to adapt their structure and mechanics to changes in ECM rigidity. Mechanistic insight into these findings comes from the observation that suppression of either a-actinin isoform reduces expression of NMMII, thereby altering mechanochemical feed- back between tumor cells and the ECM and reducing maximal traction forces. Taken together, our data support a model in which a-actinin isoforms participate in mechanochemical feedback between glioma cells and the ECM, with expression and localization of these molecules regulating NMMII-dependent processes, including adhesion, motility, stiffening, and contractil- ity. While these results are clear, two points are worth noting: First, the detailed mechanisms that link suppression of a-actinin to altered cell migration and mechanics remain to be elucidated, and it is likely that other elements of the adhesive and contractile machinery besides NMMII and vinculin play important roles. Second, we chose not to attempt to rescue effects of a-actinin- depletion by overexpressing exogenous a-actinin, both because low double-transfection efficiencies make obtaining meaningful statistics from single-cell measurements challenging, and because a-actinin overexpression has previously been observed to pheno- copy a-actinin depletion (e.g., with respect to cell migration, as described in the introduction). Thus, we cannot completely rule out the possibility that some of our observed effects may be due in part to secondary effects of a-actinin depletion. Our findings may bear implications for GBM invasion in vivo. The direct role of NMMII in GBM invasiveness was first demonstrated by studies in which treatment with MLCK inhibitors completely abrogated glioma motility [6]. Three isoforms of NMMII are known to exist (NMMIIA, B, C); NMMIIA is upregulated in human glioma xenografts, and both NMMIIA and NMMIIB are required to squeeze through pores smaller than the size of nuclei [38]. Our studies reveal a causal connection between a-actinin and NMMII in which depletion of either a-actinin isoform reduces NMMII expression. This close interplay between a-actinin and NMMII in modulating contractility is consistent with biophysical studies of gels composed of F-actin, NMMII and a- actinin, which have found that bulk contractility of the gel occurs only above a threshold a-actinin concentration [39]. The actin- crosslinking activity of NMMII in the initial stages of adhesion has been demonstrated in recent studies where overexpression of wild- type NMMII in a-actinin-depleted CHO cells was able to restore adhesion maturation [40]. In human aortic endothelial cells, the increase in cell prestress obtained with knockdown of a-actinin-1 suggests that a-actinin depletion increases actomyosin interactions and raises the possibility that a-actinin and NMMII compete for common binding sites in crosslinking F-actin [41]. The cell softening observed in our studies upon a-actinin knockdown is also consistent with a published report in which microinjection of a-actinin not only increased the stiffness of 3T3 fibroblasts but also the degree of mechanical heterogeneity of the cytoplasm, as measured by particle tracking microrheology [42]. Cell migration requires the coordinated extension of membrane protrusions, formation of transient adhesions along the membrane extensions, and NMMII-dependent detachment of the cell rear [43]. The activity of NMMII is controlled by MLC phosphory- lation, and recent efforts to elucidate regional variations in MLC phosphoregulation have suggested that MLCK preferentially regulates dynamic adhesions at the cell periphery and that ROCK regulates more stable FAs at the cell center [44,45]. We find that motility is reduced most dramatically by suppressing those isoforms and inhibiting MLCK. Because the rapid turnover of FAs at the leading edge needed for migration depends on MLC phosphorylation, we hypothesize that MLCK utilizes both a- actinin isoforms in order to promote motility. Similarly, because a Actinin and Mechanobiology the enhancement in motility observed upon ROCK inhibition is reduced by a-actinin-4 depletion, we suspect that this enhance- ment may require mobilization of a-actinin-4. Potential mecha- nisms for this effect might include increased interaction between a- actinin-4 and pMLC, which translocates to membrane protrusions in a Rac-dependent manner [46]. The adaptation of glioma cells to changes in ECM stiffness observed here is reminiscent of the finding that cultured fibroblasts match their stiffness to the underlying ECM substrate for ECM stiffnesses up to 5 kPa and reach a maximal stiffness on ECM substrates [26]. The failure of a-actinin-depleted cells to exhibit this stiffness adaptation may be due to the requirement for a- actinin to crosslink cortical actin and stabilize connections between F-actin and b integrin subunits in adhesions, which would be expected to require strongest reinforcement on high-rigidity ECMs, which support large traction forces [47]. Conversely, both a-actinin isoforms contribute most strongly to spreading area at low ECM rigidity. We speculate that this may be due to the compensatory effects of vinculin, whose expression and localiza- tion to FAs significantly increase on rigid ECMs in the setting of a- actinin suppression, thereby preserving cell shape but not necessarily cortical stiffness. This concept is supported by previous studies in which vinculin suppression reduced spreading, stress fiber formation, FA formation, and lamellipodial extension [48]. This compensatory effect may itself depend on ECM rigidity, such that a-actinin-depleted cells cultured on compliant ECMs are much less capable of recruiting additional vinculin to FAs than their counterparts on rigid ECMs. As a result, we observe negligible deficiencies in cell spreading of a-actinin depleted cells on stiff substrates but see these differences amplify as matrix stiffness is reduced. Enhanced expression of a-actinin may switch GBM tumor cells from a strongly adhesive and low-motility phenotype to a highly contractile and invasive phenotype, and the possibility that invading glioma cells may stiffen the brain parenchyma as they invade [7] suggests a complex interplay between ECM rigidity, a-actinin, and vinculin. One particularly unexpected finding from these studies is that a- actinin-depleted cells spread extensively on stiff ECMs despite having relatively low NMMII expression and low cellular stiffness and prestress. While this result may be inconsistent with the conventional notion that high cellular traction is always needed to support spreading, it is not without precedent. For example, Janmey and coworkers recently reported that under some conditions, filamin A-null cells increase their spreading in response to increasing ECM stiffness while maintaining a relatively constant cell stiffness [49], indicating that stiffness and spreading are not strictly coupled. Similarly, we recently showed that when U-373 MG cells cultured on highly compliant ECMs are treated with ROCK or NMMII inhibitors, which presumably reduce contrac- tility, they paradoxically begin to spread and migrate [7]. These and other studies suggest the possibility that specific actin crosslinking proteins may modulate the degree of coupling between cell spreading and cellular contractility. In conclusion, we have investigated the contributions of a- actinin-1 and a-actinin-4 to mechanobiological behaviors of human glioma cells and shown that suppressing each isoform bears significant but distinct implications for tumor cell morphol- ogy, motility, mechanics, rigidity-sensing, and force generation. As described earlier, expression of a-actinin has been strongly correlated with invasive behavior of tumor cells in GBM and other cancers, although the roles of each isoform remain unclear. Our findings raise the exciting possibility that enhanced expression of a-actinin isoforms in tumors could speed motility in vivo by allowing increased NMMII-generated tractional forces against the PLoS ONE | www.plosone.org 7 December 2009 | Volume 4 | Issue 12 | e8427