NEDD9 Stabilizes Focal Adhesions, Increases Binding to the Extra-Cellular Matrix and Differentially Effects 2D versus 3D Cell Migration Jessie Zhong1,2., Jaime B. Baquiran1., Navid Bonakdar3, Justin Lees1, Yu Wooi Ching 1 , Elena Pugacheva4, Ben Fabry3, Geraldine M. O’Neill1,2* 1 Children’s Cancer Research Unit, Kids Research Institute, The Children’s Hospital at Westmead, Westmead, New South Wales, Australia, 2 Discipline of Paediatrics and Child Health, The University of Sydney, Sydney, New South Wales, Australia, 3 Department of Physics, University of Erlangen-Nuremberg, Erlangen, Germany, 4 Mary Babb Randolph Cancer Center (MBRCC), West Virginia University, Morgantown, West Virginia, United States of America Abstract The speed of cell migration on 2-dimensional (2D) surfaces is determined by the rate of assembly and disassembly of clustered integrin receptors known as focal adhesions. Different modes of cell migration that have been described in 3D environments are distinguished by their dependence on integrin-mediated interactions with the extra-cellular matrix. In particular, the mesenchymal invasion mode is the most dependent on focal adhesion dynamics. The focal adhesion protein NEDD9 is a key signalling intermediary in mesenchymal cell migration, however whether NEDD9 plays a role in regulating focal adhesion dynamics has not previously been reported. As NEDD9 effects on 2D migration speed appear to depend on the cell type examined, in the present study we have used mouse embryo fibroblasts (MEFs) from mice in which the NEDD9 gene has been depleted (NEDD9 2/2 MEFs). This allows comparison with effects of other focal adhesion proteins that have previously been demonstrated using MEFs. We show that focal adhesion disassembly rates are increased in the absence of NEDD9 expression and this is correlated with increased paxillin phosphorylation at focal adhesions. NEDD92/2 MEFs have increased rates of migration on 2D surfaces, but conversely, migration of these cells is significantly reduced in 3D collagen gels. Importantly we show that myosin light chain kinase is activated in 3D in the absence of NEDD9 and is conversely inhibited in 2D cultures. Measurement of adhesion strength reveals that NEDD92/2 MEFs have decreased adhesion to fibronectin, despite upregulated a5b1 fibronectin receptor expression. We find that b1 integrin activation is significantly suppressed in the NEDD92/2, suggesting that in the absence of NEDD9 there is decreased integrin receptor activation. Collectively our data suggest that NEDD9 may promote 3D cell migration by slowing focal adhesion disassembly, promoting integrin receptor activation and increasing adhesion force to the ECM. Citation: Zhong J, Baquiran JB, Bonakdar N, Lees J, Ching YW, et al. (2012) NEDD9 Stabilizes Focal Adhesions, Increases Binding to the Extra-Cellular Matrix and Differentially Effects 2D versus 3D Cell Migration. PLoS ONE 7(4): e35058. doi:10.1371/journal.pone.0035058 Editor: Nils Cordes, Dresden University of Technology, Germany Received November 28, 2011; Accepted March 8, 2012; Published April 11, 2012 Copyright: 2012 Zhong 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 study was supported by the National Health and Medical Research (NHMRC) Australia grants 512551 and 632515 and New South Wales (NSW) Cancer Council grant RG09-16 to GO. JZ is the recipient of a post-graduate scholarship from the University of Sydney and a NSW Cancer Institute Scholar Award. 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: geraldine.oneill@health.nsw.gov.au . These authors contributed equally to this work. Introduction Cell adhesion is a prime determinant of the rate of cell migration speed on 2D surfaces [1]. This is a biphasic effect with both too much and too little adhesion resulting in decreased migration speed and persistence. In addition to the contribution that the concentration of extra-cellular matrix plays in cell speed, a role for focal adhesion dynamics has also emerged. At the focal adhesions, clusters of integrin receptors are bound on the external surface to extra-cellular matrix ligands and are associated with filaments of polymerized actin in the cytoplasm. The resulting tensile force generated at the focal adhesions in turn governs integrin activation and cell motility. Thus, the rate of focal adhesion assembly and disassembly contributes to 2D cell migration speed [2–6]. Investigations of cell migration have revealed a number of different migration strategies that are available to cells [7]. The mesenchymal mode is characterized by dependence on the dynamic formation and disassembly of focal adhesions [8]. Recent studies have revealed that NEDD9 is a major signalling intermediary in mesenchymal mode migration [9] and although it has long been know that NEDD9 localizes to focal adhesions [10,11], the role that NEDD9 plays in regulating focal adhesion dynamics has not previously been reported. NEDD9/HEF1/Cas- L is a member of the Cas family of proteins, grouped together based on a conserved overall protein-protein interaction domain structure [12]. Other members of the family include p130Cas/ BCAR1, Efs/Sin and HEPL. The Cas proteins are adhesion docking proteins and through extensive interaction with signalling partners these proteins play a role in a variety of biological process, including cell migration. In particular, elevated NEDD9 expres- sion is detected in a range of metastatic cancers and NEDD9 is considered to be a key pro-metastatic protein [13]. PLoS ONE | www.plosone.org 1 April 2012 | Volume 7 | Issue 4 | e35058