Isoform-specific contributions of alpha-actinin to glioma cell mechanobiology

Abstract

Glioblastoma Multiforme (GBM) is a malignant astrocytic tumor associated with low survival rates because of aggressive infiltration of tumor cells into the brain parenchyma. Expression of the actin binding protein alpha-actinin has been strongly correlated with the invasive phenotype of GBM in vivo. To probe the cellular basis of this correlation, we have suppressed expression of the nonmuscle isoforms alpha-actinin-1 and alpha-actinin-4 and examined the contribution of each isoform to the structure, mechanics, and motility of human glioma tumor cells in culture. While subcellular localization of each isoform is distinct, suppression of either isoform yields a phenotype that includes dramatically reduced motility, compensatory upregulation and redistribution of vinculin, reduced cortical elasticity, and reduced ability to adapt to changes in the elasticity of the extracellular matrix (ECM). Mechanistic studies reveal a relationship between alpha-actinin and non-muscle myosin II in which depletion of either alpha-actinin isoform reduces myosin expression and maximal cell-ECM tractional forces. Our results demonstrate that both alpha-actinin-1 and alpha-actinin-4 make critical and distinct contributions to cytoskeletal organization, rigidity-sensing, and motility of glioma cells, thereby yielding mechanistic insight into the observed correlation between alpha-actinin expression and GBM invasiveness in vivo.

Figure 1. Expression, localization, and suppression of α-actinin isoforms in glioma cells.

(A) Localization of α-actinin-1 and α-actinin-4 in U-373 MG cells cultured on collagen-coated glass. Solid and open arrowheads mark the localization of individual α-actinin isoforms at ruffles and along actin stress fibers, respectively. Scale Bar = 10 µm. (B) Measurement of siRNA efficacy by Western Blot of U-373 MG cells transfected with either scrambled control siRNA (siCTL) or siRNA directed against α-actinin-1 (siACTN1) or α-actinin-4 (siACTN4) (*p<0.05). (C) Subcellular localization of α-actinin-1 and α-actinin-4 following siRNA treatment. Arrowheads depict the redistribution of α-actinin-4 to FAs when α-actinin-1 is suppressed. Scale Bar = 20 µm.

Figure 2. Contributions of α-actinin isoforms to glioma cell motility.

Effect of α-actinin depletion on mean speeds (mean±SEM, *p<0.001) of cells tracked over 6 hours.

Figure 3. Contributions of α-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 α-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 α-actinin-depleted cells are statistically significant (p<0.001) for all ECM stiffness.

Figure 4. Reciprocal relationship between α-actinin and vinculin expression and recruitment to FAs.

(A) Effect of α-actinin isoform suppression on vinculin expression by Western Blot. Vinculin expression is significantly lower (*p<0.05) in both α-actinin-1 and α-actinin-4-depleted cells than in control cells. (B) Effect of α-actinin depletion on vinculin localization. Both α-actinin-1 and α-actinin-4 depleted cells contain more vinculin-positive FAs (arrows) than controls. Scale bar = 20 µm. (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 α-actinin-1 and α-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 mean±SEM.

Figure 5. Differential sensitivity of α-actinin-depleted cells to contractile inhibitors.

Mean speeds of control, α-actinin-1-depleted, and α-actinin-4-depleted cells for 6 hr following 1 hr of incubation with 10 µM ML7 or 10 µM Y27632. Compared to motility of ML7 treated control cells, knockdown of either isoform significantly reduces cell motility (**p<0.001). Depletion of α-actinin-4 (*p<0.05), but not of α-actinin-1, reduces the motility of Y27632 treated cells to levels observed in untreated cells.

Figure 6. Effect of α-actinin expression on myosin expression and contractility.

(A) Effect of α-actinin suppression on NMMII expression by Western Blot. NMMII expression is significantly lower (*p<0.05) in both α-actinin-1 and α-actinin-4-depleted cells than in controls. (B) Effect of α-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.