Inside-Out Regulation of ICAM-1 Dynamics in TNF-a- Activated Endothelium Jaap D. van Buul1*, Jos van Rijssel1, Floris P. J. van Alphen1, Mark Hoogenboezem1, Simon Tol1, Kees A. Hoeben2, Jan van Marle2, Erik P. J. Mul1, Peter L. Hordijk1 1 Sanquin Research and Landsteiner Laboratory, Department of Molecular Cell Biology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands, 2 Department of Cell Biology and Histology, Academic Medical Center, Amsterdam, The Netherlands Abstract Background: During transendothelial migration, leukocytes use adhesion molecules, such as ICAM-1, to adhere to the endothelium. ICAM-1 is a dynamic molecule that is localized in the apical membrane of the endothelium and clusters upon binding to leukocytes. However, not much is known about the regulation of ICAM-1 clustering and whether membrane dynamics are linked to the ability of ICAM-1 to cluster and bind leukocyte integrins. Therefore, we studied the dynamics of endothelial ICAM-1 under non-clustered and clustered conditions. Principal Findings: Detailed scanning electron and fluorescent microscopy showed that the apical surface of endothelial cells constitutively forms small filopodia-like protrusions that are positive for ICAM-1 and freely move within the lateral plane of the membrane. Clustering of ICAM-1, using anti-ICAM-1 antibody-coated beads, efficiently and rapidly recruits ICAM-1. Using fluorescence recovery after photo-bleaching (FRAP), we found that clustering increased the immobile fraction of ICAM-1, compared to non-clustered ICAM-1. This shift required the intracellular portion of ICAM-1. Moreover, biochemical assays showed that ICAM-1 clustering recruited beta-actin and filamin. Cytochalasin B, which interferes with actin polymerization, delayed the clustering of ICAM-1. In addition, we could show that cytochalasin B decreased the immobile fraction of clustered ICAM-1-GFP, but had no effect on non-clustered ICAM-1. Also, the motor protein myosin-II is recruited to ICAM-1 adhesion sites and its inhibition increased the immobile fraction of both non-clustered and clustered ICAM-1. Finally, blocking Rac1 activation, the formation of lipid rafts, myosin-II activity or actin polymerization, but not Src, reduced the adhesive function of ICAM-1, tested under physiological flow conditions. Conclusions: Together, these findings indicate that ICAM-1 clustering is regulated in an inside-out fashion through the actin cytoskeleton. Overall, these data indicate that signaling events within the endothelium are required for efficient ICAM-1- mediated leukocyte adhesion. Citation: van Buul JD, van Rijssel J, van Alphen FPJ, Hoogenboezem M, Tol S, et al. (2010) Inside-Out Regulation of ICAM-1 Dynamics in TNF-a-Activated Endothelium. PLoS ONE 5(6): e11336. doi:10.1371/journal.pone.0011336 Editor: Carl-Philipp Heisenberg, Max Planck Institute of Molecular Cell Biology and Genetics, Germany Received March 17, 2010; Accepted June 1, 2010; Published June 28, 2010 Copyright: 2010 Van Buul et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work has been funded by the Dutch Heart Foundation (grant no. 2005T039) and NWO (The Netherlands Organisation for Scientific Research)- ZonMW (the Netherlands Organisation for Health Research and Development) Veni grant 916.76.053. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: j.vanbuul@sanquin.nl Introduction Leukocytes use integrins to firmly adhere to activated endothelium through adhesion molecules such as VCAM-1 and ICAM-1 prior to extravasation. Over the past years, the endothelium is increasingly recognized to actively contribute to the adhesion and transmigration of leukocytes [1]. Binding of leukocyte integrin aL/Mb2 induces ICAM-1 clustering, resulting in downstream signalling in the endothelium [2–5]. This intracellular signalling induced into the endothelium regulates rearrangement of the actin cytoskeleton. We and others have shown increased stress fiber formation after ICAM-1 antibody crosslinking [6,7]. These changes in the actin cytoskeleton imply an important role for Rho-like small GTPases [8]. In agreement with this notion, various studies have shown that RhoA, which induces stress fibers and contractility, is activated downstream from ICAM-1 clustering [6,9,10]. Blocking RhoA activity by C3 toxin or overexpression of a dominant negative mutant (RhoA- T19N) prevents antibody-mediated and monocyte-induced clus- tering of ICAM-1, monocyte adhesion and subsequent stress fiber formation [11]. These data indicate that active RhoA is involved in ICAM-1 function. In addition, upon clustering, either by antibody-mediated crosslinking or by adhesion of THP-1 cells, ICAM-1 is incorporated into detergent-insoluble membrane domains, i.e. lipid rafts [12]. Interestingly, this shift to the insoluble fraction is not inhibited by cytochalasin B, an inhibitor of actin polymerization, suggesting that this response is indepen- dent from RhoA. Pioneering work from Barreiro and co-workers showed that endothelial cells actively protrude sheets of membrane around an adherent leukocyte, referred to as docking structures [13]. These authors showed that several actin-binding proteins are present in these structures, such as vinculin, paxillin, alpha-actinin, VASP and ERM-proteins. From other reports, it became clear that the PLoS ONE | www.plosone.org 1 June 2010 | Volume 5 | Issue 6 | e11336