a Actinin and Mechanobiology Figure 4. Reciprocal relationship between a-actinin and vinculin expression and recruitment to FAs. (A) Effect of a-actinin isoform suppression on vinculin expression by Western Blot. Vinculin expression is significantly lower (*p,0.05) in both a-actinin-1 and a-actinin-4-depleted cells than in control cells. (B) Effect of a-actinin depletion on vinculin localization. Both a-actinin-1 and a-actinin-4 depleted cells contain more vinculin-positive FAs (arrows) than controls. Scale bar = 20 mm. (C) Quantitative analysis of size and number of vinculin-positive adhesions. The right- shift of the siACTN1 and siACTN4 data relative to control demonstrates that both a-actinin-1 and a-actinin-4 depleted cells possess larger and more numerous adhesions than controls. (D) Circularity of vinculin-positive FAs of siCTL, siACTN1 and siACTN4 cells (*p,0.001). In all cases, data are mean6SEM. doi:10.1371/journal.pone.0008427.g004 determine if this also holds true for a-actinin-1 and -4, we tracked the localization of these two isoforms in the presence of pharmacologic inhibitors of MLCK, ROCK, and NMMII, all key components of the myosin-based contractility pathway [30,31,32]. As expected, Western Blots revealed that treatment of U-373 MG cells with ROCK and MLCK inhibitors markedly reduced levels of MLC phosphorylation relative to either untreated controls or cells in which NMMII ATPase was directly inhibited (Figure S4). While inhibition of MLCK did not lead to dramatic morphological changes (Movie S4), inhibition of either ROCK or NMMII significantly altered cell morphology, with cells exhibiting active membrane ruffles at their leading edges and extending long membrane processes at their trailing edges, consistent with previous observations in other cell systems, including glioma cells (Movie S5) [33,34]. Because both a-actinin isoforms appear to strongly regulate glioma cell motility and glioma cells are sensitive to contractility inhibitors, we reasoned that each contractility inhibitor might differentially oppose or potentiate the reduction in motility observed upon depletion of each isoform. To explore this possibility, we studied the motility of glioma cells under MLCK or ROCK inhibition and in the setting of isoform-specific a- actinin depletion (Figure 5, Movies S6, S7, S8, S9). MLCK inhibition modestly reduced (4%) migration speeds of control cells but did so much more significantly in the setting of either a- actinin-1 (,60%) or a-actinin-4 (,40%) suppression. Thus, both a-actinin isoforms act synergistically with MLCK to promote cell motility. In contrast to MLCK, ROCK inhibition enhanced mean migration speeds by ,25% relative to controls. While this enhancement of cell speed was suppressed slightly but not significantly in a-actinin-1 depleted cells, a-actinin-4 depletion reduced migration speeds to values similar to untreated cells, indicating that the enhancement in cell motility observed with ROCK inhibition may be partially attributed to a-actinin-4. Contributions of a-Actinin Isoforms to Myosin Expression and Function The above results suggest a close association between a-actinin isoforms and NMMII, whose activation is classically associated with increased contractility, increased stiffness, and generation of traction forces [35,36,37]. To test whether our observed a-actinin-dependent changes in cellular motility and mechanics were secondary to PLoS ONE | www.plosone.org 5 December 2009 | Volume 4 | Issue 12 | e8427