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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 expression lowers Rac activity and inhibits random motility of fibroblasts. (A) Primary human fibroblasts were transfected with 1 nM Rac1 siRNA or 1 nM nonspecific control pool siRNA (Contr). Pull-down assays were performed as described for Fig. 1 after 72 h of siRNA treatment, using an immunoblot of lysates for actin to verify equal protein loading. The amount of active Rac (Rac-GTP) was reduced to 67% of original levels and the total amount of Rac protein (total-Rac) was reduced to 52% after this Rac1 siRNA transfection. In contrast, levels of active Cdc42 and Rho did not change. The immunoblot for total Rac was stripped and reprobed for total Cdc42 protein, which was not altered by the Rac1 siRNA. (B and D) Composite collection of representative migration tracks of human fibroblasts starting 72 h after transfection with control compared with either 0.1 or 100 nM Rac1 siRNA, respectively, tracked at 15-min intervals over a span of 10 h. Bars, 100 μm. (C and E) Quantification of persistence of migratory directionality (D/T) and velocity of the transfected cells at the two different siRNA concentrations: (C) 0.1 nM and (E) 100 nM. Error bars indicate SEM; the differences between control and 0.1 nM or 100 nM Rac1 siRNA D/T values were significant at the P < 0.001 level. (F) Increases in D/T ratios by reduction of active Rac using Rac siRNA were observed in all (11 out of 11) independent experiments, each testing three siRNA concentrations against the control. All data were pooled together for this composite graph based on video time-lapse microscopy on a total of 933 cells.
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fig2: Suppression of Rac1 expression lowers Rac activity and inhibits random motility of fibroblasts. (A) Primary human fibroblasts were transfected with 1 nM Rac1 siRNA or 1 nM nonspecific control pool siRNA (Contr). Pull-down assays were performed as described for Fig. 1 after 72 h of siRNA treatment, using an immunoblot of lysates for actin to verify equal protein loading. The amount of active Rac (Rac-GTP) was reduced to 67% of original levels and the total amount of Rac protein (total-Rac) was reduced to 52% after this Rac1 siRNA transfection. In contrast, levels of active Cdc42 and Rho did not change. The immunoblot for total Rac was stripped and reprobed for total Cdc42 protein, which was not altered by the Rac1 siRNA. (B and D) Composite collection of representative migration tracks of human fibroblasts starting 72 h after transfection with control compared with either 0.1 or 100 nM Rac1 siRNA, respectively, tracked at 15-min intervals over a span of 10 h. Bars, 100 μm. (C and E) Quantification of persistence of migratory directionality (D/T) and velocity of the transfected cells at the two different siRNA concentrations: (C) 0.1 nM and (E) 100 nM. Error bars indicate SEM; the differences between control and 0.1 nM or 100 nM Rac1 siRNA D/T values were significant at the P < 0.001 level. (F) Increases in D/T ratios by reduction of active Rac using Rac siRNA were observed in all (11 out of 11) independent experiments, each testing three siRNA concentrations against the control. All data were pooled together for this composite graph based on video time-lapse microscopy on a total of 933 cells.

Mentions: To directly test the role of total Rac activation levels in regulating the directionality of cell migration in a range of different cell types, levels of Rac1 were knocked down using RNA interference with small interfering (si) RNA. Reductions in Rac protein levels resulted in proportional changes in Rac activity (Fig. S2 [available at http://jcb.org/cgi/content/full/jcb.200503152.DC1] and not depicted). No effects of Rac1 knockdown on Cdc42 were detected, and Rho activity levels were generally unchanged (Fig. 2 A). Random cell migration was suppressed by such Rac1 knockdown in primary human fibroblasts, which is consistent with our findings in mouse ES cell–derived GD25 cells. The cells displayed increased directionality of migration, and suppression of overall velocity occurred only with a greater extent of Rac knockdown (Fig. 2, compare C with E; compare Video 1 with 2, available at http://jcb.org/cgi/content/full/jcb.200503152.DC1). Similar results were obtained using either a pool of four Rac1 siRNA duplexes or individual Rac1 siRNA duplexes to reduce Rac1 activity (compare Fig. 2 with Fig. S3 A). Based on 11 independent experiments with primary human fibroblasts, a modest reduction in total Rac activity to 70% of original levels substantially enhanced directional persistence of migration, and 60% or lower levels produced maximal directionally persistent migration (Fig. 2 F). Conversely, overexpression of wild-type Rac1 or constitutively activated Rac1 in primary human fibroblasts promoted random cell migration with only minimal effects on overall velocity (Fig. S3, C and D). Using an independent approach to altering Rac activity, the Rac GEF inhibitor NSC 23766 reduced concentrations of active Rac in human fibroblasts and produced a loss of random motility with movements restricted to the long axis of the cells, followed by immobilization at high doses (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 expression lowers Rac activity and inhibits random motility of fibroblasts. (A) Primary human fibroblasts were transfected with 1 nM Rac1 siRNA or 1 nM nonspecific control pool siRNA (Contr). Pull-down assays were performed as described for Fig. 1 after 72 h of siRNA treatment, using an immunoblot of lysates for actin to verify equal protein loading. The amount of active Rac (Rac-GTP) was reduced to 67% of original levels and the total amount of Rac protein (total-Rac) was reduced to 52% after this Rac1 siRNA transfection. In contrast, levels of active Cdc42 and Rho did not change. The immunoblot for total Rac was stripped and reprobed for total Cdc42 protein, which was not altered by the Rac1 siRNA. (B and D) Composite collection of representative migration tracks of human fibroblasts starting 72 h after transfection with control compared with either 0.1 or 100 nM Rac1 siRNA, respectively, tracked at 15-min intervals over a span of 10 h. Bars, 100 μm. (C and E) Quantification of persistence of migratory directionality (D/T) and velocity of the transfected cells at the two different siRNA concentrations: (C) 0.1 nM and (E) 100 nM. Error bars indicate SEM; the differences between control and 0.1 nM or 100 nM Rac1 siRNA D/T values were significant at the P < 0.001 level. (F) Increases in D/T ratios by reduction of active Rac using Rac siRNA were observed in all (11 out of 11) independent experiments, each testing three siRNA concentrations against the control. All data were pooled together for this composite graph based on video time-lapse microscopy on a total of 933 cells.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171343&req=5

fig2: Suppression of Rac1 expression lowers Rac activity and inhibits random motility of fibroblasts. (A) Primary human fibroblasts were transfected with 1 nM Rac1 siRNA or 1 nM nonspecific control pool siRNA (Contr). Pull-down assays were performed as described for Fig. 1 after 72 h of siRNA treatment, using an immunoblot of lysates for actin to verify equal protein loading. The amount of active Rac (Rac-GTP) was reduced to 67% of original levels and the total amount of Rac protein (total-Rac) was reduced to 52% after this Rac1 siRNA transfection. In contrast, levels of active Cdc42 and Rho did not change. The immunoblot for total Rac was stripped and reprobed for total Cdc42 protein, which was not altered by the Rac1 siRNA. (B and D) Composite collection of representative migration tracks of human fibroblasts starting 72 h after transfection with control compared with either 0.1 or 100 nM Rac1 siRNA, respectively, tracked at 15-min intervals over a span of 10 h. Bars, 100 μm. (C and E) Quantification of persistence of migratory directionality (D/T) and velocity of the transfected cells at the two different siRNA concentrations: (C) 0.1 nM and (E) 100 nM. Error bars indicate SEM; the differences between control and 0.1 nM or 100 nM Rac1 siRNA D/T values were significant at the P < 0.001 level. (F) Increases in D/T ratios by reduction of active Rac using Rac siRNA were observed in all (11 out of 11) independent experiments, each testing three siRNA concentrations against the control. All data were pooled together for this composite graph based on video time-lapse microscopy on a total of 933 cells.
Mentions: To directly test the role of total Rac activation levels in regulating the directionality of cell migration in a range of different cell types, levels of Rac1 were knocked down using RNA interference with small interfering (si) RNA. Reductions in Rac protein levels resulted in proportional changes in Rac activity (Fig. S2 [available at http://jcb.org/cgi/content/full/jcb.200503152.DC1] and not depicted). No effects of Rac1 knockdown on Cdc42 were detected, and Rho activity levels were generally unchanged (Fig. 2 A). Random cell migration was suppressed by such Rac1 knockdown in primary human fibroblasts, which is consistent with our findings in mouse ES cell–derived GD25 cells. The cells displayed increased directionality of migration, and suppression of overall velocity occurred only with a greater extent of Rac knockdown (Fig. 2, compare C with E; compare Video 1 with 2, available at http://jcb.org/cgi/content/full/jcb.200503152.DC1). Similar results were obtained using either a pool of four Rac1 siRNA duplexes or individual Rac1 siRNA duplexes to reduce Rac1 activity (compare Fig. 2 with Fig. S3 A). Based on 11 independent experiments with primary human fibroblasts, a modest reduction in total Rac activity to 70% of original levels substantially enhanced directional persistence of migration, and 60% or lower levels produced maximal directionally persistent migration (Fig. 2 F). Conversely, overexpression of wild-type Rac1 or constitutively activated Rac1 in primary human fibroblasts promoted random cell migration with only minimal effects on overall velocity (Fig. S3, C and D). Using an independent approach to altering Rac activity, the Rac GEF inhibitor NSC 23766 reduced concentrations of active Rac in human fibroblasts and produced a loss of random motility with movements restricted to the long axis of the cells, followed by immobilization at high doses (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