Nano-twines to twine-bridges: Role of force exerting perpendicular lateral protrusions in fibroblastic cell contraction

Aligned extracellular matrix fibers enable fibroblasts to undergo myofibroblastic activation and lead to elongated cell morphology. The fibroblasts in turn contract to cause alignment of the extracellular matrix. This feedback process is critical in pathological occurrences such as desmoplasia and is not well understood. Using engineered fiber networks that serve as force sensors, we identify lateral protrusions with specific functions and morphology that are induced by elongated fibroblastic cells and which apply extracellular fiber-deflecting contractile forces. Lateral projections, named twines, produce twine bridges upon interacting with neighboring parallel fibers. These mature into \"perpendicular lateral protrusions\" (PLPs) that enable cells to spread laterally and effectively contract. Using quantitative microscopy, we show that the twines originate from the stratification of cyclic actin waves traversing the entire length of the cell. The primary twines swing freely in 3D and engage neighboring extracellular fibers. Once engaged, a lamellum extends from the primary twine and forms a second twine, which also engages with the neighboring fiber. As the lamellum fills in the space between the two twines, a sheet-like PLP is formed to contract effectively. By controlling the geometry of extracellular networks we confirm that anisotropic fibrous environments enable PLP formation, and these force-generating PLPs are oriented perpendicular to the parent cell body. PLP formation kinetics indicated mechanisms analogous to other/known actin-based structures. Our identification of force-exerting PLPs in anisotropic fibrous environments suggests an explanation for cancer-associated desmoplastic expansion at single-cell resolution, providing possible new clinical intervention opportunities.