Non-Muscle Myosin II Isoforms Have Different Functions in Matrix Rearrangement by MDA-MB-231 Cells

The role of a stiffening extra-cellular matrix (ECM) in cancer progression is documented but poorly understood. Here we use a conditioning protocol to test the role of nonmuscle myosin II isoforms in cell mediated ECM arrangement using collagen constructs seeded with breast cancer cells expressing shRNA targeted to either the IIA or IIB heavy chain isoform. While there are several methods available to measure changes in the biophysical characteristics of the ECM, we wanted to use a method which allows for the measurement of global stiffness changes as well as a dynamic response from the sample over time. The conditioning protocol used allows the direct measurement of ECM stiffness. Using various treatments, it is possible to determine the contribution of various construct and cellular components to the overall construct stiffness. Using this assay, we show that both the IIA and IIB isoforms are necessary for efficient matrix remodeling by MDA-MB-231 breast cancer cells, as loss of either isoform changes the stiffness of the collagen constructs as measured using our conditioning protocol. Constructs containing only collagen had an elastic modulus of 0.40 Pascals (Pa), parental MDA-MB-231 constructs had an elastic modulus of 9.22 Pa, while IIA and IIB KD constructs had moduli of 3.42 and 7.20 Pa, respectively. We also calculated the cell and matrix contributions to the overall sample elastic modulus. Loss of either myosin isoform resulted in decreased cell stiffness, as well as a decrease in the stiffness of the cell-altered collagen matrices. While the total construct modulus for the IIB KD cells was lower than that of the parental cells, the IIB KD cell-altered matrices actually had a higher elastic modulus than the parental cell-altered matrices (4.73 versus 4.38 Pa). These results indicate that the IIA and IIB heavy chains play distinct and non-redundant roles in matrix remodeling

Nonmuscle myosin II is responsible for maintaining endothelial cell basal tone and stress fiber integrity

Cultured confluent endothelial cells exhibit stable basal isometric tone associated with constitutive myosin II regulatory light chain (RLC) phosphorylation. Thrombin treatment causes a rapid increase in isometric tension concomitant with myosin II RLC phosphorylation, actin polymerization, and stress fiber reorganization while inhibitors of myosin light chain kinase (MLCK) and Rho-kinase prevent these responses. These findings suggest a central role for myosin II in the regulation of endothelial cell tension. The present studies examine the effects of blebbistatin, a specific inhibitor of myosin II activity, on basal tone and thrombin-induced tension development. Although blebbistatin treatment abolished basal tension, this was accompanied by an increase in myosin II RLC phosphorylation. The increase in RLC phosphorylation was Ca2+ dependent and mediated by MLCK. Similarly, blebbistatin inhibited thrombin-induced tension without interfering with the increase in RLC phosphorylation or in F-actin polymerization. Blebbistatin did prevent myosin II filament incorporation and association with polymerizing or reorganized actin filaments leading to the disappearance of stress fibers. Thus the inhibitory effects of blebbistatin on basal tone and induced tension are consistent with a requirement for myosin II activity to maintain stress fiber integrity

Knockdown of myosin II isoforms MDA-MB-231 cells expressing shRNA targeting either the IIA or IIB isoform of nonmuscle myosin II were analyzed for myosin expression levels; GAPDH was used as a loading control.

<p>Greater than 85% knockdown of myosin protein content was achieved in stable cell populations. Myosin isoform levels were assessed for every experiment to verify the level of myosin IIA and IIB knockdown.</p

Calculated elastic modulus of MDA-MB-231 cells within collagen constructs.

<p>The Construct and Matrix moduli of constructs from each experiment were used to calculate the Total Cell Modulus. This was then divided by the cell number, determined by a DNA assay for each experiment, to calculate the Single Cell Modulus. Shown are the calculated total and single cell moduli, averaged across three experiments (± SEM).</p><p>Calculated elastic modulus of MDA-MB-231 cells within collagen constructs.</p

Elastic modulus of collagen constructs seeded with MDA-MB-231 cells.

<p>Shown are the averaged elastic moduli (± SEM) for constructs across three separate experiments</p><p>Elastic modulus of collagen constructs seeded with MDA-MB-231 cells.</p

Loss of myosin II isoforms induces morphological changes in 3D Cells were added to a collagen solution, poured into Teflon molds and allowed to incubate for 4 days.

<p>Constructs were washed, fixed, permeabilized, and stained with Phalloidin-TRITC and affinity purified myosin II antibodies. Cells were examined using Two Photon Microscopy. <b>(A-F)</b> Parental MDA-MB-231 cells in three dimensions had pyramidal cell bodies with multiple projections and significant staining of both IIA <b>(B)</b> and IIB <b>(E)</b> myosin isoforms, mainly diffuse throughout the cytosol. <b>(G-L)</b> IIA KD cells had rounded cell bodies with highly branched and elongated projections in all directions and very little residual myosin IIA <b>(H)</b> staining, mostly localized to the cell bodies. The IIB <b>(K)</b> in these cells remained diffuse throughout the cytosol. <b>(M-R)</b> IIB KD cells were elongated with fewer projections and tended to be localized to a single focal plane, with residual IIB <b>(O)</b> localized at cell edges and near the nucleus. For all cell types, myosin isoform localization in 3D was mainly cytosolic.</p

Elastic recovery of collagen constructs seeded with MDA-MB-231 cells.

<p>The elastic recovery, here defined as the slope of the initial recovery of the construct after stretching, was calculated for each cell type. Shown are the averaged (±SEM) elastic recovery for constructs across three experiments.</p><p>Elastic recovery of collagen constructs seeded with MDA-MB-231 cells.</p

Knockdown of myosin II isoforms induces cytoskeletal changes in MDA-MB-231 cells in 2D Parental (A-D), IIA KD (E-H) and IIB KD (I-L) cells were fixed, permeabilized, and immunostained with affinity purified polyclonal myosin IIA and IIB primary antibodies and TRITC-Phalloidin to visualize actin filaments and myosin localization.

<p>In parental MDA-MB-231 cells, myosin IIA <b>(B)</b> localizes to stress fibers and the leading edge of cells, while myosin IIB <b>(D)</b> had cytosolic, stress fiber, and perinuclear localization. Myosin IIA KD cells had altered actin cytoskeletal structure and were slightly larger than parental controls, while the residual IIA <b>(E)</b> in these cells localized to stress fibers and myosin IIB <b>(H)</b> localization was slightly affected, displaying a microtubule-like staining pattern, thought there was still an amount remaining largely diffuse throughout the cytosol with some stress fiber and perinuclear localization. Myosin IIB KD cells exhibited a more irregular shape with short, prominent stress fibers, and the residual IIB in these cells exhibited a perinuclear localization <b>(L)</b> IIA localization was primarily to stress fibers <b>(J)</b>, as in the parental cells.</p