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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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3D fibronectin matrix reduces both Rac activity and random migration. (A) Primary human fibroblasts were plated on 2D substrates coated with fibronectin or 3D matrices rich in fibronectin and assayed for activity as in Fig. 1. The reduction of active Rac in cells plated in a 3D matrix compared with 2D was 31% (P = 0.017), whereas Cdc42 and Rho changes were not significant (Cdc42 4.5% reduction, P = 0.66; Rho 25% increase, P = 0.65). Error bars indicate SEM. (B) Cells in 2D versus 3D environments have different morphologies and locations of lamellae (arrowheads), as shown after staining for F-actin with phalloidin-Alexa 594 (left and red color in the right) and anti-fibronectin antibody (green). Bar, 20 μm. (C) Migration of cells in 2D versus 3D environments was recorded, and directionality (D/T) and velocity were calculated. (C and D) Error bars represent SEM. (D) Decreased random motility of cells within three-dimensional (3D) matrix. Primary human fibroblasts were cultured overnight within intact 3D matrices, on mechanically flattened 3D matrix (2D matrix), or on surfaces coated with solubilized 3D matrix (2D mix). Cell movements were recorded for 10 h, and the D/T ratio and velocity were calculated as described above. (E) Activated Rac in cells in 3D matrix restores random motility. Primary human fibroblasts were cotransfected with Rac Q61L VSV and Rac Q61L GFP or GFP alone (Control), sorted for low levels of GFP expression (1–50% from the peak of positive cells), and plated on 3D matrices. Cell movements were recorded for 10 h, and the D/T ratio and velocity were quantified. The bars represent the means of data pooled from two experiments, and error bars indicate SEM (n = 32–34 cells).
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fig7: 3D fibronectin matrix reduces both Rac activity and random migration. (A) Primary human fibroblasts were plated on 2D substrates coated with fibronectin or 3D matrices rich in fibronectin and assayed for activity as in Fig. 1. The reduction of active Rac in cells plated in a 3D matrix compared with 2D was 31% (P = 0.017), whereas Cdc42 and Rho changes were not significant (Cdc42 4.5% reduction, P = 0.66; Rho 25% increase, P = 0.65). Error bars indicate SEM. (B) Cells in 2D versus 3D environments have different morphologies and locations of lamellae (arrowheads), as shown after staining for F-actin with phalloidin-Alexa 594 (left and red color in the right) and anti-fibronectin antibody (green). Bar, 20 μm. (C) Migration of cells in 2D versus 3D environments was recorded, and directionality (D/T) and velocity were calculated. (C and D) Error bars represent SEM. (D) Decreased random motility of cells within three-dimensional (3D) matrix. Primary human fibroblasts were cultured overnight within intact 3D matrices, on mechanically flattened 3D matrix (2D matrix), or on surfaces coated with solubilized 3D matrix (2D mix). Cell movements were recorded for 10 h, and the D/T ratio and velocity were calculated as described above. (E) Activated Rac in cells in 3D matrix restores random motility. Primary human fibroblasts were cotransfected with Rac Q61L VSV and Rac Q61L GFP or GFP alone (Control), sorted for low levels of GFP expression (1–50% from the peak of positive cells), and plated on 3D matrices. Cell movements were recorded for 10 h, and the D/T ratio and velocity were quantified. The bars represent the means of data pooled from two experiments, and error bars indicate SEM (n = 32–34 cells).

Mentions: Cells migrating in 3D cell-derived matrices have different types of cell adhesions, morphology, and signaling when compared with cells in standard 2D tissue culture (Cukierman et al., 2001; Walpita and Hay, 2002). Human fibroblasts in such 3D matrices were found to have a partial, but highly reproducible, 30–50% reduction in active Rac, but no reduction or increase in Cdc42 or Rho activities (Fig. 7 A). This reduction in Rac activity was accompanied by decreased random migration (Fig. 7 C and Video 3), with directional persistence increasing from D/T2D = 0.48 ± 0.03 to D/T3D = 0.87 ± 0.02 (P < 0.0001). Individual cells in the 3D matrix became spindle shaped as previously described (Cukierman et al., 2001), but in addition, staining for F-actin revealed a substantial reduction in peripheral lamellae in 3D compared with 2D (Fig. 7 B). In contrast to the results in cells cultured on 2D substrates, there was a significant increase in overall cell migration velocity by 34% associated with the reductions in active Rac and random migration in a 3D environment (Fig. 7 C; P < 0.0001). These findings further indicate the separate regulation of speed and directionality.


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)

3D fibronectin matrix reduces both Rac activity and random migration. (A) Primary human fibroblasts were plated on 2D substrates coated with fibronectin or 3D matrices rich in fibronectin and assayed for activity as in Fig. 1. The reduction of active Rac in cells plated in a 3D matrix compared with 2D was 31% (P = 0.017), whereas Cdc42 and Rho changes were not significant (Cdc42 4.5% reduction, P = 0.66; Rho 25% increase, P = 0.65). Error bars indicate SEM. (B) Cells in 2D versus 3D environments have different morphologies and locations of lamellae (arrowheads), as shown after staining for F-actin with phalloidin-Alexa 594 (left and red color in the right) and anti-fibronectin antibody (green). Bar, 20 μm. (C) Migration of cells in 2D versus 3D environments was recorded, and directionality (D/T) and velocity were calculated. (C and D) Error bars represent SEM. (D) Decreased random motility of cells within three-dimensional (3D) matrix. Primary human fibroblasts were cultured overnight within intact 3D matrices, on mechanically flattened 3D matrix (2D matrix), or on surfaces coated with solubilized 3D matrix (2D mix). Cell movements were recorded for 10 h, and the D/T ratio and velocity were calculated as described above. (E) Activated Rac in cells in 3D matrix restores random motility. Primary human fibroblasts were cotransfected with Rac Q61L VSV and Rac Q61L GFP or GFP alone (Control), sorted for low levels of GFP expression (1–50% from the peak of positive cells), and plated on 3D matrices. Cell movements were recorded for 10 h, and the D/T ratio and velocity were quantified. The bars represent the means of data pooled from two experiments, and error bars indicate SEM (n = 32–34 cells).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171343&req=5

fig7: 3D fibronectin matrix reduces both Rac activity and random migration. (A) Primary human fibroblasts were plated on 2D substrates coated with fibronectin or 3D matrices rich in fibronectin and assayed for activity as in Fig. 1. The reduction of active Rac in cells plated in a 3D matrix compared with 2D was 31% (P = 0.017), whereas Cdc42 and Rho changes were not significant (Cdc42 4.5% reduction, P = 0.66; Rho 25% increase, P = 0.65). Error bars indicate SEM. (B) Cells in 2D versus 3D environments have different morphologies and locations of lamellae (arrowheads), as shown after staining for F-actin with phalloidin-Alexa 594 (left and red color in the right) and anti-fibronectin antibody (green). Bar, 20 μm. (C) Migration of cells in 2D versus 3D environments was recorded, and directionality (D/T) and velocity were calculated. (C and D) Error bars represent SEM. (D) Decreased random motility of cells within three-dimensional (3D) matrix. Primary human fibroblasts were cultured overnight within intact 3D matrices, on mechanically flattened 3D matrix (2D matrix), or on surfaces coated with solubilized 3D matrix (2D mix). Cell movements were recorded for 10 h, and the D/T ratio and velocity were calculated as described above. (E) Activated Rac in cells in 3D matrix restores random motility. Primary human fibroblasts were cotransfected with Rac Q61L VSV and Rac Q61L GFP or GFP alone (Control), sorted for low levels of GFP expression (1–50% from the peak of positive cells), and plated on 3D matrices. Cell movements were recorded for 10 h, and the D/T ratio and velocity were quantified. The bars represent the means of data pooled from two experiments, and error bars indicate SEM (n = 32–34 cells).
Mentions: Cells migrating in 3D cell-derived matrices have different types of cell adhesions, morphology, and signaling when compared with cells in standard 2D tissue culture (Cukierman et al., 2001; Walpita and Hay, 2002). Human fibroblasts in such 3D matrices were found to have a partial, but highly reproducible, 30–50% reduction in active Rac, but no reduction or increase in Cdc42 or Rho activities (Fig. 7 A). This reduction in Rac activity was accompanied by decreased random migration (Fig. 7 C and Video 3), with directional persistence increasing from D/T2D = 0.48 ± 0.03 to D/T3D = 0.87 ± 0.02 (P < 0.0001). Individual cells in the 3D matrix became spindle shaped as previously described (Cukierman et al., 2001), but in addition, staining for F-actin revealed a substantial reduction in peripheral lamellae in 3D compared with 2D (Fig. 7 B). In contrast to the results in cells cultured on 2D substrates, there was a significant increase in overall cell migration velocity by 34% associated with the reductions in active Rac and random migration in a 3D environment (Fig. 7 C; P < 0.0001). These findings further indicate the separate regulation of speed and directionality.

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