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.

Mechanistic adaptability of cancer cells strongly affects anti-migratory drug efficacy

“…Previous studies have found that nocodazole-induced depolymerization of microtubules disrupts tubulin polarity and cell-generated forces [44], and results in decreased migration velocity in cell-derived matrix [40]. In our assays, destabilization of microtubules resulted in significant cell rounding ( figure 5), but we did not find any general trend on how nocodazole treatment affected the migration speed of the cancer cells (figure 4c).…”

Microtubules in 3D cell motility

“…In parallel, a link between microtubules and 3D matrix contraction by invasive mesenchymal cells was described in studies that used collagen gels (Bell et al, 1979;Kolodney and Wysolmerski, 1992;Kraning-Rush et al, 2011). Microtubules are required to generate wide-range traction forces in adhesive and elongated cells, such as mesenchymal cells cultured in 3D matrices (Kraning-Rush et al, 2011). This might not only reflect the impact of microtubules on the regulation of adhesion and contractility, but also point to a mechanical function.…”

“…A mechanical role for microtubules in cell shape was suggested in early studies (Brown et al, 1996;Dennerll et al, 1988;Janmey et al, 1991;Rudolph and Woodward, 1978;Tomasek and Hay, 1984). In parallel, a link between microtubules and 3D matrix contraction by invasive mesenchymal cells was described in studies that used collagen gels (Bell et al, 1979;Kolodney and Wysolmerski, 1992;Kraning-Rush et al, 2011). Microtubules are required to generate wide-range traction forces in adhesive and elongated cells, such as mesenchymal cells cultured in 3D matrices (Kraning-Rush et al, 2011).…”

Measurement Systems for Cell Adhesive Forces

“…Nevertheless, these biological matrices are still used today, as a qualitative measurement of cell traction force generation can sometimes be sufficient. For example, Kraning-Rush et al showed that contractility inhibition abrogates traction force generation in both 2D and 3D, demonstrating that cytoskeletal contractility is similarly important for traction force generation in 2D and 3D [90]. Other users have modified this strategy to quantify forces underlying tissue morphogenesis, where fluorescent deformable microdroplets are functionalized with adhesive ligands and embedded inside embryonic tissue matrices [91].…”