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Raf-1 regulates Rho signaling and cell migration.

Ehrenreiter K, Piazzolla D, Velamoor V, Sobczak I, Small JV, Takeda J, Leung T, Baccarini M - J. Cell Biol. (2005)

Bottom Line: These defects are due to the hyperactivity and incorrect localization of the Rho-effector Rok-alpha to the plasma membrane.Raf-1 physically associates with Rok-alpha in wild-type (WT) cells, and reintroduction of either WT or kinase-dead Raf-1 in knockout fibroblasts rescues their defects in shape and migration.Thus, Raf-1 plays an essential, kinase-independent function as a spatial regulator of Rho downstream signaling during migration.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Genetics, Max F. Perutz Laboratories, University Departments at the Vienna Biocenter, 1030 Vienna, Austria.

ABSTRACT
Raf kinases relay signals inducing proliferation, differentiation, and survival. The Raf-1 isoform has been extensively studied as the upstream kinase linking Ras activation to the MEK/ERK module. Recently, however, genetic experiments have shown that Raf-1 plays an essential role in counteracting apoptosis, and that it does so independently of its ability to activate MEK. By conditional gene ablation, we now show that Raf-1 is required for normal wound healing in vivo and for the migration of keratinocytes and fibroblasts in vitro. Raf-1-deficient cells show a symmetric, contracted appearance, characterized by cortical actin bundles and by a disordered vimentin cytoskeleton. These defects are due to the hyperactivity and incorrect localization of the Rho-effector Rok-alpha to the plasma membrane. Raf-1 physically associates with Rok-alpha in wild-type (WT) cells, and reintroduction of either WT or kinase-dead Raf-1 in knockout fibroblasts rescues their defects in shape and migration. Thus, Raf-1 plays an essential, kinase-independent function as a spatial regulator of Rho downstream signaling during migration.

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Raf-1 ablation affects fibroblasts motility, adhesion, and shape in vitro. (A) Fibroblast motility assayed by in vitro wound healing in serum-free medium. Wound closure was photographed after 18 h. Arrows, wound margins at time 0. The percent wound closure at different times is plotted on the left. The values represent the means (±SD, vertical bars) of three independent experiments. (B) The percentage of cells adhering to different ECM components was determined in a 30-min adhesion assay as described in the legend to Fig. 2. Integrin-independent adhesion on BSA-coated surfaces was subtracted from the values plotted. Values are means (±SD, vertical bars) of five individual experiments. (C) Morphology of fibroblasts stained to visualize the actin cytoskeleton.
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fig3: Raf-1 ablation affects fibroblasts motility, adhesion, and shape in vitro. (A) Fibroblast motility assayed by in vitro wound healing in serum-free medium. Wound closure was photographed after 18 h. Arrows, wound margins at time 0. The percent wound closure at different times is plotted on the left. The values represent the means (±SD, vertical bars) of three independent experiments. (B) The percentage of cells adhering to different ECM components was determined in a 30-min adhesion assay as described in the legend to Fig. 2. Integrin-independent adhesion on BSA-coated surfaces was subtracted from the values plotted. Values are means (±SD, vertical bars) of five individual experiments. (C) Morphology of fibroblasts stained to visualize the actin cytoskeleton.

Mentions: The defects in motility, adhesion, and shape were even more dramatic in Raf-1 KO fibroblasts. These performed consistently worse than WT cells in an in vitro wound healing assay, in the presence or absence of motogenic signals (Fig. 3 A), and in a short-term adhesion assay, likely due to a delay in spreading (Fig. 3 B). Raf-1 KO fibroblasts also displayed a symmetric, contracted morphology. Longitudinal stress fibers were markedly reduced, and the actin was organized in tight cortical bundles (Fig. 3 C) reminiscent of those observed in Raf-1 KO keratinocytes. This redistribution of actin fibers to the cell cortex was not accompanied by an alteration in actin treadmilling, as the levels of F- and G-actin detected in WT and KO fibroblasts were comparable (not depicted). Thus, cell-autonomous defects in shape, spreading, and migration were a common feature of Raf-1 KO keratinocytes and fibroblasts.


Raf-1 regulates Rho signaling and cell migration.

Ehrenreiter K, Piazzolla D, Velamoor V, Sobczak I, Small JV, Takeda J, Leung T, Baccarini M - J. Cell Biol. (2005)

Raf-1 ablation affects fibroblasts motility, adhesion, and shape in vitro. (A) Fibroblast motility assayed by in vitro wound healing in serum-free medium. Wound closure was photographed after 18 h. Arrows, wound margins at time 0. The percent wound closure at different times is plotted on the left. The values represent the means (±SD, vertical bars) of three independent experiments. (B) The percentage of cells adhering to different ECM components was determined in a 30-min adhesion assay as described in the legend to Fig. 2. Integrin-independent adhesion on BSA-coated surfaces was subtracted from the values plotted. Values are means (±SD, vertical bars) of five individual experiments. (C) Morphology of fibroblasts stained to visualize the actin cytoskeleton.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171799&req=5

fig3: Raf-1 ablation affects fibroblasts motility, adhesion, and shape in vitro. (A) Fibroblast motility assayed by in vitro wound healing in serum-free medium. Wound closure was photographed after 18 h. Arrows, wound margins at time 0. The percent wound closure at different times is plotted on the left. The values represent the means (±SD, vertical bars) of three independent experiments. (B) The percentage of cells adhering to different ECM components was determined in a 30-min adhesion assay as described in the legend to Fig. 2. Integrin-independent adhesion on BSA-coated surfaces was subtracted from the values plotted. Values are means (±SD, vertical bars) of five individual experiments. (C) Morphology of fibroblasts stained to visualize the actin cytoskeleton.
Mentions: The defects in motility, adhesion, and shape were even more dramatic in Raf-1 KO fibroblasts. These performed consistently worse than WT cells in an in vitro wound healing assay, in the presence or absence of motogenic signals (Fig. 3 A), and in a short-term adhesion assay, likely due to a delay in spreading (Fig. 3 B). Raf-1 KO fibroblasts also displayed a symmetric, contracted morphology. Longitudinal stress fibers were markedly reduced, and the actin was organized in tight cortical bundles (Fig. 3 C) reminiscent of those observed in Raf-1 KO keratinocytes. This redistribution of actin fibers to the cell cortex was not accompanied by an alteration in actin treadmilling, as the levels of F- and G-actin detected in WT and KO fibroblasts were comparable (not depicted). Thus, cell-autonomous defects in shape, spreading, and migration were a common feature of Raf-1 KO keratinocytes and fibroblasts.

Bottom Line: These defects are due to the hyperactivity and incorrect localization of the Rho-effector Rok-alpha to the plasma membrane.Raf-1 physically associates with Rok-alpha in wild-type (WT) cells, and reintroduction of either WT or kinase-dead Raf-1 in knockout fibroblasts rescues their defects in shape and migration.Thus, Raf-1 plays an essential, kinase-independent function as a spatial regulator of Rho downstream signaling during migration.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Genetics, Max F. Perutz Laboratories, University Departments at the Vienna Biocenter, 1030 Vienna, Austria.

ABSTRACT
Raf kinases relay signals inducing proliferation, differentiation, and survival. The Raf-1 isoform has been extensively studied as the upstream kinase linking Ras activation to the MEK/ERK module. Recently, however, genetic experiments have shown that Raf-1 plays an essential role in counteracting apoptosis, and that it does so independently of its ability to activate MEK. By conditional gene ablation, we now show that Raf-1 is required for normal wound healing in vivo and for the migration of keratinocytes and fibroblasts in vitro. Raf-1-deficient cells show a symmetric, contracted appearance, characterized by cortical actin bundles and by a disordered vimentin cytoskeleton. These defects are due to the hyperactivity and incorrect localization of the Rho-effector Rok-alpha to the plasma membrane. Raf-1 physically associates with Rok-alpha in wild-type (WT) cells, and reintroduction of either WT or kinase-dead Raf-1 in knockout fibroblasts rescues their defects in shape and migration. Thus, Raf-1 plays an essential, kinase-independent function as a spatial regulator of Rho downstream signaling during migration.

Show MeSH
Related in: MedlinePlus