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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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Inhibition of PI 3-kinase or Cdc42 inhibits chemotaxis but not directionally persistent cell migration. (A) Inhibition of PI3K by 50 μM LY 294002 blocks primary human fibroblast chemotaxis toward 300 nM fMLP whether or not Rac is suppressed by 1 nM Rac1 siRNA. (B) Localization of GFP-tagged Akt PH domain to lamellae is suppressed by treatment with LY 294002. Insets show lower magnification views of the entire cell. Bars, 20 μm. (C) Inhibition of Cdc42 by 100 nM Cdc42 siRNA (>80% of both total Cdc42 protein and activity) blocks fMLP-stimulated chemotaxis of human fibroblasts. (D) Inhibition of PI3K by 50 μM LY 294002 does not suppress the increased directional persistence of migration induced by reduction of active Rac using 1 nM Rac1 siRNA. (A, C, and D) Error bars represent SEM.
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fig6: Inhibition of PI 3-kinase or Cdc42 inhibits chemotaxis but not directionally persistent cell migration. (A) Inhibition of PI3K by 50 μM LY 294002 blocks primary human fibroblast chemotaxis toward 300 nM fMLP whether or not Rac is suppressed by 1 nM Rac1 siRNA. (B) Localization of GFP-tagged Akt PH domain to lamellae is suppressed by treatment with LY 294002. Insets show lower magnification views of the entire cell. Bars, 20 μm. (C) Inhibition of Cdc42 by 100 nM Cdc42 siRNA (>80% of both total Cdc42 protein and activity) blocks fMLP-stimulated chemotaxis of human fibroblasts. (D) Inhibition of PI3K by 50 μM LY 294002 does not suppress the increased directional persistence of migration induced by reduction of active Rac using 1 nM Rac1 siRNA. (A, C, and D) Error bars represent SEM.

Mentions: We compared this mechanism of Rac regulation of directional persistence with chemotaxis-induced directed cell migration. Chemotactic migration is known to be regulated by PI3K and localized phosphatidylinositol lipids (Haugh et al., 2000; Srinivasan et al., 2003; Sasaki et al., 2004; Van Haastert and Devreotes, 2004). We confirmed that for human fibroblast chemotaxis toward fMLP (N-formyl-Met-Leu-Phe), inhibition of PI3K by LY 294002 with a loss of PIP3 localization, as detected by a fluorescent pleckstin homology domain probe, inhibited chemotaxis (Fig. 6, A and B). In marked contrast, inhibition of PI3K did not suppress—and, in fact, moderately promoted—intrinsic directionally persistent migration (Fig. 6 D). This increased directionality was associated with a 30–40% decrease in Rac activity (Fig. S2 B). Similar results to those were obtained with U87-MG glioblastoma cells treated with Wortmannin (unpublished data). Importantly, cells exhibiting increased directional persistence of migration because of active Rac1 reduction by siRNA treatment showed no loss of directionality after inhibition of PI3K and loss of PIP3 localization (Fig. 6 D), even though chemotaxis was blocked. Neither suppression of Rac activity nor treatment with LY 294002 had a significant effect on activation-associated phosphorylation of the MAP kinases ERK1/2 and JNK (Fig. S2). Consistent with previous data establishing a role for Cdc42 in chemotactic-directed migration, suppression of Cdc42 levels and activity by >80% suppressed fibroblast chemotaxis (Fig. 6 C).


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)

Inhibition of PI 3-kinase or Cdc42 inhibits chemotaxis but not directionally persistent cell migration. (A) Inhibition of PI3K by 50 μM LY 294002 blocks primary human fibroblast chemotaxis toward 300 nM fMLP whether or not Rac is suppressed by 1 nM Rac1 siRNA. (B) Localization of GFP-tagged Akt PH domain to lamellae is suppressed by treatment with LY 294002. Insets show lower magnification views of the entire cell. Bars, 20 μm. (C) Inhibition of Cdc42 by 100 nM Cdc42 siRNA (>80% of both total Cdc42 protein and activity) blocks fMLP-stimulated chemotaxis of human fibroblasts. (D) Inhibition of PI3K by 50 μM LY 294002 does not suppress the increased directional persistence of migration induced by reduction of active Rac using 1 nM Rac1 siRNA. (A, C, and D) Error bars represent SEM.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171343&req=5

fig6: Inhibition of PI 3-kinase or Cdc42 inhibits chemotaxis but not directionally persistent cell migration. (A) Inhibition of PI3K by 50 μM LY 294002 blocks primary human fibroblast chemotaxis toward 300 nM fMLP whether or not Rac is suppressed by 1 nM Rac1 siRNA. (B) Localization of GFP-tagged Akt PH domain to lamellae is suppressed by treatment with LY 294002. Insets show lower magnification views of the entire cell. Bars, 20 μm. (C) Inhibition of Cdc42 by 100 nM Cdc42 siRNA (>80% of both total Cdc42 protein and activity) blocks fMLP-stimulated chemotaxis of human fibroblasts. (D) Inhibition of PI3K by 50 μM LY 294002 does not suppress the increased directional persistence of migration induced by reduction of active Rac using 1 nM Rac1 siRNA. (A, C, and D) Error bars represent SEM.
Mentions: We compared this mechanism of Rac regulation of directional persistence with chemotaxis-induced directed cell migration. Chemotactic migration is known to be regulated by PI3K and localized phosphatidylinositol lipids (Haugh et al., 2000; Srinivasan et al., 2003; Sasaki et al., 2004; Van Haastert and Devreotes, 2004). We confirmed that for human fibroblast chemotaxis toward fMLP (N-formyl-Met-Leu-Phe), inhibition of PI3K by LY 294002 with a loss of PIP3 localization, as detected by a fluorescent pleckstin homology domain probe, inhibited chemotaxis (Fig. 6, A and B). In marked contrast, inhibition of PI3K did not suppress—and, in fact, moderately promoted—intrinsic directionally persistent migration (Fig. 6 D). This increased directionality was associated with a 30–40% decrease in Rac activity (Fig. S2 B). Similar results to those were obtained with U87-MG glioblastoma cells treated with Wortmannin (unpublished data). Importantly, cells exhibiting increased directional persistence of migration because of active Rac1 reduction by siRNA treatment showed no loss of directionality after inhibition of PI3K and loss of PIP3 localization (Fig. 6 D), even though chemotaxis was blocked. Neither suppression of Rac activity nor treatment with LY 294002 had a significant effect on activation-associated phosphorylation of the MAP kinases ERK1/2 and JNK (Fig. S2). Consistent with previous data establishing a role for Cdc42 in chemotactic-directed migration, suppression of Cdc42 levels and activity by >80% suppressed fibroblast chemotaxis (Fig. 6 C).

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