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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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Mutations in PLS affect cell process growth. (A) p190A−/− MEFs expressing the indicated HA-tagged p190A constructs were plated overnight on fibronectin, fixed, and stained for nucleus (Hoechst, blue), cortactin (red), focal adhesions (vinculin, gray), and HA (green). Arrowheads show cortactin-rich edges, and arrows show focal adhesions. (B) Quantification of focal adhesion area in cells transfected as described in A (n = 15 cells per condition). Inset represents the number of focal adhesions (FA) per cell in the same experiment. (C) Quantification of ruffling cell process length. Graph presents the mean distance between nucleus and ruffle edges in cells transfected as described in A (n = 15 cells per condition). (D) Transfilter chemokinesis of cells transfected as described in A. Results are expressed as a percentage of basal migration without chemoattractant (n > 100 cells). For all graphs, values are expressed as the mean ± SEM of three (B and C) or five (D) independent experiments. Statistical significance was calculated relative to p190AWT condition. *, P < 0.05; **, P < 0.01; ***, P < 0.001. NT, nontransfected cells.
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fig7: Mutations in PLS affect cell process growth. (A) p190A−/− MEFs expressing the indicated HA-tagged p190A constructs were plated overnight on fibronectin, fixed, and stained for nucleus (Hoechst, blue), cortactin (red), focal adhesions (vinculin, gray), and HA (green). Arrowheads show cortactin-rich edges, and arrows show focal adhesions. (B) Quantification of focal adhesion area in cells transfected as described in A (n = 15 cells per condition). Inset represents the number of focal adhesions (FA) per cell in the same experiment. (C) Quantification of ruffling cell process length. Graph presents the mean distance between nucleus and ruffle edges in cells transfected as described in A (n = 15 cells per condition). (D) Transfilter chemokinesis of cells transfected as described in A. Results are expressed as a percentage of basal migration without chemoattractant (n > 100 cells). For all graphs, values are expressed as the mean ± SEM of three (B and C) or five (D) independent experiments. Statistical significance was calculated relative to p190AWT condition. *, P < 0.05; **, P < 0.01; ***, P < 0.001. NT, nontransfected cells.

Mentions: To get more insights into the function of cancer-associated p190A mutations, we then used mouse embryonic fibroblasts (MEFs), which display a spindle shape with a ruffling cell process easier to identify and measure than that of Huh7 cells. Moreover, tension applied by stress fibers induces focal adhesion formation in this model. HA-tagged versions of p190A were expressed in p190A knockout MEFs to avoid additive effect of endogenous p190A, and the number of focal adhesions was evaluated. A decreased focal adhesion area was observed in p190A−/− MEFs transfected with HA-p190AWT compared with nontransfected cells after overnight plating on fibronectin (Fig. 7, A and B). Expression of the mutants with predicted high GAP activity led to an even more dramatic decrease of focal adhesion area and a marginal decrease of the number of focal adhesions per cell. Focal adhesions regulate ruffles and lamellipodia stability (Schwarz and Gardel, 2012). Because persistent membrane ruffling favors cell process growth, we hypothesized that p190A mutants may affect cell process length. To test this, we measured the distance between the nucleus and ruffle edges of spindle shape MEFs plated overnight on fibronectin (Fig. 7 C). MEFs transfected with p190AWT displayed a cell process length similar to nontransfected cells, arguing that overexpression of p190AWT does not disturb cell process stability in MEFs. However, p190AΔPLS, S866F, and Δ(865–870) significantly decreased the cell process length, indicating that their higher GAP activity reduces lamellipodial persistence, a mechanism involving cortactin. As a consequence, we found that p190AΔPLS, S866F, and Δ(865–870) expression in MEFs increase random cell migration in chemokinesis assays when compared with untransfected or p190AWT-expressing cells (Fig. 7 D).


Cancer-associated mutations in the protrusion-targeting region of p190RhoGAP impact tumor cell migration
Mutations in PLS affect cell process growth. (A) p190A−/− MEFs expressing the indicated HA-tagged p190A constructs were plated overnight on fibronectin, fixed, and stained for nucleus (Hoechst, blue), cortactin (red), focal adhesions (vinculin, gray), and HA (green). Arrowheads show cortactin-rich edges, and arrows show focal adhesions. (B) Quantification of focal adhesion area in cells transfected as described in A (n = 15 cells per condition). Inset represents the number of focal adhesions (FA) per cell in the same experiment. (C) Quantification of ruffling cell process length. Graph presents the mean distance between nucleus and ruffle edges in cells transfected as described in A (n = 15 cells per condition). (D) Transfilter chemokinesis of cells transfected as described in A. Results are expressed as a percentage of basal migration without chemoattractant (n > 100 cells). For all graphs, values are expressed as the mean ± SEM of three (B and C) or five (D) independent experiments. Statistical significance was calculated relative to p190AWT condition. *, P < 0.05; **, P < 0.01; ***, P < 0.001. NT, nontransfected cells.
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fig7: Mutations in PLS affect cell process growth. (A) p190A−/− MEFs expressing the indicated HA-tagged p190A constructs were plated overnight on fibronectin, fixed, and stained for nucleus (Hoechst, blue), cortactin (red), focal adhesions (vinculin, gray), and HA (green). Arrowheads show cortactin-rich edges, and arrows show focal adhesions. (B) Quantification of focal adhesion area in cells transfected as described in A (n = 15 cells per condition). Inset represents the number of focal adhesions (FA) per cell in the same experiment. (C) Quantification of ruffling cell process length. Graph presents the mean distance between nucleus and ruffle edges in cells transfected as described in A (n = 15 cells per condition). (D) Transfilter chemokinesis of cells transfected as described in A. Results are expressed as a percentage of basal migration without chemoattractant (n > 100 cells). For all graphs, values are expressed as the mean ± SEM of three (B and C) or five (D) independent experiments. Statistical significance was calculated relative to p190AWT condition. *, P < 0.05; **, P < 0.01; ***, P < 0.001. NT, nontransfected cells.
Mentions: To get more insights into the function of cancer-associated p190A mutations, we then used mouse embryonic fibroblasts (MEFs), which display a spindle shape with a ruffling cell process easier to identify and measure than that of Huh7 cells. Moreover, tension applied by stress fibers induces focal adhesion formation in this model. HA-tagged versions of p190A were expressed in p190A knockout MEFs to avoid additive effect of endogenous p190A, and the number of focal adhesions was evaluated. A decreased focal adhesion area was observed in p190A−/− MEFs transfected with HA-p190AWT compared with nontransfected cells after overnight plating on fibronectin (Fig. 7, A and B). Expression of the mutants with predicted high GAP activity led to an even more dramatic decrease of focal adhesion area and a marginal decrease of the number of focal adhesions per cell. Focal adhesions regulate ruffles and lamellipodia stability (Schwarz and Gardel, 2012). Because persistent membrane ruffling favors cell process growth, we hypothesized that p190A mutants may affect cell process length. To test this, we measured the distance between the nucleus and ruffle edges of spindle shape MEFs plated overnight on fibronectin (Fig. 7 C). MEFs transfected with p190AWT displayed a cell process length similar to nontransfected cells, arguing that overexpression of p190AWT does not disturb cell process stability in MEFs. However, p190AΔPLS, S866F, and Δ(865–870) significantly decreased the cell process length, indicating that their higher GAP activity reduces lamellipodial persistence, a mechanism involving cortactin. As a consequence, we found that p190AΔPLS, S866F, and Δ(865–870) expression in MEFs increase random cell migration in chemokinesis assays when compared with untransfected or p190AWT-expressing cells (Fig. 7 D).

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