Figure 5. Differential sensitivity of a-actinin-depleted cells to contractile inhibitors. Mean speeds of control, a-actinin-1-depleted, and a-actinin-4-depleted cells for 6 hr following 1 hr of incubation with 10 mM ML7 or 10 mM Y27632. Compared to motility of ML7 treated control cells, knockdown of either isoform significantly reduces cell motility (**p,0.001). Depletion of a-actinin-4 (*p,0.05), but not of a- actinin-1, reduces the motility of Y27632 treated cells to levels observed in untreated cells. doi:10.1371/journal.pone.0008427.g005 changes in myosin activity, we compared the levels of NMMII expression and phosphorylation in control and a-actinin-depleted cells (Figure 6A, Figures S5, S6). Indeed, suppression of either a- actinin-1 or a-actinin-4 reduced myosin expression, with a reduction of ,66% in a-actinin-1 knockdown cells and ,60% in a-actinin-4 depleted cells relative to control. As expected, suppression of either isoform also led to reduced levels of MLC phosphorylation (pMLC) (Figure S5). To further determine the functional effect of this reduced NMMII expression in a-actinin depleted cells on traction force generation, we used traction force microscopy to compare cell-ECM tractional forces between control and a-actinin-depleted cells on ECMs with elasticites of 2 kPa and 18 kPa, respectively (Figure 6B). This method measures cellular tractional forces (a measure of contractility) by tracking relaxation of ECM-embedded fiduciary markers following cell detachment. On the soft (2 kPa) gels, the mean traction force of control cells, a-actinin-1, and a-actinin-4- depleted cells were 150 Pa, 85 Pa, and 95 Pa, respectively, with knockdown of either subunit eliminating the high-traction (.250 Pa) population observed in control cells (Figure S7). Interestingly, the reduction in mean traction forces closely mirrored the 40% reduction in cell stiffness of actinin-depleted cells observed on 2 kPa gels (Fig. 3). On the stiffer (18 kPa) gels, the mean traction force values shifted to 700 Pa, 540 Pa, and 580 Pa, respectively, and knockdown of either subunit rendered cells unable to generate tractional forces .1.2 kPa. Taken together, these results show that depletion of either a-actinin isoform leads to suppression of NMMII expression and function. Discussion We have examined the contributions of the non-muscle a- actinin isoforms 1 and 4 to the mechanobiology of human glioma cells. We find that these two isoforms localize to different subcellular compartments, and that depletion of either isoform leads to an unexpected increase in vinculin expression and localization to adhesions, which is accompanied by reduced motility. We also show that both isoforms serve mechanosenory functions, with depletion compromising the ability of glioma cells PLoS ONE | www.plosone.org 6 December 2009 | Volume 4 | Issue 12 | e8427 a Actinin and Mechanobiology Figure 6. Effect of a-actinin expression on myosin expression and contractility. (A) Effect of a-actinin suppression on NMMII expression by Western Blot. NMMII expression is significantly lower (*p,0.05) in both a- actinin-1 and a-actinin-4-depleted cells than in controls. (B) Effect of a- actinin depletion on traction force generation measured by traction force microscopy. Box-whisker plots of root-mean squared (RMS) traction of siCTL, siACTN1 and siACTN4 cells on 2 kPa and 18 kPa ECM substrates. The error bars mark the maximum and minimum of the data sets, the three horizontal lines mark the 25th, 50th, and 75th percentile of the data, and the point marks the mean. N.10 cells in all cases. doi:10.1371/journal.pone.0008427.g006