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

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

Morphological characteristics of cells lacking myosin II isoforms Collagen constructs containing cells were prepared and imaged as outlined.

<p>IMARIS image analysis software was used to quantify the observations made on cell morphology in 3D collagen gels across three separate experiments. <b>(A)</b> The average number of protrusions per cell were measured using the Filaments function in IMARIS. Statistical significance was calculated using one-way ANOVA with a Tukey post-test. The difference between parental and IIA KD cell types was significant (p < 0.01), as was the difference between IIA and IIB KD cells (p < 0.001). <b>(B)</b> The sphericity of the cell bodies was calculated using the Surfaces function in IMARIS. IIA KD cells were slightly more spherical than parental or IIB KD cells, though the difference was not statistically significant. <b>(C)</b> The elongation factor of the cell bodies was calculated using the measurements function in IMARIS. We defined the elongation factor as the measurement of the longest dimension of the cell body, divided by the measurement of the shortest dimension. IIB KD cells had a slightly higher elongation factor than the parental or IIA KD cells, though the difference was not statistically significant.</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

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

Loss of myosin II isoforms inhibits the ability of MDA-MB-231 cells to compress a collagen gel MDA-MB-231 cells or 1 μm fluorescent beads were mixed with a collagen solution and poured into a Teflon mold with a central mandrel.

<p>Constructs were incubated for 1 to 4 days to allow the cells to compress the collagen gel. MDA-MB-231 constructs were washed, fixed, permeabilized, and stained with TRITC-Phalloidin and Hoescht 33258 dye. Cells were examined using Two Photon Microscopy. <b>(A)</b> Z-stacks were taken from the top to the bottom of the constructs and the thickness recorded. Representative z-stacks of each cell type, stained with Hoescht 33258 dye to ensure even distribution of the cells through the depth of the collagen gel, are shown. Images were examined using IMARIS software and maximum intensity projections were generated using the Volume function. <b>(B)</b>. Data shown are average collagen gel thickness measurements (error bars are SD) from three separate experiments. Parental MDA-MB-231 cells were able to constrict the collagen gel by over 50%, as were IIB KD cells, while IIA KD cells were only able to constrict the gel by 15%. The statistical significance was calculated using one-way analysis of variance with a Tukey post-test on both the 1 and 4 day measurements. The difference between collagen with fluorescent beads alone and parental and IIB KD cell constructs (p < 0.0001) was significant, as was the difference between the beads alone and IIA KD constructs (p < 0.05). In addition, the difference between parental and IIA KD constructs was significant (p < 0.0001).</p

Matrix rigidity of MDA-MB-231 cells Representative collagen construct stress calculations from a single experiment for the initial, untreated stretch (A) and the deoxycholate treated stretch (B) plotted as Pa versus percent strain.

<p>For both, myosin II KD cell constructs had a decreased stiffness in response to strain. Because the stiffness calculation takes the cross sectional area of the constructs into account, and the IIA constructs are much larger, the difference between the parental and IIA KD constructs is enhanced when compared to the differences seen when the strain response is plotted as mN versus Strain (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131920#pone.0131920.g006" target="_blank">Fig 6</a>). Loss of myosin IIA had a more significant effect on force in response to strain than did loss of myosin IIB.</p