A) The expression of endogenous p190A and cortactin was assayed by immunoblotting with an anti-p190A antibody and two antibodies from different species (mAb from mouse and rbAb from rabbit) against cortactin. β-Actin is used as a loading control. (B) Immunofluorescence staining of cortactin (red) and endogenous p190A (green) was performed on Huh7 cells transfected with siControl or siCortactin1. Asterisk indicates cells with absence of cortactin staining. (C) Huh7 cells transfected with siControl or siCortactin1 were immunostained as indicated on each panel. Graphs under each panel represent the quantification of the relative staining of cortactin (red), actin (gray), and p190A, Arp3, or α-actinin–GFP (green) determined inside the white rectangle of 2 × 5 µm. Results are expressed as mean value of the height. (D) Correlation of cortactin staining with p190A, Arp3, and α-actinin–GFP at the lamellipodium edge. Mean values of membrane areas of 2 × 0.28 µm are plotted on the graph. Closed and open circles represent measurement in cells transfected with control siRNA and siCortactin1, respectively (n = 30/condition). Linear regression is presented when Spearman correlation is significant. ***, P < 0.001; n.s, not significant.
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fig4: Cortactin recruits p190A to the lamellipodium’s edge. Huh7 cells were transfected with control siRNA (siControl) or siRNA targeting cortactin (siCortactin1 and 2) and treated with 100 µM C8N6-BPA to induce lamellipodia growth. (A) The expression of endogenous p190A and cortactin was assayed by immunoblotting with an anti-p190A antibody and two antibodies from different species (mAb from mouse and rbAb from rabbit) against cortactin. β-Actin is used as a loading control. (B) Immunofluorescence staining of cortactin (red) and endogenous p190A (green) was performed on Huh7 cells transfected with siControl or siCortactin1. Asterisk indicates cells with absence of cortactin staining. (C) Huh7 cells transfected with siControl or siCortactin1 were immunostained as indicated on each panel. Graphs under each panel represent the quantification of the relative staining of cortactin (red), actin (gray), and p190A, Arp3, or α-actinin–GFP (green) determined inside the white rectangle of 2 × 5 µm. Results are expressed as mean value of the height. (D) Correlation of cortactin staining with p190A, Arp3, and α-actinin–GFP at the lamellipodium edge. Mean values of membrane areas of 2 × 0.28 µm are plotted on the graph. Closed and open circles represent measurement in cells transfected with control siRNA and siCortactin1, respectively (n = 30/condition). Linear regression is presented when Spearman correlation is significant. ***, P < 0.001; n.s, not significant.

Mentions: To test whether cortactin is required for p190A targeting to membrane protrusions, we studied the impact of cortactin silencing on p190A subcellular localization. To study this mechanism in a well-defined structure, we induced lamellipodia with C8N6-BPA in Huh7 cells. Silencing cortactin with two different siRNAs produced a similar and dramatic decrease of cortactin expression without affecting p190A level (Fig. 4 A). Lamellipodia induced in Huh7 cells transfected with control siRNA presented both cortactin and p190A at their edge (Fig. 4 B). When siRNA targeting cortactin is transfected, cells exhibiting the most efficient silencing of cortactin displayed a defect in p190A staining at lamellipodia’s edge. We quantified the effect of cortactin knockdown on the localization of p190A at the cell periphery (Fig. 4, C and D; and Fig. S4 C). Arp3, a known interacting partner of cortactin, and α-actinin were used as positive and negative controls, respectively. Measurement of relative staining intensities in cells treated with control siRNA showed peaks of actin, cortactin, Arp3, and α-actinin–GFP at the lamellipodium’s edge, as expected. Downregulation of cortactin erased the peak of cortactin without affecting actin or α-actinin–GFP expression at the cell edge. However, it reduced the peak of Arp3 and p190A. These results, together with the highly significant correlation found between cortactin staining and both Arp3 and p190A at lamellipodium’s edge (Fig. 4 D and Fig. S4 C), confirm the role of cortactin in Arp3 recruitment (Weaver et al., 2002) and reveal the requirement of cortactin for p190A targeting (Fig. 4 C).


Cancer-associated mutations in the protrusion-targeting region of p190RhoGAP impact tumor cell migration
Cortactin recruits p190A to the lamellipodium’s edge. Huh7 cells were transfected with control siRNA (siControl) or siRNA targeting cortactin (siCortactin1 and 2) and treated with 100 µM C8N6-BPA to induce lamellipodia growth. (A) The expression of endogenous p190A and cortactin was assayed by immunoblotting with an anti-p190A antibody and two antibodies from different species (mAb from mouse and rbAb from rabbit) against cortactin. β-Actin is used as a loading control. (B) Immunofluorescence staining of cortactin (red) and endogenous p190A (green) was performed on Huh7 cells transfected with siControl or siCortactin1. Asterisk indicates cells with absence of cortactin staining. (C) Huh7 cells transfected with siControl or siCortactin1 were immunostained as indicated on each panel. Graphs under each panel represent the quantification of the relative staining of cortactin (red), actin (gray), and p190A, Arp3, or α-actinin–GFP (green) determined inside the white rectangle of 2 × 5 µm. Results are expressed as mean value of the height. (D) Correlation of cortactin staining with p190A, Arp3, and α-actinin–GFP at the lamellipodium edge. Mean values of membrane areas of 2 × 0.28 µm are plotted on the graph. Closed and open circles represent measurement in cells transfected with control siRNA and siCortactin1, respectively (n = 30/condition). Linear regression is presented when Spearman correlation is significant. ***, P < 0.001; n.s, not significant.
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fig4: Cortactin recruits p190A to the lamellipodium’s edge. Huh7 cells were transfected with control siRNA (siControl) or siRNA targeting cortactin (siCortactin1 and 2) and treated with 100 µM C8N6-BPA to induce lamellipodia growth. (A) The expression of endogenous p190A and cortactin was assayed by immunoblotting with an anti-p190A antibody and two antibodies from different species (mAb from mouse and rbAb from rabbit) against cortactin. β-Actin is used as a loading control. (B) Immunofluorescence staining of cortactin (red) and endogenous p190A (green) was performed on Huh7 cells transfected with siControl or siCortactin1. Asterisk indicates cells with absence of cortactin staining. (C) Huh7 cells transfected with siControl or siCortactin1 were immunostained as indicated on each panel. Graphs under each panel represent the quantification of the relative staining of cortactin (red), actin (gray), and p190A, Arp3, or α-actinin–GFP (green) determined inside the white rectangle of 2 × 5 µm. Results are expressed as mean value of the height. (D) Correlation of cortactin staining with p190A, Arp3, and α-actinin–GFP at the lamellipodium edge. Mean values of membrane areas of 2 × 0.28 µm are plotted on the graph. Closed and open circles represent measurement in cells transfected with control siRNA and siCortactin1, respectively (n = 30/condition). Linear regression is presented when Spearman correlation is significant. ***, P < 0.001; n.s, not significant.
Mentions: To test whether cortactin is required for p190A targeting to membrane protrusions, we studied the impact of cortactin silencing on p190A subcellular localization. To study this mechanism in a well-defined structure, we induced lamellipodia with C8N6-BPA in Huh7 cells. Silencing cortactin with two different siRNAs produced a similar and dramatic decrease of cortactin expression without affecting p190A level (Fig. 4 A). Lamellipodia induced in Huh7 cells transfected with control siRNA presented both cortactin and p190A at their edge (Fig. 4 B). When siRNA targeting cortactin is transfected, cells exhibiting the most efficient silencing of cortactin displayed a defect in p190A staining at lamellipodia’s edge. We quantified the effect of cortactin knockdown on the localization of p190A at the cell periphery (Fig. 4, C and D; and Fig. S4 C). Arp3, a known interacting partner of cortactin, and α-actinin were used as positive and negative controls, respectively. Measurement of relative staining intensities in cells treated with control siRNA showed peaks of actin, cortactin, Arp3, and α-actinin–GFP at the lamellipodium’s edge, as expected. Downregulation of cortactin erased the peak of cortactin without affecting actin or α-actinin–GFP expression at the cell edge. However, it reduced the peak of Arp3 and p190A. These results, together with the highly significant correlation found between cortactin staining and both Arp3 and p190A at lamellipodium’s edge (Fig. 4 D and Fig. S4 C), confirm the role of cortactin in Arp3 recruitment (Weaver et al., 2002) and reveal the requirement of cortactin for p190A targeting (Fig. 4 C).

View Article: PubMed Central - HTML - PubMed

ABSTRACT

p190RhoGAP (p190A) is a negative regulator of RhoA and localizes to membrane protrusions, where its GAP activity is required for directional migration. Here, Binam&eacute; et al. identify the protrusion-localization sequence in p190A and show that cancer-associated mutations in this region affect p190A localization and function as well as tumor cell migration.

No MeSH data available.


Related in: MedlinePlus