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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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Effects of RhoA, Rac1, Cdc42, and inhibitors on cell displacement. A shows trajectories of untreated (control) cells (left plot), cells expressing N19RhoA (middle plot) and cells pretreated with Y-27632 (right plot) under static conditions. The cells were tracked for 5 h in static conditions. B and C show total cell displacement in static conditions (B) and under shear stress (C). Total cell displacement is a percentage of the total number of cells that migrated over a preset horizon of 50 μm during 5 h of experiment (n = 90). In A, each plot shows trajectories of 30 endothelial cells in static conditions.
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fig8: Effects of RhoA, Rac1, Cdc42, and inhibitors on cell displacement. A shows trajectories of untreated (control) cells (left plot), cells expressing N19RhoA (middle plot) and cells pretreated with Y-27632 (right plot) under static conditions. The cells were tracked for 5 h in static conditions. B and C show total cell displacement in static conditions (B) and under shear stress (C). Total cell displacement is a percentage of the total number of cells that migrated over a preset horizon of 50 μm during 5 h of experiment (n = 90). In A, each plot shows trajectories of 30 endothelial cells in static conditions.

Mentions: To study the ability of the cells to move effectively in one direction, we determined the percentage of cells that had translocated a distance of 50 μm or more from their starting point in each experiment. Because over 20% of cells in all experimental conditions managed to migrate over this distance, it was chosen as suitable for comparisons of cell displacement. For groups of cells that migrate at similar speeds, cell displacement can be used as a measure for persistence of cell movement as only cells that migrate persistently in one direction manage to migrate over a preset horizon. In static conditions, HUVECs frequently changed direction and only 47% of cells migrated over 50 μm in 5 h (Fig. 8, A and B) . Cells expressing N19RhoA or treated with Y-27632 showed a significant increase in total cell displacement (Fig. 8 B; P < 0.01), changed direction less frequently than control cells, and had straighter trajectories, particularly in cells treated with Y-27632 (Fig. 8 A).


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)

Effects of RhoA, Rac1, Cdc42, and inhibitors on cell displacement. A shows trajectories of untreated (control) cells (left plot), cells expressing N19RhoA (middle plot) and cells pretreated with Y-27632 (right plot) under static conditions. The cells were tracked for 5 h in static conditions. B and C show total cell displacement in static conditions (B) and under shear stress (C). Total cell displacement is a percentage of the total number of cells that migrated over a preset horizon of 50 μm during 5 h of experiment (n = 90). In A, each plot shows trajectories of 30 endothelial cells in static conditions.
© Copyright Policy
Related In: Results  -  Collection

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

fig8: Effects of RhoA, Rac1, Cdc42, and inhibitors on cell displacement. A shows trajectories of untreated (control) cells (left plot), cells expressing N19RhoA (middle plot) and cells pretreated with Y-27632 (right plot) under static conditions. The cells were tracked for 5 h in static conditions. B and C show total cell displacement in static conditions (B) and under shear stress (C). Total cell displacement is a percentage of the total number of cells that migrated over a preset horizon of 50 μm during 5 h of experiment (n = 90). In A, each plot shows trajectories of 30 endothelial cells in static conditions.
Mentions: To study the ability of the cells to move effectively in one direction, we determined the percentage of cells that had translocated a distance of 50 μm or more from their starting point in each experiment. Because over 20% of cells in all experimental conditions managed to migrate over this distance, it was chosen as suitable for comparisons of cell displacement. For groups of cells that migrate at similar speeds, cell displacement can be used as a measure for persistence of cell movement as only cells that migrate persistently in one direction manage to migrate over a preset horizon. In static conditions, HUVECs frequently changed direction and only 47% of cells migrated over 50 μm in 5 h (Fig. 8, A and B) . Cells expressing N19RhoA or treated with Y-27632 showed a significant increase in total cell displacement (Fig. 8 B; P < 0.01), changed direction less frequently than control cells, and had straighter trajectories, particularly in cells treated with Y-27632 (Fig. 8 A).

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