and comparing mean migration speeds for each condition (Figure 2). We found that knockdown of either isoform caused a ,35% reduction in mean cell speed relative to control, indicating that both a-actinin-1 and a-actinin-4 contribute to glioma cell motility. a-Actinin Isoforms Contribute to ECM Rigidity Sensing by Glioma Cells To test the hypothesis that a-actinin-1 and a-actinin-4 participate in sensing mechanical cues from the ECM and regulating the shape plasticity of glioma tumor cells, we cultured control, a-actinin-1-depleted, and a-actinin-4-depleted cells on a series of collagen I-coated polyacrylamide ECMs spanning a range of elasticities and compared the dependence of projected cell spreading area and cortical stiffness on ECM elasticity (Figure 3). Similar to other cell systems [21,24,25], control cells spread poorly Figure 3. Contributions of a-actinin isoforms to cell-ECM rigidity sensing. (A) Projected cell-ECM adhesion area of siCTL (squares), siACTN1 (circles), and siACTN4 (triangles) cells on collagen I- coated polyacrylamide ECMs of varying elasticity. Cell spreading differences between control and a-actinin depleted cells are statistically significant (p,0.001) for both isoforms on 2 kPa and 8 kPa ECMs. (B) Cortical cell elasticity of siCTL, siACTN1, and siACTN4 cells on variable- rigidity ECMs by AFM. Differences in cortical elasticity between control and a-actinin-depleted cells are statistically significant (p,0.001) for all ECM stiffness. doi:10.1371/journal.pone.0008427.g003 a Actinin and Mechanobiology on relatively compliant ECMs (,5 kPa), more extensively on stiffer ECMs (8–20 kPa), and to a maximal area above a threshold ECM stiffness (.20 kPa). (Figure 3A). In contrast, both a-actinin-1 and a-actinin-4 depleted cells spread less extensively than controls at low ECM stiffness, with up to ,25% reduction in spreading area on the most compliant ECMs. On intermediate-stiffness (,8 kPa) gels, both a-actinin-1 and a-actinin-4 depleted cells were less spread compared to controls, with a-actinin-4 depleted cells spreading ,40% more than a-actinin-1 depleted cells. On the stiffest gels and on glass, both a-actinin-1 and a-actinin-4-depleted cells achieved spreading areas comparable to control cells. Consistent with the increase in spreading area from soft (,2 kPa) gels to intermediate-compliance (,8 kPa) gels, the cortical stiffness of control cells increased from ,2.6 kPa on the soft gels to ,6 kPa on 8 kPa gels, without any further increases in cortical stiffness with increasing ECM stiffness (Figure 3B). This adaptation of cortical stiffness to ECM stiffness has been observed previously in both cultured fibroblasts [26] and mesenchymal stem cells [27]. Knockdown of either a-actinin isoform blunted this stiffness adaptation and significantly reduced cortical stiffness for all ECM stiffnesses. On the most compliant (2 kPa) ECMs, the stiffnesses of a-actinin-1 and a-actinin-4 depleted cells were ,1.75 kPa and ,1.5 kPa respectively, representing a ,40% reduction in stiffness compared to controls. On the stiffest ECMs (.18 kPa), the cortical stiffness of both a-actinin-1 and a-actinin-4 depleted cells achieved a plateau stiffness of ,2.7 kPa, represent- ing a ,55% deficiency in cortical stiffness relative to control cells. Taken together, these results demonstrate that each a-actinin isoform plays a critical role in the shape plasticity and adaptation of glioma cells to micromechanical cues, with both isoforms contributing optimally to cell spreading on compliant ECMs and cortical stiffness on stiff ECMs. a-Actinin Suppression Increases Vinculin Expression and Recruitment to FAs The previous results illustrate that while depletion of either a- actinin isoform significantly reduces cortical stiffness and disrupts actin cytoskeletal architecture, the effects on cell spreading area are surprisingly modest, particularly at high ECM stiffness. To test if this might be explained by compensatory upregulation and redistribution of other FA proteins, we tracked the expression and subcellular localization of vinculin following knockdown of each isoform (Figure 4A, Figures S2 and S6). Depletion of either a- actinin-1 or a-actinin-4 led to enhanced vinculin expression by Western Blot, with an increase of ,40% in a-actinin-4 knockdown cells, and a more robust increase of ,55% in a-actinin-1-depleted cells relative to control. Moreover, confocal imaging revealed differences in the number and distribution of vinculin-positive FAs, with a-actinin-1-depleted cells assembling significantly greater numbers of vinculin-positive FAs relative to controls (Figure 4B). Although depletion of a-actinin-4 also enhanced vinculin-positive adhesions relative to control, morphometric analysis revealed that these FAs were smaller and less numerous than in a-actinin-1 depleted cells (Figure 4C). Interestingly, FAs in a-actinin-1-depleted and a-actinin-4-depleted cells were also more elongated than in controls, suggesting that a-actinin depletion yields more mature adhesions (Figure 4D) that also contain pY397FAK (Figure S3). Interactions between a-Actinin Isoforms and Activators of Myosin-Based Contractility The localization of many mechanosensory FA proteins has been shown to specifically depend on activation of myosin motors, which in turn governs cytoskeletal contractility or prestress [28,29]. To PLoS ONE | www.plosone.org 4 December 2009 | Volume 4 | Issue 12 | e8427