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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.

No MeSH data available.


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p190A localizes to membrane protrusions in Huh7 cells. (A) As a standard condition, Huh7 cells were plated on glass coverslips and then fixed after overnight culture. Lamellipodium growth was either stimulated by spreading on fibronectin for 30 min (top) or by treatment with 100 µM C8N6-BPA (middle). Huh7 cells were transfected with α-actinin–GFP used as a lamellipodium marker, fixed, and stained for p190A (green), F-actin (red), and nuclei (blue). Bottom panel shows enlargement of C8N6-BPA condition. (B) Huh7 cells were transfected with HA-p190AWT and α-actinin–GFP, fixed, and stained for HA tag (green), F-actin (red), and nuclei (blue). (C) Huh7 cells were transfected with HA-p190AWT, fixed, and stained for HA tag (green), VASP (red) or F-actin (red), and nuclei (blue). Bottom panel shows enlargement of C8N6-BPA condition. (A and B) Arrowheads show membrane ruffles with p190A/HA-p190AWT localization.
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fig1: p190A localizes to membrane protrusions in Huh7 cells. (A) As a standard condition, Huh7 cells were plated on glass coverslips and then fixed after overnight culture. Lamellipodium growth was either stimulated by spreading on fibronectin for 30 min (top) or by treatment with 100 µM C8N6-BPA (middle). Huh7 cells were transfected with α-actinin–GFP used as a lamellipodium marker, fixed, and stained for p190A (green), F-actin (red), and nuclei (blue). Bottom panel shows enlargement of C8N6-BPA condition. (B) Huh7 cells were transfected with HA-p190AWT and α-actinin–GFP, fixed, and stained for HA tag (green), F-actin (red), and nuclei (blue). (C) Huh7 cells were transfected with HA-p190AWT, fixed, and stained for HA tag (green), VASP (red) or F-actin (red), and nuclei (blue). Bottom panel shows enlargement of C8N6-BPA condition. (A and B) Arrowheads show membrane ruffles with p190A/HA-p190AWT localization.

Mentions: In human hepatocellular carcinoma Huh7 cells, endogenous p190A localizes to cell edges such as membrane ruffles and lamellipodia (Fig. 1 A), as previously described in other cell types (Tsubouchi et al., 2002; Bass et al., 2008; Guegan et al., 2008). In standard conditions in which cells were plated overnight on glass coverslips, p190A colocalizes with cortical F-actin at the cell periphery. To unambiguously show the localization of p190A to actin-rich protrusions, we induced lamellipodium outgrowth by short-term spreading on fibronectin or by chemical treatment with the lamellipodial growth promoter C8N6-branched polyamine (BPA; Nedeva et al., 2013). Endogenous p190A colocalizes along lamellipodium’s edge with actin and α-actinin, an F-actin cross-linking protein found at the leading edge of migrating cells (Langanger et al., 1984; Knight et al., 2000). When expressed in cells, wild-type (WT) HA-tagged p190A (HA-p190AWT) localized similarly to the endogenous protein in standard and stimulated conditions (Fig. 1 B). Moreover, costaining with anti–vasodilator-stimulated phosphoprotein (VASP) antibodies revealed that HA-tagged p190A localized at the very edge of lamellipodia (Fig. 1 C).


Cancer-associated mutations in the protrusion-targeting region of p190RhoGAP impact tumor cell migration
p190A localizes to membrane protrusions in Huh7 cells. (A) As a standard condition, Huh7 cells were plated on glass coverslips and then fixed after overnight culture. Lamellipodium growth was either stimulated by spreading on fibronectin for 30 min (top) or by treatment with 100 µM C8N6-BPA (middle). Huh7 cells were transfected with α-actinin–GFP used as a lamellipodium marker, fixed, and stained for p190A (green), F-actin (red), and nuclei (blue). Bottom panel shows enlargement of C8N6-BPA condition. (B) Huh7 cells were transfected with HA-p190AWT and α-actinin–GFP, fixed, and stained for HA tag (green), F-actin (red), and nuclei (blue). (C) Huh7 cells were transfected with HA-p190AWT, fixed, and stained for HA tag (green), VASP (red) or F-actin (red), and nuclei (blue). Bottom panel shows enlargement of C8N6-BPA condition. (A and B) Arrowheads show membrane ruffles with p190A/HA-p190AWT localization.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5037408&req=5

fig1: p190A localizes to membrane protrusions in Huh7 cells. (A) As a standard condition, Huh7 cells were plated on glass coverslips and then fixed after overnight culture. Lamellipodium growth was either stimulated by spreading on fibronectin for 30 min (top) or by treatment with 100 µM C8N6-BPA (middle). Huh7 cells were transfected with α-actinin–GFP used as a lamellipodium marker, fixed, and stained for p190A (green), F-actin (red), and nuclei (blue). Bottom panel shows enlargement of C8N6-BPA condition. (B) Huh7 cells were transfected with HA-p190AWT and α-actinin–GFP, fixed, and stained for HA tag (green), F-actin (red), and nuclei (blue). (C) Huh7 cells were transfected with HA-p190AWT, fixed, and stained for HA tag (green), VASP (red) or F-actin (red), and nuclei (blue). Bottom panel shows enlargement of C8N6-BPA condition. (A and B) Arrowheads show membrane ruffles with p190A/HA-p190AWT localization.
Mentions: In human hepatocellular carcinoma Huh7 cells, endogenous p190A localizes to cell edges such as membrane ruffles and lamellipodia (Fig. 1 A), as previously described in other cell types (Tsubouchi et al., 2002; Bass et al., 2008; Guegan et al., 2008). In standard conditions in which cells were plated overnight on glass coverslips, p190A colocalizes with cortical F-actin at the cell periphery. To unambiguously show the localization of p190A to actin-rich protrusions, we induced lamellipodium outgrowth by short-term spreading on fibronectin or by chemical treatment with the lamellipodial growth promoter C8N6-branched polyamine (BPA; Nedeva et al., 2013). Endogenous p190A colocalizes along lamellipodium’s edge with actin and α-actinin, an F-actin cross-linking protein found at the leading edge of migrating cells (Langanger et al., 1984; Knight et al., 2000). When expressed in cells, wild-type (WT) HA-tagged p190A (HA-p190AWT) localized similarly to the endogenous protein in standard and stimulated conditions (Fig. 1 B). Moreover, costaining with anti–vasodilator-stimulated phosphoprotein (VASP) antibodies revealed that HA-tagged p190A localized at the very edge of lamellipodia (Fig. 1 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, 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.

No MeSH data available.


Related in: MedlinePlus