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Shear stress-induced endothelial cell polarization is mediated by Rho and Rac but not Cdc42 or PI 3-kinases.

Wojciak-Stothard B, Ridley AJ - J. Cell Biol. (2003)

Bottom Line: Instead, Rho and Rac1 regulated directionality of cell movement.Inhibition of Rho or Rho-kinase did not affect the cell speed but significantly increased cell displacement.Our results show that endothelial cells reorient in response to shear stress by a two-step process involving Rho-induced depolarization, followed by Rho/Rac-mediated polarization and migration in the direction of flow.

View Article: PubMed Central - PubMed

Affiliation: Ludwig Institute for Cancer Research, Royal Free and University College School of Medicine, 91 Riding House St., London W1W 7BS, UK. beata@ludwig.ucl.ac.uk

ABSTRACT
Shear stress induces endothelial polarization and migration in the direction of flow accompanied by extensive remodeling of the actin cytoskeleton. The GTPases RhoA, Rac1, and Cdc42 are known to regulate cell shape changes through effects on the cytoskeleton and cell adhesion. We show here that all three GTPases become rapidly activated by shear stress, and that each is important for different aspects of the endothelial response. RhoA was activated within 5 min after stimulation with shear stress and led to cell rounding via Rho-kinase. Subsequently, the cells respread and elongated within the direction of shear stress as RhoA activity returned to baseline and Rac1 and Cdc42 reached peak activation. Cell elongation required Rac1 and Cdc42 but not phosphatidylinositide 3-kinases. Cdc42 and PI3Ks were not required to establish shear stress-induced polarity although they contributed to optimal migration speed. Instead, Rho and Rac1 regulated directionality of cell movement. Inhibition of Rho or Rho-kinase did not affect the cell speed but significantly increased cell displacement. Our results show that endothelial cells reorient in response to shear stress by a two-step process involving Rho-induced depolarization, followed by Rho/Rac-mediated polarization and migration in the direction of flow.

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The effects of dominant negative RhoA, Rac1, and Cdc42 and inhibitors on the direction of cell migration. Cell trajectories during a 12-h experiment are shown in static conditions (A and C) or under shear stress (B and D). The starting point of each cell trajectory is plotted at the intersection of the X and Y axes (C and D). Circular histograms (E and F) show the proportion of cells migrating into each of 20 equal segments, measured when each cell had migrated 50 μm from its starting point. Cells that migrated <50 μm are excluded from this analysis. Arrow indicates the mean direction of the cell population where this is significant (P values indicate significance), and the green shaded areas mark the 95% confidence intervals of statistical significance, as calculated using a Rayleigh test. The direction of shear stress in all cases is toward the bottom of the histograms (B, arrow). For each condition, 90 cells in three independent experiments were analyzed.
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fig7: The effects of dominant negative RhoA, Rac1, and Cdc42 and inhibitors on the direction of cell migration. Cell trajectories during a 12-h experiment are shown in static conditions (A and C) or under shear stress (B and D). The starting point of each cell trajectory is plotted at the intersection of the X and Y axes (C and D). Circular histograms (E and F) show the proportion of cells migrating into each of 20 equal segments, measured when each cell had migrated 50 μm from its starting point. Cells that migrated <50 μm are excluded from this analysis. Arrow indicates the mean direction of the cell population where this is significant (P values indicate significance), and the green shaded areas mark the 95% confidence intervals of statistical significance, as calculated using a Rayleigh test. The direction of shear stress in all cases is toward the bottom of the histograms (B, arrow). For each condition, 90 cells in three independent experiments were analyzed.

Mentions: As described above (Fig. 6), in the absence of shear stress endothelial cells in sparse cultures moved randomly, whereas cells under shear stress migrated predominantly within the direction of the flow (Fig. 7 , A–D). During chemotaxis, directionality has been shown to depend on Cdc42 and PI3Ks (Wang et al., 2002). In contrast, directional migration induced by shear stress was unaffected by either N17Cdc42 or LY294002/wortmannin (Fig. 7 E). Instead, orientation of cell movement was abolished in cells expressing N17Rac1 or N19RhoA, or cells treated with Y-27632 or ML-7 (Fig. 7 E), correlating with their effect on shear stress–induced cell alignment.


Shear stress-induced endothelial cell polarization is mediated by Rho and Rac but not Cdc42 or PI 3-kinases.

Wojciak-Stothard B, Ridley AJ - J. Cell Biol. (2003)

The effects of dominant negative RhoA, Rac1, and Cdc42 and inhibitors on the direction of cell migration. Cell trajectories during a 12-h experiment are shown in static conditions (A and C) or under shear stress (B and D). The starting point of each cell trajectory is plotted at the intersection of the X and Y axes (C and D). Circular histograms (E and F) show the proportion of cells migrating into each of 20 equal segments, measured when each cell had migrated 50 μm from its starting point. Cells that migrated <50 μm are excluded from this analysis. Arrow indicates the mean direction of the cell population where this is significant (P values indicate significance), and the green shaded areas mark the 95% confidence intervals of statistical significance, as calculated using a Rayleigh test. The direction of shear stress in all cases is toward the bottom of the histograms (B, arrow). For each condition, 90 cells in three independent experiments were analyzed.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172912&req=5

fig7: The effects of dominant negative RhoA, Rac1, and Cdc42 and inhibitors on the direction of cell migration. Cell trajectories during a 12-h experiment are shown in static conditions (A and C) or under shear stress (B and D). The starting point of each cell trajectory is plotted at the intersection of the X and Y axes (C and D). Circular histograms (E and F) show the proportion of cells migrating into each of 20 equal segments, measured when each cell had migrated 50 μm from its starting point. Cells that migrated <50 μm are excluded from this analysis. Arrow indicates the mean direction of the cell population where this is significant (P values indicate significance), and the green shaded areas mark the 95% confidence intervals of statistical significance, as calculated using a Rayleigh test. The direction of shear stress in all cases is toward the bottom of the histograms (B, arrow). For each condition, 90 cells in three independent experiments were analyzed.
Mentions: As described above (Fig. 6), in the absence of shear stress endothelial cells in sparse cultures moved randomly, whereas cells under shear stress migrated predominantly within the direction of the flow (Fig. 7 , A–D). During chemotaxis, directionality has been shown to depend on Cdc42 and PI3Ks (Wang et al., 2002). In contrast, directional migration induced by shear stress was unaffected by either N17Cdc42 or LY294002/wortmannin (Fig. 7 E). Instead, orientation of cell movement was abolished in cells expressing N17Rac1 or N19RhoA, or cells treated with Y-27632 or ML-7 (Fig. 7 E), correlating with their effect on shear stress–induced cell alignment.

Bottom Line: Instead, Rho and Rac1 regulated directionality of cell movement.Inhibition of Rho or Rho-kinase did not affect the cell speed but significantly increased cell displacement.Our results show that endothelial cells reorient in response to shear stress by a two-step process involving Rho-induced depolarization, followed by Rho/Rac-mediated polarization and migration in the direction of flow.

View Article: PubMed Central - PubMed

Affiliation: Ludwig Institute for Cancer Research, Royal Free and University College School of Medicine, 91 Riding House St., London W1W 7BS, UK. beata@ludwig.ucl.ac.uk

ABSTRACT
Shear stress induces endothelial polarization and migration in the direction of flow accompanied by extensive remodeling of the actin cytoskeleton. The GTPases RhoA, Rac1, and Cdc42 are known to regulate cell shape changes through effects on the cytoskeleton and cell adhesion. We show here that all three GTPases become rapidly activated by shear stress, and that each is important for different aspects of the endothelial response. RhoA was activated within 5 min after stimulation with shear stress and led to cell rounding via Rho-kinase. Subsequently, the cells respread and elongated within the direction of shear stress as RhoA activity returned to baseline and Rac1 and Cdc42 reached peak activation. Cell elongation required Rac1 and Cdc42 but not phosphatidylinositide 3-kinases. Cdc42 and PI3Ks were not required to establish shear stress-induced polarity although they contributed to optimal migration speed. Instead, Rho and Rac1 regulated directionality of cell movement. Inhibition of Rho or Rho-kinase did not affect the cell speed but significantly increased cell displacement. Our results show that endothelial cells reorient in response to shear stress by a two-step process involving Rho-induced depolarization, followed by Rho/Rac-mediated polarization and migration in the direction of flow.

Show MeSH
Related in: MedlinePlus