We demonstrate that four functionally distinct tropomyosins are required for assembly of stress fibers in cultured osteosarcoma cells. Tm1, Tm2/3, and Tm5NM1/2 stabilize actin filaments at distinct stress fiber regions. In contrast, Tm4 promotes stress fiber assembly by recruiting myosin II to stress fiber precursors. Elimination of any one of the tropomyosins fatally compromises stress fiber formation. Importantly, Dia2 formin is critical to stress fiber assembly by nucleating Tm4-decorated actin filaments at the cell cortex. Myosin II is specifically recruited through a Tm4-dependent mechanism to the Dia2-nucleated filaments, which subsequently assemble endwise with Arp2/3-nucleated actin filament structures to yield contractile stress fibers.
These experiments identified a pathway, involving Dia2- and Arp2/3-promoted actin filament nucleation and several functionally distinct tropomyosins, that is required for generation of contractile actomyosin arrays in cells.