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

Aligned extracellular matrix fibers impart an elongated cell morphology and enable fibroblasts to undergo myofibroblastic activation. Yet the biomechanical intricacies that are associated with this microenvironmental phenomenon are unknown. Here, we identify a new role of lateral protrusions, originating anywhere along elongated fibroblastic cells, which apply fiber-deflecting contractile forces. Using aligned fiber networks that serve as force sensors, we quantitate the role of lateral nano-projections (twines) that mature into perpendicular lateral protrusions (PLPs) through the formation of twine-bridges, thus allowing elongated cells to spread laterally and effectively contract. Using quantitative microscopy at high spatiotemporal resolution, we show that twines of varying lengths can originate from stratification of cyclic actin waves traversing along the entire length of the cell. Primary twines swing freely in 3D and engage with neighboring extracellular fibers in interaction times of seconds. Once engaged, an actin lamellum grows along the length of primary twine and re-stratifies to form a secondary twine. Engagement of secondary twine with the neighboring fiber leads to the formation of twine-bridge, a critical step in providing a conduit for actin to advance along and populate the twine-bridge. Through arrangement of fiber networks in varying configurations, we confirm that anisotropic fibrous environments are fertile for PLP dynamics, and importantly force-generating PLPs are oriented perpendicular to the parent cell body. Furthermore, cell spreading onto multiple fibers and myofibroblastic-like contraction occur through PLPs. Our identification of force exertion by PLPs identifies a possible explanation for cancer-associated desmoplastic expansion, at single-cell resolution, thus providing new clinical intervention opportunities.