Stress fibers are generated by two distinct actin assembly mechanisms in motile cells. Pirta Hotulainen and Pekka Lappalainen from the Institute of Biotechnology, University of Helsinki, Finland, investigated the mechanisms of stress fiber assembly in motile cells using live cell microscopy. Stress fibers play a central role in cell adhesion, motility, and morphogenesis. The study revealed that stress fibers are generated by two distinct mechanisms: (1) dorsal stress fibers, which are connected to the substrate via focal adhesions, are assembled through formin (mDia1/DRF1)-driven actin polymerization at focal adhesions. (2) Transverse arcs, which are not directly anchored to the substrate, are generated by endwise annealing of actin and myosin bundles. The study used multicolor live cell microscopy and FRAP methods to examine the assembly of actin stress fibers in cells. The results showed that actin filaments for stress fibers are derived from two different sources. Dorsal stress fibers are generated by formin-driven actin polymerization at focal adhesions, while transverse arcs are formed by the endwise annealing of actin and myosin bundles. The study also demonstrated that myosin can be incorporated into dorsal stress fibers after they have associated with transverse arcs. The study further showed that transverse arcs are formed by the endwise annealing of cortical Arp2/3-nucleated actin bundles and myosin bundles. Experiments using blebbistatin, an inhibitor of myosin ATPase activity, demonstrated a critical role of myosin II activity in the transverse arc assembly and maintenance. The study also revealed that the dynamics of stress fiber components, including actin, α-actinin, and myosin, are highly dynamic, with α-actinin showing a particularly high degree of dynamic association with stress fibers. The study provides a model for the assembly of dorsal, transverse, and ventral stress fibers, showing that dorsal stress fibers are generated by formin-driven actin polymerization at focal adhesions, while transverse arcs are formed by the endwise annealing of actin and myosin bundles. Ventral stress fibers are generated from preexisting transverse arc/dorsal stress fiber networks. The study also highlights the importance of dynamic cross-linking of actin filaments during stress fiber formation and maintenance, as well as the role of α-actinin in myosin incorporation and stress fiber contraction. The findings suggest that stress fibers are generated through two distinct mechanisms, with formin-driven actin polymerization at focal adhesions being essential for dorsal stress fiber formation, while endwise annealing of actin and myosin bundles is crucial for transverse arc formation.Stress fibers are generated by two distinct actin assembly mechanisms in motile cells. Pirta Hotulainen and Pekka Lappalainen from the Institute of Biotechnology, University of Helsinki, Finland, investigated the mechanisms of stress fiber assembly in motile cells using live cell microscopy. Stress fibers play a central role in cell adhesion, motility, and morphogenesis. The study revealed that stress fibers are generated by two distinct mechanisms: (1) dorsal stress fibers, which are connected to the substrate via focal adhesions, are assembled through formin (mDia1/DRF1)-driven actin polymerization at focal adhesions. (2) Transverse arcs, which are not directly anchored to the substrate, are generated by endwise annealing of actin and myosin bundles. The study used multicolor live cell microscopy and FRAP methods to examine the assembly of actin stress fibers in cells. The results showed that actin filaments for stress fibers are derived from two different sources. Dorsal stress fibers are generated by formin-driven actin polymerization at focal adhesions, while transverse arcs are formed by the endwise annealing of actin and myosin bundles. The study also demonstrated that myosin can be incorporated into dorsal stress fibers after they have associated with transverse arcs. The study further showed that transverse arcs are formed by the endwise annealing of cortical Arp2/3-nucleated actin bundles and myosin bundles. Experiments using blebbistatin, an inhibitor of myosin ATPase activity, demonstrated a critical role of myosin II activity in the transverse arc assembly and maintenance. The study also revealed that the dynamics of stress fiber components, including actin, α-actinin, and myosin, are highly dynamic, with α-actinin showing a particularly high degree of dynamic association with stress fibers. The study provides a model for the assembly of dorsal, transverse, and ventral stress fibers, showing that dorsal stress fibers are generated by formin-driven actin polymerization at focal adhesions, while transverse arcs are formed by the endwise annealing of actin and myosin bundles. Ventral stress fibers are generated from preexisting transverse arc/dorsal stress fiber networks. The study also highlights the importance of dynamic cross-linking of actin filaments during stress fiber formation and maintenance, as well as the role of α-actinin in myosin incorporation and stress fiber contraction. The findings suggest that stress fibers are generated through two distinct mechanisms, with formin-driven actin polymerization at focal adhesions being essential for dorsal stress fiber formation, while endwise annealing of actin and myosin bundles is crucial for transverse arc formation.