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Actomyosin stress fibers (SFs) support cell shape and migration by directing intracellular tension to the extracellular matrix (ECM) via focal adhesions. Migrating cells exhibit three SF subtypes (dorsal SFs, transverse arcs, and ventral SFs), which differ in their origin, location, and ECM connectivity. While each subtype is hypothesized to play unique structural roles, this idea has not been directly tested at the single-SF level. Here, we interrogate the mechanical properties of single SFs of each subtype based on their retraction kinetics following laser incision. While each SF subtype bears distinct mechanical properties, these properties are highly interdependent, with incision of dorsal fibers producing centripetal recoil of adjacent transverse arcs and the retraction of incised transverse arcs being limited by attachment points to dorsal SFs. These observations hold whether cells are allowed to spread freely or are confined to crossbow ECM patterns. Consistent with this interdependence, subtype-specific knockdown of dorsal SFs (palladin) or transverse arcs (mDia2) influences ventral SF retraction. These altered mechanics are partially phenocopied in cells cultured on ECM microlines that preclude assembly of dorsal SFs and transverse arcs. Our findings directly demonstrate that different SF subtypes play distinct roles in generating tension and form a mechanically interdependent network.

Actomyosin stress fiber subtypes have unique viscoelastic properties and roles in tension generation