The Role of GEF Trio in ICAM-1-mediated leukocyte transendothelial migration Jaap D. van Buul 1 , Jos van Rijssel 1 , Floris PJ van Alphen 1 , Judy Geissler 2 , Mark Hoogenboezem 1 , and Peter L. Hordijk 1 1 Dept. of Molecular Cell Biology, 2 Dept. of Blood Cell Research, Sanquin Research and Landsteiner Laboratory, University of Amsterdam, Amsterdam, The Netherlands During inflammation, leukocytes transmigrate across the endothelium towards the site of injury. This process of extravasation requires integrin-mediated adhesion to endothelial adhesion receptors such as ICAM-1. Upon binding and clustering of ICAM-1, the small GTPases RhoA and Rac1 are activated, which induce endothelial cell shape changes to allow passage of transmigrating leukocytes. In addition, we demonstrated that another Rho family GTPase, RhoG, is activated after ICAM-1 engagement, controlling the formation of endothelial apical cup structures that surround adhering and transmigrating leukocytes. However, the signalling pathways that are triggered downstream of ICAM-1 leading to the activation of these small GTPases remain largely unclear. We have shown that the F-actin crosslinker protein Filamin interacts directly with the intracellular domain of ICAM-1 and controls ICAM-1-driven leukocyte transendothelial migration. The Filamin-interacting guanine nucleotide exchange factor (GEF) Trio is able to exhange both RhoG, Rac1 and RhoA and is therefore a potential candidate for playing a role in the downstream ICAM-1 signalling. Here we show preliminary data on the involvement of Trio in this process. Introduction  j.vanbuul@sanquin.nl; www.mcb.sanquin.nl ; www.debsociety.nl Conclusions: 5μm Trio is an approximately 350 kD protein comprising three enzymatic domains: a GEF domain specific for RhoG and Rac1 (Trio-D1), a GEF domain specific for RhoA (Trio-D2) and a serine kinase domain at the C-terminus. (A). Endogenous Trio protein of 350 kD is present in HUVEC, but also in brain (HCMEC) and microvascular (MVEC) ECs (B). Endogenous Trio is expressed in all tested cell lines as indicated. (C). Full-length GFP-Trio or (D). GFP-Trio-D1 with ICAM-1-mCherry overexpressed in HeLa cells co-colocalise around αICAM-1 antibody-coated 10 µm beads (arrow). Bar, 5 µm. A. C. Trio expression increases under inflammatory conditions B. (A). To mimic ICAM-1-mediated leukocyte adhesion on ECs, α-ICAM-1 antibody-coated beads were used. Activated Trio binds to nucleotide-free RhoG (RhoG-G15A) after adhesion of α-ICAM-1 antibody-coated beads on HUVEC. Lower panels show activation of RhoG after adhesion of αICAM-1 antibody-coated beads or full-length GFP-Trio. (B). ECs were cultured on Transwell-filters and treated with shRNA against Trio or CTRL. Neutrophils were allowed to migrate in time across the endothelial monolayers to fMLP. Reduced Trio expression promotes fMLP-induced neutrophil migration across EC-monolayer. (C). Permeability of endothelial monolayer was measured by the diffusion of a fluorescent marker Dextran 3000MW. Reduction of Trio expression induced the permeability of the EC-monolayer. (D). Efficient knock-down of Trio using shRNA constructs delivered into ECs using lentivirus. A. C. αICAM-1 antibody-coated beads induce activation of Trio Trio is expressed in endothelial cells and is upregulated under inflammatory conditions Full-length Trio and its first GEF domain (Trio D1) interact with the intracellular domain of ICAM-1 Full-length Trio and Trio-D1 co-localize with clustered ICAM-1 around α-ICAM-1 antibody-coated beads Adhesion of α-ICAM-1 antibody-coated beads induces activation of Trio Reduction of Trio promotes neutrophil transendothelial migration. Acknowledgements: We thank Philippe Fort and Anne Debant (CRBM, Montpellier, France) for supplying Trio plasmids, Betty Eipper (University of Connecticut Health Center, Farmington , USA) for Trio antibody and plasmids and Dirk Geerts (AMC Amsterdam, the Netherlands) for the shRNA constructs. This work was supported by the Netherlands Heart Foundation grant #2005T039 (JDvB Dekker fellow) and NWO-ZonMW-Veni grant #916.76.053 B. (A). Inflammatory stimuli TNF-α, IL1β and LPS, but not IFN-γ upregulate Trio protein expression in HUVEC. (B). TNF-α does not upregulate rio protein expression in HeLa, A549 or Cos7 cells. (C). TNF-α does not upregulate protein expression of other tested GEFs in HUVEC. (D). TNF-α upregulates Trio, but not Vav2 mRNA levels in HUVEC. N=4. A. B. C. D. Trio-FL and Trio-D1 interact with the intracellular domain of ICAM-1 Myc-Trio full-length (A) and Myc-Trio D1 (B) overexpressed in Cos7 cells interact with a peptide comprising the intracellular C-terminal domain of ICAM-1, but not with the intracellular domain of VCAM-1. Interaction of Filamin A is shown as positive control. A. B. Trio Structure Trio is expressed in endothelial cells SH3 C C C C C SH3 ICAM-1 VCAM-1 PDZ C C C C C C SH3 C C C C C SH3 SH3 C C C C C SH3 SH3 C C C C C SH3 SH3 C C C C C SH3 SH3 C C C C C SH3 SH3 C C C C C SH3 Clustering LFA-1/MAC-1 PDZ C C C C C C PDZ C C C C C C PDZ C C C C C C PDZ C C C C C C Clustering VLA-4 Leukocyte Endothelium RhoG Rac1 RhoA F-Actin Linking to Actin cytoskeleton Filamin Actinin ERM Cortactin Linking to Actin cytoskeleton RhoGEFs: Trio ICAM-1 Rac1 Leukocyte VE-cadherin Leukocyte Endotheel Endotheel Signaling… 0 300 600 900 1200 1500 1800 time in seconds 0 10 20 30 40 50 shCTRL + TNF shTrio + TNF shCTRL - TNF shTrio - TNF * * * * % Migrated Neutrophils shCtrl shTrio 0 10 20 30 (Thousands) Fluorescence (AU) D. Trio Vav2 0 1 2 3 4 0 hr TNF α 20 hr TNF α mRNA fold change opyright pr ster Bank. Copyright protected. F100 ed. F1000 Poster Bank. Copyright protected. F1000 Poster Ban pyright protected. F1000 Poster Bank. Copyright protected. F1000 Poster Bank. 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