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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 Rac1 results in increased directional persistence of migration in human epithelial and glioblastoma cells. (A) Comparison of total and active Rac levels in three cell lines and effects of knockdown by Rac1 siRNA. Primary human foreskin fibroblasts (HFF), U87-MG human glioblastoma cells, and MCF-10A human mammary epithelial cells were transfected with 50 nM Rac1 pool siRNA or control siRNA, and then assayed for total and active Rac levels. (B) MCF-10A cells transfected with Rac1 siRNA migrate with much higher directional persistence with no change in velocity. MCF-10A cells were transfected with 50 nM Rac1 or control siRNA, detached with trypsin 72 h after transfection, replated at single-cell density, and recorded by time-lapse video microscopy. Suppression of Rac1 expression (Rac1 siRNA) induced straightening of cell migration tracks when compared with the cells transfected with the nonspecific siRNA (Control siRNA). Bar, 100 μm. The quantified directionality of Rac1 siRNA transfected cells (Rac1) increased threefold compared with cells transfected with control siRNA (Contr) (D/TControl = 0.26 ± 0.02 vs. D/TRac1 = 0.76 ± 0.05; P < 0.0001), whereas the velocities remained comparable (VContr = 15.3 ± 0.8 μm/h vs. VRac1 = 15.7 ± 1.3 μm/h; P = 0.79). (C) U87-MG cells transfected with Rac1 siRNA lose random motility and migrate with greater directionality. U87-MG cells were transfected with 200 nM Rac1 siRNA, and after 3 d, cell migration was recorded. Bar, 100 μm. Rac1 suppression resulted in a significant increase in directionality (D/TControl = 0.31 ± 0.03 vs. D/TRac1 = 0.54 ± 0.04; P < 0.0001) and a slight decrease in velocity (VContr = 31.6 ± 1.0 μm/h vs. VRac1 = 25.5 ± 1.6 μm/h; P < 0.005).
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fig4: Suppression of Rac1 results in increased directional persistence of migration in human epithelial and glioblastoma cells. (A) Comparison of total and active Rac levels in three cell lines and effects of knockdown by Rac1 siRNA. Primary human foreskin fibroblasts (HFF), U87-MG human glioblastoma cells, and MCF-10A human mammary epithelial cells were transfected with 50 nM Rac1 pool siRNA or control siRNA, and then assayed for total and active Rac levels. (B) MCF-10A cells transfected with Rac1 siRNA migrate with much higher directional persistence with no change in velocity. MCF-10A cells were transfected with 50 nM Rac1 or control siRNA, detached with trypsin 72 h after transfection, replated at single-cell density, and recorded by time-lapse video microscopy. Suppression of Rac1 expression (Rac1 siRNA) induced straightening of cell migration tracks when compared with the cells transfected with the nonspecific siRNA (Control siRNA). Bar, 100 μm. The quantified directionality of Rac1 siRNA transfected cells (Rac1) increased threefold compared with cells transfected with control siRNA (Contr) (D/TControl = 0.26 ± 0.02 vs. D/TRac1 = 0.76 ± 0.05; P < 0.0001), whereas the velocities remained comparable (VContr = 15.3 ± 0.8 μm/h vs. VRac1 = 15.7 ± 1.3 μm/h; P = 0.79). (C) U87-MG cells transfected with Rac1 siRNA lose random motility and migrate with greater directionality. U87-MG cells were transfected with 200 nM Rac1 siRNA, and after 3 d, cell migration was recorded. Bar, 100 μm. Rac1 suppression resulted in a significant increase in directionality (D/TControl = 0.31 ± 0.03 vs. D/TRac1 = 0.54 ± 0.04; P < 0.0001) and a slight decrease in velocity (VContr = 31.6 ± 1.0 μm/h vs. VRac1 = 25.5 ± 1.6 μm/h; P < 0.005).

Mentions: The mode of cell migration was found to depend on the relative level of Rac activity for two other types of cells (Fig. 4). After siRNA-induced reduction of Rac activity in the nontransformed human epithelial cell line MCF-10A, directional persistence was markedly increased (Videos 4 and 5, available at http://jcb.org/cgi/content/full/jcb.200503152.DC1) with the D/T ratio increasing threefold (Fig. 4, A and B). In addition, the tumor cell line U87-MG also showed enhanced directional persistence of migration (Videos 6 and 7, available at http://jcb.org/cgi/content/full/jcb.200503152.DC1) with a 74% increase in the D/T ratio after Rac knockdown (Fig. 4, A and C). The three cell types showed minimal to moderate decreases in migration speed after partial Rac suppression. However, strong reductions in Rac activity resulted in the suppression of lamella formation and the failure of cells to migrate as noted in many other studies (Nobes and Hall, 1995; Allen et al., 1998; Glogauer et al., 2003; Pradip et al., 2003; unpublished data).


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 Rac1 results in increased directional persistence of migration in human epithelial and glioblastoma cells. (A) Comparison of total and active Rac levels in three cell lines and effects of knockdown by Rac1 siRNA. Primary human foreskin fibroblasts (HFF), U87-MG human glioblastoma cells, and MCF-10A human mammary epithelial cells were transfected with 50 nM Rac1 pool siRNA or control siRNA, and then assayed for total and active Rac levels. (B) MCF-10A cells transfected with Rac1 siRNA migrate with much higher directional persistence with no change in velocity. MCF-10A cells were transfected with 50 nM Rac1 or control siRNA, detached with trypsin 72 h after transfection, replated at single-cell density, and recorded by time-lapse video microscopy. Suppression of Rac1 expression (Rac1 siRNA) induced straightening of cell migration tracks when compared with the cells transfected with the nonspecific siRNA (Control siRNA). Bar, 100 μm. The quantified directionality of Rac1 siRNA transfected cells (Rac1) increased threefold compared with cells transfected with control siRNA (Contr) (D/TControl = 0.26 ± 0.02 vs. D/TRac1 = 0.76 ± 0.05; P < 0.0001), whereas the velocities remained comparable (VContr = 15.3 ± 0.8 μm/h vs. VRac1 = 15.7 ± 1.3 μm/h; P = 0.79). (C) U87-MG cells transfected with Rac1 siRNA lose random motility and migrate with greater directionality. U87-MG cells were transfected with 200 nM Rac1 siRNA, and after 3 d, cell migration was recorded. Bar, 100 μm. Rac1 suppression resulted in a significant increase in directionality (D/TControl = 0.31 ± 0.03 vs. D/TRac1 = 0.54 ± 0.04; P < 0.0001) and a slight decrease in velocity (VContr = 31.6 ± 1.0 μm/h vs. VRac1 = 25.5 ± 1.6 μm/h; P < 0.005).
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fig4: Suppression of Rac1 results in increased directional persistence of migration in human epithelial and glioblastoma cells. (A) Comparison of total and active Rac levels in three cell lines and effects of knockdown by Rac1 siRNA. Primary human foreskin fibroblasts (HFF), U87-MG human glioblastoma cells, and MCF-10A human mammary epithelial cells were transfected with 50 nM Rac1 pool siRNA or control siRNA, and then assayed for total and active Rac levels. (B) MCF-10A cells transfected with Rac1 siRNA migrate with much higher directional persistence with no change in velocity. MCF-10A cells were transfected with 50 nM Rac1 or control siRNA, detached with trypsin 72 h after transfection, replated at single-cell density, and recorded by time-lapse video microscopy. Suppression of Rac1 expression (Rac1 siRNA) induced straightening of cell migration tracks when compared with the cells transfected with the nonspecific siRNA (Control siRNA). Bar, 100 μm. The quantified directionality of Rac1 siRNA transfected cells (Rac1) increased threefold compared with cells transfected with control siRNA (Contr) (D/TControl = 0.26 ± 0.02 vs. D/TRac1 = 0.76 ± 0.05; P < 0.0001), whereas the velocities remained comparable (VContr = 15.3 ± 0.8 μm/h vs. VRac1 = 15.7 ± 1.3 μm/h; P = 0.79). (C) U87-MG cells transfected with Rac1 siRNA lose random motility and migrate with greater directionality. U87-MG cells were transfected with 200 nM Rac1 siRNA, and after 3 d, cell migration was recorded. Bar, 100 μm. Rac1 suppression resulted in a significant increase in directionality (D/TControl = 0.31 ± 0.03 vs. D/TRac1 = 0.54 ± 0.04; P < 0.0001) and a slight decrease in velocity (VContr = 31.6 ± 1.0 μm/h vs. VRac1 = 25.5 ± 1.6 μm/h; P < 0.005).
Mentions: The mode of cell migration was found to depend on the relative level of Rac activity for two other types of cells (Fig. 4). After siRNA-induced reduction of Rac activity in the nontransformed human epithelial cell line MCF-10A, directional persistence was markedly increased (Videos 4 and 5, available at http://jcb.org/cgi/content/full/jcb.200503152.DC1) with the D/T ratio increasing threefold (Fig. 4, A and B). In addition, the tumor cell line U87-MG also showed enhanced directional persistence of migration (Videos 6 and 7, available at http://jcb.org/cgi/content/full/jcb.200503152.DC1) with a 74% increase in the D/T ratio after Rac knockdown (Fig. 4, A and C). The three cell types showed minimal to moderate decreases in migration speed after partial Rac suppression. However, strong reductions in Rac activity resulted in the suppression of lamella formation and the failure of cells to migrate as noted in many other studies (Nobes and Hall, 1995; Allen et al., 1998; Glogauer et al., 2003; Pradip et al., 2003; unpublished data).

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