The Integrin-Ligand Interaction Regulates Adhesion and Migration through a Molecular Clutch Lingfeng Chen 1 , Miguel Vicente-Manzanares1,2, Laurent Potvin-Trottier3, Paul W. Wiseman3,4, Alan Rick Horwitz1* Abstract 1 Department of Cell Biology, University of Virginia, Charlottesville, Virginia, United States of America, 2 School of Medicine at the Hospital de la Princesa, Universidad Autonoma de Madrid, Madrid, Spain, 3 Department of Physics, McGill University, Montreal, Quebec, Canada, 4 Department of Chemistry, McGill University, Montreal, Quebec, Canada Adhesive and migratory behavior can be cell type, integrin, and substrate dependent. We have compared integrin and substrate differences using three integrin receptors: a5b1, a6b1, and aLb2 expressed in a common cell type, CHO.B2 cells, which lack integrin a subunits, as well as in different cell types that express one or more of these integrins. We find that CHO.B2 cells expressing either a6b1 or aLb2 integrins migrate and protrude faster and are more directionally persistent on laminin or ICAM-1, respectively, than CHO.B2 cells expressing a5b1 on fibronectin. Despite rapid adhesion maturation and the presence of large adhesions in both the a6b1- and aLb2-expressing cells, they display robust tyrosine phosphorylation. In addition, whereas myosin II regulates adhesion maturation and turnover, protrusion rates, and polarity in cells migrating on fibronectin, surprisingly, it does not have comparable effects in cells expressing a6b1 or aLb2. This apparent difference in the integration of myosin II activity, adhesion, and migration arises from alterations in the ligand–integrin–actin linkage (molecular clutch). The elongated adhesions in the protrusions of the a6b1-expressing cells on laminin or the aLb2- expressing cells on ICAM-1 display a novel, rapid retrograde flux of integrin; this was largely absent in the large adhesions in protrusions of a5b1-expressing cells on fibronectin. Furthermore, the force these adhesions exert on the substrate in protrusive regions is reduced compared to similar regions in a5-expressing cells, and the adhesion strength is reduced. This suggests that intracellular forces are not efficiently transferred from actomyosin to the substratum due to altered adhesion strength, that is, avidity, affinity, or the ligand-integrin-actin interaction. Finally, we show that the migration of fast migrating leukocytes on fibronectin or ICAM-1 is also largely independent of myosin II; however, their adhesions are small and do not show retrograde fluxing suggesting other intrinsic factors determine their migration differences. Citation: Chen L, Vicente-Manzanares M, Potvin-Trottier L, Wiseman PW, Horwitz AR (2012) The Integrin-Ligand Interaction Regulates Adhesion and Migration through a Molecular Clutch. PLoS ONE 7(7): e40202. doi:10.1371/journal.pone.0040202 Editor: Erik H. J. Danen, Leiden University, The Netherlands Received February 16, 2012; Accepted June 4, 2012; Published July 6, 2012 Copyright: 2012 Chen 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: ARH was supported by National Institutes of Health grants GM23244 and the Cell Migration Consortium (U54 GM064346). MVM is supported by the Ramon y Cajal program (RYC-2010-06094) from the Spanish Ministerio de Econom´ıa y Competividad (MINECO). PWW acknowledges funding support from the Natural Sciences and Engineering Council of Canada (NSERC) and the Canadian Institutes of Health Research. LPT acknowledges fellowship support from Fonds que´be´cois de la recherche sur la nature et les technologies (FQRNT) and NSERC. 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: Horwitz@virginia.edu Introduction Cell migration is a complex process comprised of multiple integrated and regulated steps [1,2]. During migration, actin polymerization produces the forces that drive protrusion and retrograde flow of F-actin at the leading edge [3–5]. These forces are coupled to the substratum through integrin-based adhesions, which serve as traction points over which the cell moves as well as sources of migration-related regulatory signals [2,6–8]. The efficiency of force transmission from the force generating systems in the cell to the substratum depends on the efficiency of a molecular clutch that connects adhesions to actin filaments [9,10]. Myosin II has emerged as a critical regulator and integrator of cell migration [11]. By organizing the actomyosin cytoskeleton and generating contractile forces, it determines front-back polarity, regulates adhesion and the signals they produce, and mediates rear retraction. It also integrates the spatially separated processes that comprise migration and interprets the pliability of the substratum through a poorly understood signaling loop [11,12]. While a picture of adhesion function and the pivotal role of myosin II and actin polymerization in cell migration are clear [2,8], most of the data have been generated in fibroblasts adhering to fibronectin or vitronectin using either the a5b1 or avb3 integrins. However, other cell types, integrins and substrates have not been studied in comparable detail and may be different, since cells utilizing them have different migratory properties and adhesions. For example, many cells migrate on laminin, a process mediated mainly by the a6b1 integrin [13]. Also, leukocyte migration on ICAM-1 is characterized by high cellular speed, short and rapidly extending protrusions, and small, almost undetectable adhesions [14]. To investigate migration mechanisms using different integrins and substrates, we have used CHO.B2 cells, a CHO cell variant that expresses the integrin b1 subunit but almost no a subunit and therefore does not adhere or migrate on substrates like fibronectin PLoS ONE | www.plosone.org 1 July 2012 | Volume 7 | Issue 7 | e40202