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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 Rac reduces the number of peripheral lamellae and changes in direction of cell migration. (A) Primary human fibroblasts transfected with control or Rac1 siRNA were recorded by time-lapse video microscopy. Phase-contrast micrographs and migration paths of two representative cells are shown at 1-h intervals. Green arrowheads indicate lamellae extending in the direction of cell movement. Yellow arrowheads show lamellar protrusions in other directions. Green arrows in the diagrams to the right of the micrographs indicate the direction and distance of cell movement each hour. (B and C) Quantification of Rac-dependent lamellae in an axial versus a peripheral location. The main cell axis was determined as the longest distance between the ends of each cell (continuous red line in the image of a cell stained for F-actin). Two zones were defined as follows: “axial” was within 20° of the main axis (area between the dashed white lines) and “peripheral” was anything lateral to this zone. The length of total membrane in all lamellae of each cell and the number of lamellae per cell were determined for axial (green) compared with peripheral (yellow) lamellae in cells transfected with control (Contr) and Rac1 (Rac1) siRNA and presented as mean values ± SEM. Bar, 20 μm.
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fig5: Suppression of Rac reduces the number of peripheral lamellae and changes in direction of cell migration. (A) Primary human fibroblasts transfected with control or Rac1 siRNA were recorded by time-lapse video microscopy. Phase-contrast micrographs and migration paths of two representative cells are shown at 1-h intervals. Green arrowheads indicate lamellae extending in the direction of cell movement. Yellow arrowheads show lamellar protrusions in other directions. Green arrows in the diagrams to the right of the micrographs indicate the direction and distance of cell movement each hour. (B and C) Quantification of Rac-dependent lamellae in an axial versus a peripheral location. The main cell axis was determined as the longest distance between the ends of each cell (continuous red line in the image of a cell stained for F-actin). Two zones were defined as follows: “axial” was within 20° of the main axis (area between the dashed white lines) and “peripheral” was anything lateral to this zone. The length of total membrane in all lamellae of each cell and the number of lamellae per cell were determined for axial (green) compared with peripheral (yellow) lamellae in cells transfected with control (Contr) and Rac1 (Rac1) siRNA and presented as mean values ± SEM. Bar, 20 μm.

Mentions: The cell biological mechanism underlying this Rac-dependent regulation of migration became apparent from time-lapse video recordings, where each change in the direction of migration was accompanied by a change in the leading edge of the cell: a new lamella elsewhere on the cell became dominant (Fig. 5 A). In contrast, cells with reduced active Rac were more elongated and had lamellae confined to only one or both ends of the cell. We tested the hypothesis that Rac1 regulates peripheral versus such axial lamellae by quantifying these locomotory structures. Staining of F-actin with phalloidin allowed us to identify morphologically distinct, ruffling lamellae (Wu et al., 2003; Fig. S4, available at http://jcb.org/cgi/content/full/jcb.200503152.DC1). The presence of lamellae at an end of the long axis of the cell (within 20° of the longest dimension of each cell) was compared with the number of more peripheral lamellae located outside of this central zone (Fig. 5 B). Knockdown of Rac1 strongly inhibited only the formation of peripheral lamellae. There were threefold fewer peripheral lamellae per cell and a fivefold reduction in lamellar membrane; i.e., the total length of cell membrane associated with these structures per cell (Fig. 5 C). In contrast, there were no detectable effects of Rac reduction on lamellae located at the ends of the long axis of cells, as determined both by the number of lamellae per cell and by the total length of lamella membrane (Fig. 5 C). As a direct consequence of this selective loss of peripheral lamellae, the total number of lamellae per cell was reduced by half, and overall lamella length was decreased by 57% in human fibroblasts with Rac1 knockdown and by 66% in the integrin mutant cells (Table I). Restoration of Rac activity levels in the integrin mutant cells by expression of activated Akt restored normal numbers of lamellae and lamellar length, whereas overexpression of activated Rac1 enhanced numbers of lamellae and length accompanied by an elevation of random motility as reflected by a low D/T ratio (compare Table I and Fig. 1, D and E, with Fig. S1 C and Fig. S3 D). Collectively, these results demonstrate that active Rac induces random motility by promoting peripheral lamellae oriented in directions different from the direction of migration along the main cell axis.


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 Rac reduces the number of peripheral lamellae and changes in direction of cell migration. (A) Primary human fibroblasts transfected with control or Rac1 siRNA were recorded by time-lapse video microscopy. Phase-contrast micrographs and migration paths of two representative cells are shown at 1-h intervals. Green arrowheads indicate lamellae extending in the direction of cell movement. Yellow arrowheads show lamellar protrusions in other directions. Green arrows in the diagrams to the right of the micrographs indicate the direction and distance of cell movement each hour. (B and C) Quantification of Rac-dependent lamellae in an axial versus a peripheral location. The main cell axis was determined as the longest distance between the ends of each cell (continuous red line in the image of a cell stained for F-actin). Two zones were defined as follows: “axial” was within 20° of the main axis (area between the dashed white lines) and “peripheral” was anything lateral to this zone. The length of total membrane in all lamellae of each cell and the number of lamellae per cell were determined for axial (green) compared with peripheral (yellow) lamellae in cells transfected with control (Contr) and Rac1 (Rac1) siRNA and presented as mean values ± SEM. Bar, 20 μm.
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fig5: Suppression of Rac reduces the number of peripheral lamellae and changes in direction of cell migration. (A) Primary human fibroblasts transfected with control or Rac1 siRNA were recorded by time-lapse video microscopy. Phase-contrast micrographs and migration paths of two representative cells are shown at 1-h intervals. Green arrowheads indicate lamellae extending in the direction of cell movement. Yellow arrowheads show lamellar protrusions in other directions. Green arrows in the diagrams to the right of the micrographs indicate the direction and distance of cell movement each hour. (B and C) Quantification of Rac-dependent lamellae in an axial versus a peripheral location. The main cell axis was determined as the longest distance between the ends of each cell (continuous red line in the image of a cell stained for F-actin). Two zones were defined as follows: “axial” was within 20° of the main axis (area between the dashed white lines) and “peripheral” was anything lateral to this zone. The length of total membrane in all lamellae of each cell and the number of lamellae per cell were determined for axial (green) compared with peripheral (yellow) lamellae in cells transfected with control (Contr) and Rac1 (Rac1) siRNA and presented as mean values ± SEM. Bar, 20 μm.
Mentions: The cell biological mechanism underlying this Rac-dependent regulation of migration became apparent from time-lapse video recordings, where each change in the direction of migration was accompanied by a change in the leading edge of the cell: a new lamella elsewhere on the cell became dominant (Fig. 5 A). In contrast, cells with reduced active Rac were more elongated and had lamellae confined to only one or both ends of the cell. We tested the hypothesis that Rac1 regulates peripheral versus such axial lamellae by quantifying these locomotory structures. Staining of F-actin with phalloidin allowed us to identify morphologically distinct, ruffling lamellae (Wu et al., 2003; Fig. S4, available at http://jcb.org/cgi/content/full/jcb.200503152.DC1). The presence of lamellae at an end of the long axis of the cell (within 20° of the longest dimension of each cell) was compared with the number of more peripheral lamellae located outside of this central zone (Fig. 5 B). Knockdown of Rac1 strongly inhibited only the formation of peripheral lamellae. There were threefold fewer peripheral lamellae per cell and a fivefold reduction in lamellar membrane; i.e., the total length of cell membrane associated with these structures per cell (Fig. 5 C). In contrast, there were no detectable effects of Rac reduction on lamellae located at the ends of the long axis of cells, as determined both by the number of lamellae per cell and by the total length of lamella membrane (Fig. 5 C). As a direct consequence of this selective loss of peripheral lamellae, the total number of lamellae per cell was reduced by half, and overall lamella length was decreased by 57% in human fibroblasts with Rac1 knockdown and by 66% in the integrin mutant cells (Table I). Restoration of Rac activity levels in the integrin mutant cells by expression of activated Akt restored normal numbers of lamellae and lamellar length, whereas overexpression of activated Rac1 enhanced numbers of lamellae and length accompanied by an elevation of random motility as reflected by a low D/T ratio (compare Table I and Fig. 1, D and E, with Fig. S1 C and Fig. S3 D). Collectively, these results demonstrate that active Rac induces random motility by promoting peripheral lamellae oriented in directions different from the direction of migration along the main cell axis.

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