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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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Suppression of Cdc42 or RhoA expression does not affect directional persistence of cell migration. (A and B) Specificity of Cdc42 and RhoA knockdowns. (A) 3 d after transfection of primary human fibroblasts with 100 nM Cdc42 siRNA, total Cdc42 protein levels were assayed by Western blotting with antibodies against Cdc42 in lysates of the transfected (Cdc42) and control cells treated with nonspecific siRNA (Control). Cdc42 siRNA led to a substantial decrease of both Cdc42 protein levels (total-Cdc42) and Cdc42 activity (Cdc42-GTP) by pull-down assay, whereas the activity and total amount of Rac in the same lysates (Rac-GTP and total-Rac) were not affected. (B) Primary human fibroblasts were transfected with 100 nM RhoA siRNA (RhoA) or nonspecific siRNA (Control). 3 d later, Rho protein levels were determined by Western blotting with antibodies against Rho. Although the levels of other Rho family members were not affected, the amount and activity of RhoA were substantially decreased. (C) After knockdown of Cdc42 by >80%, there were no significant changes in the D/T directionality ratio (D/TControl = 0.46 ± 0.03 and D/TCdc42 = 0.46 ± 0.05; PD/T = 0.89) or the velocity (VContr = 44.4 ± 3.9 μm/h and VCdc42 = 38.6 ± 2.1 μm/h; PV = 0.18). (C and D) Error bars represent the SEM. (D) Suppression of RhoA expression affects the velocity of migration but not directionality. After reducing RhoA levels by at least 60% using siRNA, velocity of cell migration was reduced by 40% (VContr = 34.4 ± 2.4 μm/h and VRhoA = 20.8 ± 1.4 μm/h; P < 0.0001), but directional persistence of migration was unaffected (D/TControl = 0.44 ± 0.03 and D/TRhoA = 0.42 ± 0.03; P = 0.74).
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fig3: Suppression of Cdc42 or RhoA expression does not affect directional persistence of cell migration. (A and B) Specificity of Cdc42 and RhoA knockdowns. (A) 3 d after transfection of primary human fibroblasts with 100 nM Cdc42 siRNA, total Cdc42 protein levels were assayed by Western blotting with antibodies against Cdc42 in lysates of the transfected (Cdc42) and control cells treated with nonspecific siRNA (Control). Cdc42 siRNA led to a substantial decrease of both Cdc42 protein levels (total-Cdc42) and Cdc42 activity (Cdc42-GTP) by pull-down assay, whereas the activity and total amount of Rac in the same lysates (Rac-GTP and total-Rac) were not affected. (B) Primary human fibroblasts were transfected with 100 nM RhoA siRNA (RhoA) or nonspecific siRNA (Control). 3 d later, Rho protein levels were determined by Western blotting with antibodies against Rho. Although the levels of other Rho family members were not affected, the amount and activity of RhoA were substantially decreased. (C) After knockdown of Cdc42 by >80%, there were no significant changes in the D/T directionality ratio (D/TControl = 0.46 ± 0.03 and D/TCdc42 = 0.46 ± 0.05; PD/T = 0.89) or the velocity (VContr = 44.4 ± 3.9 μm/h and VCdc42 = 38.6 ± 2.1 μm/h; PV = 0.18). (C and D) Error bars represent the SEM. (D) Suppression of RhoA expression affects the velocity of migration but not directionality. After reducing RhoA levels by at least 60% using siRNA, velocity of cell migration was reduced by 40% (VContr = 34.4 ± 2.4 μm/h and VRhoA = 20.8 ± 1.4 μm/h; P < 0.0001), but directional persistence of migration was unaffected (D/TControl = 0.44 ± 0.03 and D/TRhoA = 0.42 ± 0.03; P = 0.74).

Mentions: Cdc42 regulates filopodia formation (Nobes and Hall, 1995), and although it is involved in determining cell polarity (Nobes and Hall, 1999; Palazzo et al., 2001; Etienne-Manneville and Hall, 2003) and in regulating the directional migration of astrocytes in serum-free culture (Etienne-Manneville and Hall, 2001), knockdown experiments in human fibroblasts to selectively suppress Cdc42 levels and activity produced no loss of directional persistence of migration. Reducing the level of fibroblast Cdc42 protein and activity by >80% did not change Rac or Rho activity and also did not change either directionality or velocity of migration (Fig. 3, A and C, and not depicted). Rho stimulates cell contractility and adhesion by inducing the formation of actin stress fibers and focal adhesions, inhibition of Rho suppresses cell migration, and Rho can also affect directional persistence of migration in an epithelial cell system if cells express β1 rather than β3 integrins (Allen et al., 1998; Ridley et al., 2003; Danen et al., 2005). Although knocking down the RhoA level in human fibroblasts by >60% slowed migration speed by 40%, again there was no effect on Rac activity or on directionality, and the D/T ratio remained unchanged (Fig. 3, B and D). In addition, knockdown of Rac1 activity was not accompanied by any changes in the levels of β1 or β3 integrins (Fig. S4 A, available at http://jcb.org/cgi/content/full/jcb.200503152.DC1), ruling out a mechanism involving integrin switching. Thus, only the overall levels of Rac activity could be linked specifically to the regulation of random versus directionally persistent migration under regular cell culture conditions.


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)

Suppression of Cdc42 or RhoA expression does not affect directional persistence of cell migration. (A and B) Specificity of Cdc42 and RhoA knockdowns. (A) 3 d after transfection of primary human fibroblasts with 100 nM Cdc42 siRNA, total Cdc42 protein levels were assayed by Western blotting with antibodies against Cdc42 in lysates of the transfected (Cdc42) and control cells treated with nonspecific siRNA (Control). Cdc42 siRNA led to a substantial decrease of both Cdc42 protein levels (total-Cdc42) and Cdc42 activity (Cdc42-GTP) by pull-down assay, whereas the activity and total amount of Rac in the same lysates (Rac-GTP and total-Rac) were not affected. (B) Primary human fibroblasts were transfected with 100 nM RhoA siRNA (RhoA) or nonspecific siRNA (Control). 3 d later, Rho protein levels were determined by Western blotting with antibodies against Rho. Although the levels of other Rho family members were not affected, the amount and activity of RhoA were substantially decreased. (C) After knockdown of Cdc42 by >80%, there were no significant changes in the D/T directionality ratio (D/TControl = 0.46 ± 0.03 and D/TCdc42 = 0.46 ± 0.05; PD/T = 0.89) or the velocity (VContr = 44.4 ± 3.9 μm/h and VCdc42 = 38.6 ± 2.1 μm/h; PV = 0.18). (C and D) Error bars represent the SEM. (D) Suppression of RhoA expression affects the velocity of migration but not directionality. After reducing RhoA levels by at least 60% using siRNA, velocity of cell migration was reduced by 40% (VContr = 34.4 ± 2.4 μm/h and VRhoA = 20.8 ± 1.4 μm/h; P < 0.0001), but directional persistence of migration was unaffected (D/TControl = 0.44 ± 0.03 and D/TRhoA = 0.42 ± 0.03; P = 0.74).
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fig3: Suppression of Cdc42 or RhoA expression does not affect directional persistence of cell migration. (A and B) Specificity of Cdc42 and RhoA knockdowns. (A) 3 d after transfection of primary human fibroblasts with 100 nM Cdc42 siRNA, total Cdc42 protein levels were assayed by Western blotting with antibodies against Cdc42 in lysates of the transfected (Cdc42) and control cells treated with nonspecific siRNA (Control). Cdc42 siRNA led to a substantial decrease of both Cdc42 protein levels (total-Cdc42) and Cdc42 activity (Cdc42-GTP) by pull-down assay, whereas the activity and total amount of Rac in the same lysates (Rac-GTP and total-Rac) were not affected. (B) Primary human fibroblasts were transfected with 100 nM RhoA siRNA (RhoA) or nonspecific siRNA (Control). 3 d later, Rho protein levels were determined by Western blotting with antibodies against Rho. Although the levels of other Rho family members were not affected, the amount and activity of RhoA were substantially decreased. (C) After knockdown of Cdc42 by >80%, there were no significant changes in the D/T directionality ratio (D/TControl = 0.46 ± 0.03 and D/TCdc42 = 0.46 ± 0.05; PD/T = 0.89) or the velocity (VContr = 44.4 ± 3.9 μm/h and VCdc42 = 38.6 ± 2.1 μm/h; PV = 0.18). (C and D) Error bars represent the SEM. (D) Suppression of RhoA expression affects the velocity of migration but not directionality. After reducing RhoA levels by at least 60% using siRNA, velocity of cell migration was reduced by 40% (VContr = 34.4 ± 2.4 μm/h and VRhoA = 20.8 ± 1.4 μm/h; P < 0.0001), but directional persistence of migration was unaffected (D/TControl = 0.44 ± 0.03 and D/TRhoA = 0.42 ± 0.03; P = 0.74).
Mentions: Cdc42 regulates filopodia formation (Nobes and Hall, 1995), and although it is involved in determining cell polarity (Nobes and Hall, 1999; Palazzo et al., 2001; Etienne-Manneville and Hall, 2003) and in regulating the directional migration of astrocytes in serum-free culture (Etienne-Manneville and Hall, 2001), knockdown experiments in human fibroblasts to selectively suppress Cdc42 levels and activity produced no loss of directional persistence of migration. Reducing the level of fibroblast Cdc42 protein and activity by >80% did not change Rac or Rho activity and also did not change either directionality or velocity of migration (Fig. 3, A and C, and not depicted). Rho stimulates cell contractility and adhesion by inducing the formation of actin stress fibers and focal adhesions, inhibition of Rho suppresses cell migration, and Rho can also affect directional persistence of migration in an epithelial cell system if cells express β1 rather than β3 integrins (Allen et al., 1998; Ridley et al., 2003; Danen et al., 2005). Although knocking down the RhoA level in human fibroblasts by >60% slowed migration speed by 40%, again there was no effect on Rac activity or on directionality, and the D/T ratio remained unchanged (Fig. 3, B and D). In addition, knockdown of Rac1 activity was not accompanied by any changes in the levels of β1 or β3 integrins (Fig. S4 A, available at http://jcb.org/cgi/content/full/jcb.200503152.DC1), ruling out a mechanism involving integrin switching. Thus, only the overall levels of Rac activity could be linked specifically to the regulation of random versus directionally persistent migration under regular cell culture conditions.

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