JOURNAL OF CELLULAR PHYSIOLOGY 201:55–70 (2004) Microtubule Disassembly Induces Cytoskeletal Remodeling and Lung Vascular Barrier Dysfunction: Role of Rho-Dependent Mechanisms ANNA A. BIRUKOVA, 1 KSENYA SMUROVA, 1,2 KONSTANTIN G. BIRUKOV, 1 PETER USATYUK, 1 FENG LIU, 1 KOZO KAIBUCHI, 3 ANILA RICKS-CORD, 1 VISWANATHAN NATARAJAN, 1 IRINA ALIEVA, 2 JOE G.N. GARCIA, 1 AND ALEXANDER D. VERIN 1 * 1 Division of Pulmonary and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 2 Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Vorobievy Gory, Russia 3 Department of Cell Pharmacology, Nagoya University Graduate School of Medicine, Nagoya, Japan Barrier dysfunction of pulmonary endothelial monolayer is associated with dra- matic cytoskeletal reorganization, activation of actomyosin contractility, and gap formation. The linkage between the microtubule (MT) network and the contractile cytoskeleton has not been fully explored, however, clinical observations suggest that intravenous administration of anti-cancer drugs and MT inhibitors (such as the vinca alkaloids) can lead to the sudden development of pulmonary edema in breast cancer patients. In this study, we investigated the crosstalk between MT and actomyosin cytoskeleton and characterized specific molecular mechanisms of endothelial cells (EC) barrier dysfunction induced by MT inhibitor nocodazole (ND). Our results demonstrate that MT disassembly by ND induced rapid decreases in transendothelial electrical resistance (TER) and actin cytoskeletal remodeling, indicating EC barrier dysfunction. These effects involved ND-induced activation of Rho GTPase. Rho-mediated activation of its downstream target, Rho-kinase, induced phosphorylation of Rho-kinase effector EC MLC phosphatase (MYPT1) at Thr 696 and Thr 850 resulting in MYPT1 inactivation. Phosphatase inhibition leaded to accumulation of diphospho-MLC, which induced acto-myosin polymerization, stress fiber formation and gap formation. Inhibition of Rho-kinase by Y27632 abolished ND-induced MYPT1 phosphorylation, MLC phosphorylation, and stress fiber formation. In addition, MT preservation via the MT stabilizer paclitaxel, Rho inhibition (via C3 exotoxin, or dominant negative (DN)-Rho, or DN-Rho-kinase) attenuated ND-induced TER decreases, stress fiber formation and MLC phosphor- ylation. Collectively, our results demonstrate a leading role for Rho-dependent mechanisms in crosstalk between the MT and actomyosin cytoskeleton, and suggest Rho-kinase and MYPT1 as major Rho effectors mediating pulmonary EC barrier disruption in response to ND-induced MT disassembly. J. Cell. Physiol. 201: 55–70, 2004. ß 2004 Wiley-Liss, Inc. A key vascular endothelial cells (EC) function is to regulate the exchanges across the capillary wall between circulating blood and the interstitial fluid. Reorganization of the endothelial cytoskeleton, which is composed of actin filaments, microtubules (MT), and intermediate filaments, leads to alteration in cell shape and provides a structural basis for increase of vascular permeability, which has been implicated in the patho- genesis of many diseases including asthma, sepsis, and the acute lung injury (Garcia et al., 1995; Lum and Malik, 1996; Dudek and Garcia, 2001). While the importance of MT component in the maintenance of EC barrier function remains largely unexplored, intra- venous administration of anti-cancer drugs and MT ß 2004 WILEY-LISS, INC. Contract grant sponsor: National Heart, Lung, and Blood Institutes; Contract grant numbers: HL67307, HL68062, HL58064; Contract grant sponsor: American Heart Association (to ADV); Contract grant sponsor: Russian Foundation of Biomedical Research (to IBA); Contract grant numbers: RFBR- 03-04-48035, MD-244.2003.04. *Correspondence to: Alexander D. Verin, Division of Pulmonary and Critical Care Medicine, Johns Hopkins University School of Medicine, 5200 Eastern Avenue, MFL Center Tower, Room 676, Baltimore, MD 21224. E-mail: averin1@jhmi.edu Received 1 October 2003; Accepted 12 December 2003 DOI: 10.1002/jcp.20055