importance of each isoform to underlying cellular mechanobiolo- gical properties remains unclear. For example, siRNA-mediated knockdown of a-actinin-1 increases motility and tumorigenicity of fibroblasts, consistent with a role in stabilizing cell-ECM adhesive contacts [16,17]. In intestinal epithelial cells, suppression of a-actinin-1, but not a-actinin-4, inhibits deformation-induced ERK phosphorylation and proliferation [18], reflecting the role of a-actinin-1 in linking the cytoskeleton to the extracellular matrix (ECM). In ovarian carcinoma cells, a-actinin-4 knockdown leads to reduced motility and invasion [13], whereas mice genetically deficient in a-actinin-4 exhibit increased lymphocyte chemotaxis [19]. This heterogeneity of findings illustrates two broader points: First, the role of each isoform is highly cell-type specific, making it difficult to extrapolate these studies to human glioma cells. Second, particularly if a-actinin is to be pursued as a drug target, the field could benefit from additional quantitative and molecular-scale insight into how each isoform contributes to biophysical interactions between tumor cells and the ECM, including adhesion, contractility, and mechanotransduction. Here we seek to address both of these gaps in our understanding of a-actinin function by investigating the contributions of a- actinin-1 and a-actinin-4 to the structure, mechanics, and motility of human glioma cells. We find that siRNA-mediated suppression of either isoform reduces random cell migration speed and alters the expression and localization of the FA protein vinculin. Strikingly, suppression of either a-actinin isoform reduces ECM rigidity sensitivity and alters responses to pharmacologic inhibition of MLCK and Rho-associated kinase (ROCK). We attribute these differences to alterations in NMMII expression and modulation of contractility through interactions between MLCK and individual a-actinin isoforms. Taken together, 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. Materials and Methods Cell Culture and RNA Interference U373-MG human glioma cells were obtained and cultured as previously described [7]. Cells were plated either on glass coverslips or tissue culture polystyrene coated with bovine collagen I (Inamed Biosciences) or on collagen-coated polyacrylamide (PA) hydrogels attached to glass coverslips (see below). To suppress a- actinin expression, cells were transfected with isoform-specific a- actinin siRNA sequences (siACTN1 or siACTN4) or non-targeting sequences (siCTL) (Santa Cruz, CA) per manufacturer’s specifi- cations. For MLCK and ROCK inhibition studies, cells were incubated with either ML7 (Calbiochem), Y27632 (Calbiochem) or blebbistatin (Sigma) at 10 mM final concentration. For cell motility experiments, cells were incubated with drug for 1 hour prior to acquisition of time-lapse images. Immunofluorescence and Western Blots Immunofluorescence [7,20] utilized previously validated primary (mouse anti-a-actinin-1 (1:200, Santa Cruz); mouse anti-a-actinin-4 (1:250, Abcam); mouse anti-vinculin (1:200, Sigma)) and secondary antibodies (Alexa Fluor 543 goat anti-mouse IgG; Alexa Fluor 543 donkey-anti-goat IgG (1:500, Invitrogen)). F-actin and nuclear stains utilized Alexa Fluor 488-phalloidin (1:200, Invitrogen) and DAPI (1:500, Invitrogen), respectively. Immunoblots followed manufacturer specifications (Invitrogen Western Blot kit), using previously validated primary antibodies ((a-actinin-1 (1:500, Santa Cruz); a-actinin-4 (1:500, Santa Cruz); NMMII (1:500, Santa Cruz); vinculin (1:500, Sigma); phosphorylated MLC (pMLC) a Actinin and Mechanobiology (1:500, Cell Signaling); pY397FAK (1:500, Invitrogen); GAPDH (1:20000, Sigma)) and HRP-conjugated secondary antibodies (goat- anti-mouse, donkey-anti-goat or goat-anti-rabbit (Invitrogen)), followed by TMB chromogenic substrate (Invitrogen). For pMLC blots, chemiluminescent rather than colorimetric detection was performed according to manufacturer protocols. After development and scanning, band intensities were quantified using ImageJ (NIH). Image Acquisition and Analysis Imaging was performed using a Nikon TE2000E2 microscope equipped with an incubator chamber for controlled temperature, humidity, and CO2. Images were recorded with a CCD camera (Photometrics CoolSNAP HQ2) interfaced to image acquisition software (Compix SimplePCI). Cell spreading areas were measured using ImageJ (NIH) by manually tracing the projected outlines of 50–100 cells per condition. Confocal images were obtained with a Zeiss LSM510 microscope. For quantifying size distribution of FAs, confocal images of vinculin stained cells (10 cells per condition) were thresholded (ImageJ) and quantified for number and size of FAs. FA circularity, defined as 4p * (area/perimeter2), ranges between 0 and 1, with a value close to 0 indicating an oblong morphology and a value of 1 indicating a perfectly circular morphology. For motility measurements, cells were imaged every 15 minutes at 106 magnification for .6 hours and quantified using manual tracking in ImageJ. The data was further processed to obtain mean speed for a given condition. Synthesis of Polyacrylamide ECMs and Traction Force Microscopy (TFM) Polyacrylamide ECMs of defined stiffness were synthesized by polymerizing and crosslinking predetermined ratios of acrylamide and bisacrylamide [7,21]. For TFM experiments [22,23], Texas red-labeled microspheres (1 mm diameter, Invitrogen) were includ- ed in the acrylamide/bisacrylamide solution prior to polymeriza- tion. Gels were then covalently decorated with full-length bovine collagen I at a fixed density using the photoactivatable crosslinker sulfo-SANPAH. For TFM, maps of substrate displacement and strain were computed from bead positions before and after cell detachment using Fourier transform traction cytometry [22]. Atomic Force Microscopy (AFM) The indentational elasticity of cells cultured on either collagen- coated PA hydrogels or glass coverslips were measured with an Asylum MFP 3-D AFM (Asylum Research, CA) as described previously [20]. Results Localization and Suppression of a-Actinin Isoforms in Cultured Glioma Cells To establish baseline expression and localization of a-actinin isoforms in a human glioma cell line we had previously demonstrated to be mechanosensitive [7], we cultured U-373 MG human glioma cells on collagen I-coated glass coverslips and examined isoform distributions by immunofluorescence. While U-373 MG glioma cells were found to exhibit a wide heterogeneity in cell morphology, consistent with our and others’ previous reports, membrane ruffles were observed in ,48% of cells. In this population of cells, both a- actinin-1 and a-actinin-4 localized strongly to membrane ruffles (Figure 1A). In addition, a-actinin-1 stained in a focal adhesion-like distribution in ,70% of cells and colocalized with stress fibers in ,35% of cells. In comparison to a-actinin-1, a-actinin-4 localized along stress fibers in ,45% of cells. We next suppressed expression PLoS ONE | www.plosone.org 2 December 2009 | Volume 4 | Issue 12 | e8427