The role of the cytoskeleton in cellular force generation in 2D and 3D environments

Abstract

To adhere and migrate, cells generate forces through the cytoskeleton that are transmitted to the surrounding matrix. While cellular force generation has been studied on 2D substrates, less is known about cytoskeletal-mediated traction forces of cells embedded in more in vivo-like 3D matrices. Recent studies have revealed important differences between the cytoskeletal structure, adhesion, and migration of cells in 2D and 3D. Because the cytoskeleton mediates force, we sought to directly compare the role of the cytoskeleton in modulating cell force in 2D and 3D. MDA-MB-231 cells were treated with agents that perturbed actin, microtubules, or myosin, and analyzed for changes in cytoskeletal organization and force generation in both 2D and 3D. To quantify traction stresses in 2D, traction force microscopy was used; in 3D, force was assessed based on single cell-mediated collagen fibril reorganization imaged using confocal reflectance microscopy. Interestingly, even though previous studies have observed differences in cell behaviors like migration in 2D and 3D, our data indicate that forces generated on 2D substrates correlate with forces within 3D matrices. Disruption of actin, myosin or microtubules in either 2D or 3D microenvironments disrupts cell-generated force. These data suggest that despite differences in cytoskeletal organization in 2D and 3D, actin, microtubules and myosin contribute to contractility and matrix reorganization similarly in both microenvironments.

Fig. 1

Cytoskeletal effectors affect cell morphology and cytoskeletal organization of MDA-MB-231 cells in 2D. Fluorescent staining of actin (left), microtubules (MT, middle), and merge (right), with actin (red), MT (green), and nucleus (blue), scale bar is 50 μm. Cells were treated with DMSO control (a); actin-acting agent cytochalasin D [CD] (b); microtubule-acting agents nocodazole [Noco] (c) and paclitaxel [Ptx] (d); and myosin-acting agents blebbistatin [Bleb] (e) and calyculin A [CLA] (f).

Fig. 2

Quantification of average fluorescence intensities of actin and MTs in treated cells. Actin (a) and MT (b) fluorescence was imaged and the average intensity of each treatment was quantified by measuring integrated density. Average intensities were normalized to control. Dashed line indicates control value. CD, cytochalasin D; Noco, nocodazole; Ptx, paclitaxel; Bleb, blebbistatin; CLA, calyculin A; a.u., arbitrary units. Mean + SEM. (*) indicates statistical difference from control.

Fig. 3

Effect of cytoskeletal agents on 2D traction force generation and morphology. Traction stress, T, distribution in cytochalasin D [CD]-, nocodazole [Noco]-, paclitaxel [Ptx]-, blebbistatin [Bleb]-, and calyculin A [CLA]- treated cells and controls (a). Force maps (left) and phase images (right) shown, scale bar = 50 μm. Average total traction force magnitude, |F|, in CD-, Noco-, Ptx-, Bleb-, and CLA- treated cells (b). Plot of the relative percentage each projection (long, black bars; short, white bars) contributed to the total of both traction integrals (c). The circularity of treated cells (d) was calculated (4πArea / Perimeter²). Dashed line indicates control value. Mean + SEM. (*) indicates statistical difference from control.

Fig. 4

Effect of cytoskeletal agents on 3D collagen contraction. Cells were seeded in collagen gels and treated with pharmacological agents over 24 hours. Original gel area (thick dashed line) and area after 24 hours of contraction (thin dashed line) were measured, scale bar is 1 cm (A). Relative collagen matrix contraction was quantified by normalizing the change in area of each condition to the change in area of the control (B). Dashed line indicates control value. Mean + SEM. (*) indicates statistical difference from control. CD, cytochalasin D; Noco, nocodazole; Ptx, paclitaxel; Bleb, blebbistatin; CLA, calyculin A.

Fig. 5

Cartoon schematic of ECM remodeling as a metric of force generation in 3D. Cells (red outline) generate contractile forces (white arrows), which develop over time and contribute to local ECM remodeling, as indicated by increased collagen fiber density and orientation relative to the cell body. Collagen organization and density can by visualized using Confocal Reflectance Microscopy.

Fig. 6

Effect of cytoskeletal agents on cell morphology and ECM reorganization in 3D collagen matrices. Fluorescent staining of actin and microtubules (MT) in MDA-MB-231 cells embedded in 3D collagen matrices (A), and confocal reflectance of surrounding collagen fibers (B). Cells were treated with DMSO control; actin-acting agent cytochalasin D [CD]; microtubule-acting agents nocodazole [Noco] and paclitaxel [Ptx]; and myosin-acting agents blebbistatin [Bleb] and calyculin A [CLA] and imaged after 24 hours. Scale bar is 20 μm. Merge image pseudocolored red (actin), green (MTs), blue (nuclei).

Fig. 7

Quantification of cell-mediated collagen remodeling. Quantification of confocal reflectance imaging of collagen fibers. Collagen intensity decreases exponentially as a function of distance from the cell membrane, and decreases differentially upon treatment with pharmacological agents (A). Each treatment is fit with an exponential decay model, from which tau (τ) is extracted as a descriptive parameter of decay of collagen intensity (B). Control (▲); CD, cytochalasin D (◆); Noco, nocodazole (+); Ptx, paclitaxel (×); Bleb, blebbistatin (■); CLA, calyculin A (●). Mean + SEM. (*) indicates statistical difference from control.