pubmed.ncbi.nlm.nih.gov

Flow-induced endothelial cell alignment requires the RhoGEF Trio as a scaffold protein to polarize active Rac1 distribution - PubMed

  • ️Sun Jan 01 2017

Flow-induced endothelial cell alignment requires the RhoGEF Trio as a scaffold protein to polarize active Rac1 distribution

Jeffrey Kroon et al. Mol Biol Cell. 2017.

Abstract

Endothelial cells line the lumen of the vessel wall and are exposed to flow. In linear parts of the vessel, the endothelial cells experience laminar flow, resulting in endothelial cell alignment in the direction of flow, thereby protecting the vessel wall from inflammation and permeability. In order for endothelial cells to align, they undergo rapid remodeling of the actin cytoskeleton by local activation of the small GTPase Rac1. However, it is not clear whether sustained and local activation of Rac1 is required for long-term flow-induced cell alignment. Using a FRET-based DORA Rac1 biosensor, we show that local Rac1 activity remains for 12 h upon long-term flow. Silencing studies show that the RhoGEF Trio is crucial for keeping active Rac1 at the downstream side of the cell and, as a result, for long-term flow-induced cell alignment. Surprisingly, Trio appears to be not involved in flow-induced activation of Rac1. Our data show that flow induces Rac1 activity at the downstream side of the cell in a Trio-dependent manner and that Trio functions as a scaffold protein rather than a functional GEF under long-term flow conditions.

© 2017 Kroon et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

PubMed Disclaimer

Figures

FIGURE 1:
FIGURE 1:

Active Rac1 is required for flow-induced alignment. (A) Left, time-lapse Venus/Cer3 ratio images of the Rac1 DORA biosensor simultaneously recorded with an epifluorescence microscope, showing spatiotemporal Rac1 activation under static conditions or upon flow (arrowheads; total 12 h; arrow shows direction of flow; flow speed, 10 dynes/cm2). Direction of flow is from top to bottom. Bar, 25 μm. Calibration bar (LUT) shows Rac1 activation (red) relative to basal Rac1 activity (blue). Right, activation ratio of the Rac1 biosensor in time. Top, static conditions; bottom, flow conditions. Data are mean of three independent experiments ± SEM. Significance compared with 0 h. *p < 0.05, **p < 0.01. (B) Rac1 activity measured with G-LISA at different shear stress times (30 min and 1, 2, 6, and 12 h). *p < 0.05. (C) FRET ratio measured in upstream (red) and downstream (green) sides of the cell upon the induction of flow. Rac1 activity was particularly detected at the downstream side. Data are mean of three independent experiments ± SEM. Significance compared with 0 h. *p < 0.05; **p < 0.01; ****p < 0.001. (D) Left, inhibition of Rac1 activity by EHT 1864 blocks alignment under flow, whereas solvent control-treated ECs are aligned in the direction of flow. Note that the inhibitor was present throughout the experiment due to the closed system used for long-term flow experiments. Right, percentage of aligned cells under static and flow conditions for both EHT 1864–treated and solvent-treated Ctrl ECs. ECs orientated with a 0–45° angle are quantified as being aligned. Data are mean of three independent experiments ± SEM. ***p < 0.001. Bar, 25 μm. (E) Left, long-term flow results in linearized VE-cadherin–based cell–cell junctions. F-actin in red and VE-cadherin in green. ROI, region of interest. Bar, 25 μm. Right, junction linearization index. Per experiment, three fields of view were quantified for junction linearization after 12 h of 10 dynes/cm2 compared with 12 h of static conditions. Data are mean of three independent experiments ± SEM. *p < 0.05. (F) Resistance measurements using ECIS under long-term flow conditions show an increase in monolayer integrity under long-term flow conditions (10 dynes/cm2; green), whereas the resistance did not change under static (red) conditions. Data are mean of three independent experiments ± SEM. *p < 0.05.

FIGURE 2:
FIGURE 2:

Trio silencing inhibits flow-induced EC alignment. (A) Left, HUVECs treated with Ctrl and Trio shRNA (shCtrl and shTrio) were applied to flow for 12 h. Direction of flow is from top to bottom. Trio-deficient ECs failed to align. Bar, 25 μm. Middle, quantification of EC alignment upon flow vs. static conditions. Cells orientated between 0 and 45° are quantified as aligned. Data are mean of three independent experiments ± SEM. *p < 0.05. Right, Trio depletion with shRNA analyzed by Western blotting; actin is used as loading control. (B) Magnification of EC–cell junctions. Flow induces linear junction (open arrowhead), marked by VE-cadherin in green and F-actin in red. Depletion of Trio (shTrio) results in unstable, zipper-like junctions (closed arrowheads). Bar, 25 μm. (C) Resistance measurements using ECIS under flow conditions as indicated show that flow promotes EC resistance in time (green), whereas ECs depleted for Trio failed to increase flow-induced barrier resistance in time. Data are mean of three independent experiments ± SEM. *p < 0.05; **p < 0.01.

FIGURE 3:
FIGURE 3:

The N-terminal part of Trio rescues alignment and loss in resistance in Trio-deficient ECs. (A) Schematic overview of shRNA-insensitive GFP-tagged mutants GFP-TrioN, GFP-TrioGEF1, and GFP-TrioGEF2. Trio consists of three catalytic domains. GEF domain 1 activates Rac1 and RhoG, and GEF domain 2 activates RhoA and a serine/threonine kinase domain at the C-terminus. (B) Left, immunofluorescence staining of shTrio-treated ECs and overexpression of GFP-TrioGEF1, GFP-TrioGEF2, and GFP-TrioN and were subjected to 12 h of flow. Direction of flow is from top to bottom. GFP is shown in green, VE-cadherin in red, and F-actin in white. Right, quantification of aligned cells upon rescue of Trio expression indicates that TrioN expression rescues flow-induced alignment. Data are mean of three independent experiments ± SEM. *p < 0.05. (C) ECIS under flow was used to measure the EC monolayer resistance in control and Trio-knockdown conditions. Normalized resistance after 12 h of flow. Data are mean of three independent experiments ± SEM. *p < 0.05. (D) Western blot analysis confirmed the knockdown of Trio and subsequent overexpression of GFP-TrioN. VE-cadherin expression is not affected; actin is shown as loading control.

FIGURE 4:
FIGURE 4:

The activity of TrioGEF1 is not necessary for flow-induced alignment. (A) Left, TrioGEF1 activity was blocked by ITX3. Inhibition of GEF1 activity does not interfere with flow-induced alignment. VE-cadherin is shown in green and F-actin in red. ROI shows zoom of EC–cell junction region. Direction of flow is from top to bottom. Bar, 25 μm. Right, quantification of EC alignment. Data are mean of three independent experiments ± SEM. NS, not significant. (B) GFP-tagged Trio constructs were transfected in HEK293 cells and subjected to biochemical Rac1 and RhoG pull-down assays. GFP-TrioN-GEF N1406A/D1407A mutant (cat. dead) failed to induce Rac1 (GTP; top) and RhoG (GTP; third panel) activity, whereas GFP-TrioGEF1 and GFP-TrioN did. Second and fourth panels show Rac1 and RhoG protein expression as input controls. Bottom, expression of GFP-tagged constructs in cell lysates. (C) shTrio-tagRFP ECs were transduced with GFP-TrioN-N1406A/D1407A (cat. dead) and subjected to 12 h of flow. Rescue of Trio expression with GFP-TrioN catalytic-dead mutant rescued flow-induced alignment. Data are mean of three independent experiments ± SEM. *p < 0.05. (D) Data obtained from Rac1 G-LISA experiments show that flow (measured at different time points as indicated) increases Rac1 activation (Rac1.GTP) in both control (shCtrl) and Trio-deficient (shTrio) cells. Significance is indicated time point compared with 0 h. *p < 0.05.

FIGURE 5:
FIGURE 5:

Flow promotes Trio immobilization at junction regions and Trio colocalization with VE-cadherin. (A) Left, ECs were transfected with GFP-TrioFL and subjected to flow (12 h) or left untreated. Flow induces colocalization of GFP-TrioFL (green) with VE-cadherin (white). Red, F-actin. Right, fluorescence intensity of the bar (6 μm in length) on the main figure, showing increased colocalization between TrioFL (green) and VE-cadherin (red). Bar, 25 μm. (B) Pixel overlap between VE-cadherin and GFP-TrioFL under static conditions or flow (12 h) conditions. **p < 0.01. (C) FRAP was performed on GFP-Trio under static or flow (12 h) conditions at junctional regions or cytosolic areas, as indicated. Flow increases the immobile fraction of Trio at junctions, whereas no difference was detected in the cytosol. VE-cadherin–GFP FRAP analysis showed no difference in mobility under static and flow conditions. Data are mean of three independent experiments ± SEM. ***p < 0.001. ns, not significant.

FIGURE 6:
FIGURE 6:

Trio regulates the localization of active Rac1. (A) HUVECs were first transduced with shRNA targeting Trio containing an RFP tag (shTrio-tagRFP) and subsequently transfected with the FRET-based DORA Rac1 biosensor. Right, differential interference contrast (DIC). Bar, 25 μm. (B) Top, ECs were subsequently exposed to flow (12 h), and Rac1 activity and localization were monitored. Calibration bar (LUT) shows Rac1 activation (red) relative to basal Rac1 activity (blue). Bottom, quantification of ratiometric imaging analysis (Venus/Cer3) of the whole cell upon exposure to flow. Even in the absence of Trio, flow rapidly activates Rac1 at 30 min, followed by a decline and a second activation increase.*p < 0.05; **p < 0.01. (C) Activation ratio of the Rac1 biosensor in time at the downstream (green) and upstream (red) sides of the EC side of the cell. No significance is calculated between downstream and upstream regions for Rac1 activation. However, overall Rac1 activity is increased significantly compared with 0-h time point. Data are mean of three independent experiments ± SEM. *p < 0.05.

Similar articles

Cited by

References

    1. Ballermann BJ, Dardik A, Eng E, Liu A. Shear stress and the endothelium. Kidney Int Suppl. 1998;67:S100–S108. - PubMed
    1. Blangy A, Vignal E, Schmidt S, Debant A, Gauthier-Rouviere C, Fort P. TrioGEF1 controls Rac- and Cdc42-dependent cell structures through the direct activation of rhoG. J Cell Sci. 2000;113:729–739. - PubMed
    1. Boon RA, Leyen TA, Fontijn RD, Fledderus JO, Baggen JM, Volger OL, van Nieuw Amerongen GP, Horrevoets AJ. KLF2-induced actin shear fibers control both alignment to flow and JNK signaling in vascular endothelium. Blood. 2010;115:2533–2542. - PubMed
    1. Bouquier N, Vignal E, Charrasse S, Weill M, Schmidt S, Leonetti JP, Blangy A, Fort P. A cell active chemical GEF inhibitor selectively targets the Trio/RhoG/Rac1 signaling pathway. Chem Biol. 2009;16:657–666. - PubMed
    1. Chappell DC, Varner SE, Nerem RM, Medford RM, Alexander RW. Oscillatory shear stress stimulates adhesion molecule expression in cultured human endothelium. Circ Res. 1998;82:532–539. - PubMed

MeSH terms

Substances

LinkOut - more resources