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A Rac switch regulates random versus directionally persistent cell migration.

Pankov R, Endo Y, Even-Ram S, Araki M, Clark K, Cukierman E, Matsumoto K, Yamada KM - J. Cell Biol. (2005)

Bottom Line: In three-dimensional rather than traditional two-dimensional cell culture, cells had a lower level of Rac activity that was associated with rapid, directional migration.In contrast to the directed migration of chemotaxis, this intrinsic directional persistence of migration was not mediated by phosphatidylinositol 3'-kinase lipid signaling.Total Rac1 activity can therefore provide a regulatory switch between patterns of cell migration by a mechanism distinct from chemotaxis.

View Article: PubMed Central - PubMed

Affiliation: Craniofacial Developmental Biology and Regeneration Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
Directional migration moves cells rapidly between points, whereas random migration allows cells to explore their local environments. We describe a Rac1 mechanism for determining whether cell patterns of migration are intrinsically random or directionally persistent. Rac activity promoted the formation of peripheral lamellae that mediated random migration. Decreasing Rac activity suppressed peripheral lamellae and switched the cell migration patterns of fibroblasts and epithelial cells from random to directionally persistent. In three-dimensional rather than traditional two-dimensional cell culture, cells had a lower level of Rac activity that was associated with rapid, directional migration. In contrast to the directed migration of chemotaxis, this intrinsic directional persistence of migration was not mediated by phosphatidylinositol 3'-kinase lipid signaling. Total Rac1 activity can therefore provide a regulatory switch between patterns of cell migration by a mechanism distinct from chemotaxis.

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Related in: MedlinePlus

Integrin mutation inhibits Rac signaling and suppresses random cell motility. (A) GD25 cells with a point mutation in the β1 integrin cytoplasmic domain (W775A) have a selective defect in Rac activation. Activities in pull-down assays for Rac-GTP, Cdc42-GTP, and Rho-GTP and total amounts of each protein in lysates of cells cultured on 1 μg/ml fibronectin were analyzed using antibodies against Rac, Cdc42, and Rho. Densitometry for each GTP-bound protein was normalized to the amount of the total protein, and results are presented as fold change compared with cells expressing wild-type integrin. The reduction of active Rac was 75% (P < 0.0001), whereas active Cdc42 and active Rho were not significantly reduced (by 10%, P = 0.31 and 8%, P = 0.79, respectively). In this and subsequent figures, all adjacent gel lanes are from the same gel and blot, but their order may be rearranged for clarity as indicated by white lines between the lanes. (B) Representative examples of migration tracks of cells expressing wild-type β1 (β1wt) or mutant (W775A) integrins cultured on fibronectin and tracked for 12 h. In these and subsequent composite migration figures, randomly selected individual migration tracks were copied and combined into a single figure to avoid empty spaces. Bar, 100 μm. (C) Quantification of the persistence of migratory directionality and velocity. Cell movements were recorded by time-lapse video microscopy and quantified by MetaMorph software. D/T ratios represent the ratio of the direct distance from start to end point [D] divided by the total track distance [T]. Motility was calculated as velocity (μm/h). Data were pooled from four independent experiments; error bars indicate SEM based on n = 42–45 cells. (D) Activated Akt can restore Rac activity. W775A cells were transfected with constitutively activated Akt, and stable clones were selected by serum starvation for 1 wk to induce apoptosis in cells with low Akt activity. The activity of Akt in the isolated clones (Akt clones 1–3) was assayed by immunoblotting with antibodies against phosphorylated Akt (p-Akt). Densitometry values for phospho-Akt were normalized to actin in the lysates, and the changes were calculated as fold increases compared with the parental W775A cells. The activity of Rac in the isolated Akt clones was calculated as the fold increase above W775A cell values after normalization of densitometry of the pull-down samples (Rac-GTP) to the total Rac in the lysates. (E) Activated Akt restores random motility. Quantification of cell movements of the Akt clones was performed as described in C. The decreases in D/T ratio after activated Akt expression were all significant (Akt clone 1, P < 0.01; clone 2, P < 0.001; and clone 3, P < 0.001), as were the decreases in velocity (clone 1, P < 0.01; and clones 2 and 3, P < 0.001). Error bars indicate SEM.
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fig1: Integrin mutation inhibits Rac signaling and suppresses random cell motility. (A) GD25 cells with a point mutation in the β1 integrin cytoplasmic domain (W775A) have a selective defect in Rac activation. Activities in pull-down assays for Rac-GTP, Cdc42-GTP, and Rho-GTP and total amounts of each protein in lysates of cells cultured on 1 μg/ml fibronectin were analyzed using antibodies against Rac, Cdc42, and Rho. Densitometry for each GTP-bound protein was normalized to the amount of the total protein, and results are presented as fold change compared with cells expressing wild-type integrin. The reduction of active Rac was 75% (P < 0.0001), whereas active Cdc42 and active Rho were not significantly reduced (by 10%, P = 0.31 and 8%, P = 0.79, respectively). In this and subsequent figures, all adjacent gel lanes are from the same gel and blot, but their order may be rearranged for clarity as indicated by white lines between the lanes. (B) Representative examples of migration tracks of cells expressing wild-type β1 (β1wt) or mutant (W775A) integrins cultured on fibronectin and tracked for 12 h. In these and subsequent composite migration figures, randomly selected individual migration tracks were copied and combined into a single figure to avoid empty spaces. Bar, 100 μm. (C) Quantification of the persistence of migratory directionality and velocity. Cell movements were recorded by time-lapse video microscopy and quantified by MetaMorph software. D/T ratios represent the ratio of the direct distance from start to end point [D] divided by the total track distance [T]. Motility was calculated as velocity (μm/h). Data were pooled from four independent experiments; error bars indicate SEM based on n = 42–45 cells. (D) Activated Akt can restore Rac activity. W775A cells were transfected with constitutively activated Akt, and stable clones were selected by serum starvation for 1 wk to induce apoptosis in cells with low Akt activity. The activity of Akt in the isolated clones (Akt clones 1–3) was assayed by immunoblotting with antibodies against phosphorylated Akt (p-Akt). Densitometry values for phospho-Akt were normalized to actin in the lysates, and the changes were calculated as fold increases compared with the parental W775A cells. The activity of Rac in the isolated Akt clones was calculated as the fold increase above W775A cell values after normalization of densitometry of the pull-down samples (Rac-GTP) to the total Rac in the lysates. (E) Activated Akt restores random motility. Quantification of cell movements of the Akt clones was performed as described in C. The decreases in D/T ratio after activated Akt expression were all significant (Akt clone 1, P < 0.01; clone 2, P < 0.001; and clone 3, P < 0.001), as were the decreases in velocity (clone 1, P < 0.01; and clones 2 and 3, P < 0.001). Error bars indicate SEM.

Mentions: A potential mechanism for regulating intrinsic cell migration involving Rac was identified in our ongoing studies of an integrin point mutation that selectively affects an Akt–PKB pathway (Pankov et al., 2003). Fig. 1 shows that this specific integrin mutation also selectively suppresses downstream Rac activity, which is accompanied by a substantial suppression of random motility and enhanced persistence of migratory directionality. Specifically, Rac-GTP levels were decreased by 75% with no differences in total Rac protein (Fig. 1 A).


A Rac switch regulates random versus directionally persistent cell migration.

Pankov R, Endo Y, Even-Ram S, Araki M, Clark K, Cukierman E, Matsumoto K, Yamada KM - J. Cell Biol. (2005)

Integrin mutation inhibits Rac signaling and suppresses random cell motility. (A) GD25 cells with a point mutation in the β1 integrin cytoplasmic domain (W775A) have a selective defect in Rac activation. Activities in pull-down assays for Rac-GTP, Cdc42-GTP, and Rho-GTP and total amounts of each protein in lysates of cells cultured on 1 μg/ml fibronectin were analyzed using antibodies against Rac, Cdc42, and Rho. Densitometry for each GTP-bound protein was normalized to the amount of the total protein, and results are presented as fold change compared with cells expressing wild-type integrin. The reduction of active Rac was 75% (P < 0.0001), whereas active Cdc42 and active Rho were not significantly reduced (by 10%, P = 0.31 and 8%, P = 0.79, respectively). In this and subsequent figures, all adjacent gel lanes are from the same gel and blot, but their order may be rearranged for clarity as indicated by white lines between the lanes. (B) Representative examples of migration tracks of cells expressing wild-type β1 (β1wt) or mutant (W775A) integrins cultured on fibronectin and tracked for 12 h. In these and subsequent composite migration figures, randomly selected individual migration tracks were copied and combined into a single figure to avoid empty spaces. Bar, 100 μm. (C) Quantification of the persistence of migratory directionality and velocity. Cell movements were recorded by time-lapse video microscopy and quantified by MetaMorph software. D/T ratios represent the ratio of the direct distance from start to end point [D] divided by the total track distance [T]. Motility was calculated as velocity (μm/h). Data were pooled from four independent experiments; error bars indicate SEM based on n = 42–45 cells. (D) Activated Akt can restore Rac activity. W775A cells were transfected with constitutively activated Akt, and stable clones were selected by serum starvation for 1 wk to induce apoptosis in cells with low Akt activity. The activity of Akt in the isolated clones (Akt clones 1–3) was assayed by immunoblotting with antibodies against phosphorylated Akt (p-Akt). Densitometry values for phospho-Akt were normalized to actin in the lysates, and the changes were calculated as fold increases compared with the parental W775A cells. The activity of Rac in the isolated Akt clones was calculated as the fold increase above W775A cell values after normalization of densitometry of the pull-down samples (Rac-GTP) to the total Rac in the lysates. (E) Activated Akt restores random motility. Quantification of cell movements of the Akt clones was performed as described in C. The decreases in D/T ratio after activated Akt expression were all significant (Akt clone 1, P < 0.01; clone 2, P < 0.001; and clone 3, P < 0.001), as were the decreases in velocity (clone 1, P < 0.01; and clones 2 and 3, P < 0.001). Error bars indicate SEM.
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Related In: Results  -  Collection

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fig1: Integrin mutation inhibits Rac signaling and suppresses random cell motility. (A) GD25 cells with a point mutation in the β1 integrin cytoplasmic domain (W775A) have a selective defect in Rac activation. Activities in pull-down assays for Rac-GTP, Cdc42-GTP, and Rho-GTP and total amounts of each protein in lysates of cells cultured on 1 μg/ml fibronectin were analyzed using antibodies against Rac, Cdc42, and Rho. Densitometry for each GTP-bound protein was normalized to the amount of the total protein, and results are presented as fold change compared with cells expressing wild-type integrin. The reduction of active Rac was 75% (P < 0.0001), whereas active Cdc42 and active Rho were not significantly reduced (by 10%, P = 0.31 and 8%, P = 0.79, respectively). In this and subsequent figures, all adjacent gel lanes are from the same gel and blot, but their order may be rearranged for clarity as indicated by white lines between the lanes. (B) Representative examples of migration tracks of cells expressing wild-type β1 (β1wt) or mutant (W775A) integrins cultured on fibronectin and tracked for 12 h. In these and subsequent composite migration figures, randomly selected individual migration tracks were copied and combined into a single figure to avoid empty spaces. Bar, 100 μm. (C) Quantification of the persistence of migratory directionality and velocity. Cell movements were recorded by time-lapse video microscopy and quantified by MetaMorph software. D/T ratios represent the ratio of the direct distance from start to end point [D] divided by the total track distance [T]. Motility was calculated as velocity (μm/h). Data were pooled from four independent experiments; error bars indicate SEM based on n = 42–45 cells. (D) Activated Akt can restore Rac activity. W775A cells were transfected with constitutively activated Akt, and stable clones were selected by serum starvation for 1 wk to induce apoptosis in cells with low Akt activity. The activity of Akt in the isolated clones (Akt clones 1–3) was assayed by immunoblotting with antibodies against phosphorylated Akt (p-Akt). Densitometry values for phospho-Akt were normalized to actin in the lysates, and the changes were calculated as fold increases compared with the parental W775A cells. The activity of Rac in the isolated Akt clones was calculated as the fold increase above W775A cell values after normalization of densitometry of the pull-down samples (Rac-GTP) to the total Rac in the lysates. (E) Activated Akt restores random motility. Quantification of cell movements of the Akt clones was performed as described in C. The decreases in D/T ratio after activated Akt expression were all significant (Akt clone 1, P < 0.01; clone 2, P < 0.001; and clone 3, P < 0.001), as were the decreases in velocity (clone 1, P < 0.01; and clones 2 and 3, P < 0.001). Error bars indicate SEM.
Mentions: A potential mechanism for regulating intrinsic cell migration involving Rac was identified in our ongoing studies of an integrin point mutation that selectively affects an Akt–PKB pathway (Pankov et al., 2003). Fig. 1 shows that this specific integrin mutation also selectively suppresses downstream Rac activity, which is accompanied by a substantial suppression of random motility and enhanced persistence of migratory directionality. Specifically, Rac-GTP levels were decreased by 75% with no differences in total Rac protein (Fig. 1 A).

Bottom Line: In three-dimensional rather than traditional two-dimensional cell culture, cells had a lower level of Rac activity that was associated with rapid, directional migration.In contrast to the directed migration of chemotaxis, this intrinsic directional persistence of migration was not mediated by phosphatidylinositol 3'-kinase lipid signaling.Total Rac1 activity can therefore provide a regulatory switch between patterns of cell migration by a mechanism distinct from chemotaxis.

View Article: PubMed Central - PubMed

Affiliation: Craniofacial Developmental Biology and Regeneration Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
Directional migration moves cells rapidly between points, whereas random migration allows cells to explore their local environments. We describe a Rac1 mechanism for determining whether cell patterns of migration are intrinsically random or directionally persistent. Rac activity promoted the formation of peripheral lamellae that mediated random migration. Decreasing Rac activity suppressed peripheral lamellae and switched the cell migration patterns of fibroblasts and epithelial cells from random to directionally persistent. In three-dimensional rather than traditional two-dimensional cell culture, cells had a lower level of Rac activity that was associated with rapid, directional migration. In contrast to the directed migration of chemotaxis, this intrinsic directional persistence of migration was not mediated by phosphatidylinositol 3'-kinase lipid signaling. Total Rac1 activity can therefore provide a regulatory switch between patterns of cell migration by a mechanism distinct from chemotaxis.

Show MeSH
Related in: MedlinePlus