Isoform-Specific Contributions of a-Actinin to Glioma Cell Mechanobiology Shamik Sen1, Meimei Dong1,2, Sanjay Kumar1* 1 Department of Bioengineering, University of California, Berkeley, California, United States of America, 2 Institute of Molecular and Cell Biology, Mannheim University of Applied Sciences, Mannheim, Germany Abstract Glioblastoma Multiforme (GBM) is a malignant astrocytic tumor associated with low survival rates because of aggressive infiltration of tumor cells into the brain parenchyma. Expression of the actin binding protein a-actinin has been strongly correlated with the invasive phenotype of GBM in vivo. To probe the cellular basis of this correlation, we have suppressed expression of the nonmuscle isoforms a-actinin-1 and a-actinin-4 and examined the contribution of each isoform to the structure, mechanics, and motility of human glioma tumor cells in culture. While subcellular localization of each isoform is distinct, suppression of either isoform yields a phenotype that includes dramatically reduced motility, compensatory upregulation and redistribution of vinculin, reduced cortical elasticity, and reduced ability to adapt to changes in the elasticity of the extracellular matrix (ECM). Mechanistic studies reveal a relationship between a-actinin and non-muscle myosin II in which depletion of either a-actinin isoform reduces myosin expression and maximal cell-ECM tractional forces. Our results demonstrate that both a-actinin-1 and a-actinin-4 make critical and distinct contributions to cytoskeletal organization, rigidity-sensing, and motility of glioma cells, thereby yielding mechanistic insight into the observed correlation between a-actinin expression and GBM invasiveness in vivo. Citation: Sen S, Dong M, Kumar S (2009) Isoform-Specific Contributions of a-Actinin to Glioma Cell Mechanobiology. PLoS ONE 4(12): e8427. doi:10.1371/ journal.pone.0008427 Editor: Laurent Kreplak, Dalhousie University, Canada Received July 22, 2009; Accepted December 1, 2009; Published December 23, 2009 Copyright: 2009 Sen 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: S.K. gratefully acknowledges the support of the University of California, the Arnold and Mabel Beckman Young Investigator Award, and the National Institutes of Health (NIH) Director’s New Innovator Award (1DP2OD004213), a part of the NIH Roadmap for Medical Research. 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: skumar@berkeley.edu Introduction Glioblastoma Multiforme (GBM) is a high-grade astrocytoma characterized by aggressive invasion of individual tumor cells into the brain parenchyma [1]. The diffuse infiltration of GBM tumors along vasculature and white matter tracts in the central nervous system makes complete resection virtually impossible, giving rise to a mean survival time from diagnosis of only 1–2 years, even with aggressive therapy. The remarkable invasiveness of GBM tumors is attributed in part to the capacity of the constituent tumor cells to remodel the extracellular matrix (ECM), which is made possible by integrin upregulation [2], matrix metalloprotease (MMP)-mediat- ed proteolysis [3], and de novo secretion of ECM proteins [4]. This remodeling also depends on the ability of the tumor cells to generate actomyosin-based contractile forces, which have been observed in other systems to facilitate ECM fibril remodeling during migration, thereby providing contact guidance cues to invasive cells [5]. The importance of non-muscle myosin II (NMMII) in glioma invasiveness has been demonstrated by studies where inhibition of myosin light chain kinase (MLCK) completely abrogated glioma motility [6]. To explore potential connections between ECM-encoded signals, cellular contractility, and tumor progression, we recently investigated the role of ECM rigidity (stiffness) in controlling behaviors of glioma cells relevant to growth and spread [7]. We demonstrated that the adhesion and cytoarchitecture of a variety of glioma cell culture models are indeed sensitive to ECM stiffness, and that this microenvironmental parameter can profoundly influence cell motility and proliferation. Moreover, NMMII is specifically required for this rigidity sensitivity, with inhibition of NMMII abrogating stiffness-dependent differences in adhesion and rescuing cell motility on highly compliant ECMs. While this study clearly established a connection between ECM-based mechanical cues, tumor cell adhesion and migration, and NMMII activity, the underlying molecular mechanisms remain incom- pletely understood. Focal adhesions (FAs) play a central role in transducing mechanical signals between the cytoskeleton and ECM [8]. In the case of GBM specifically, a comparative study of in vitro migration and invasion across ten human GBM cell lines revealed that levels of the FA and actin-binding protein a-actinin correlates directly with biological aggressiveness [9]. The field’s understand- ing of a-actinin function in this context has been complicated by the relatively recent discovery that four distinct isoforms exist in humans: the nonmuscle isoforms 1 and 4 and the muscle-specific isoforms 2 and 3. While both a-actinin-1 and a-actinin-4 have been reported to localize along stress fibers [10], a-actinin-1 also localizes to FAs and cell-cell contacts [11], and a-actinin-4 is also enriched at the leading edges of invading cells [12]. Further, immunohistochemical analysis of human tumors demonstrates that the cytoplasmic localization of a-actinin-4 accurately predicts an infiltrative phenotype and poor clinical prognosis [13,14,15]. While the above studies clearly establish that both a-actinin-1 and -4 contribute to tumor invasion and metastasis, the PLoS ONE | www.plosone.org 1 December 2009 | Volume 4 | Issue 12 | e8427