Figure 1. Transverse arcs fuse during centripetal flow to generate a contractile ventral stress fiber.
(A) U2OS cells have three subtypes of stress fibers: Dorsal stress fibers (red arrowheads), which are attached to focal adhesion at their distal end; Transverse arcs (yellow arrowheads), which are curved actomyosin bundles oriented parallel to the leading edge; Ventral stress fibers (orange arrowheads), which are thick contractile bundles connected to focal adhesions at both ends. All three stress fiber categories are present in cells grown on glass or on stiff (E = 64 kPa) silicone matrix, whereas assembly of contractile ventral stress fibers is compromised in cells grown on soft (0.5 kPa) matrix. (B) Live-imaging of U2OS cells expressing GFP-calponin-3 (CaP3) revealed that transverse arcs fuse with each other during centripetal flow to form thicker actomyosin bundles. Red and yellow brackets highlight fusing arcs, and the orange dashed line indicates the thick ventral stress fiber derived from the fusing arcs. (C) Arc fusion often initiates at the connection points of dorsal stress fibers and transverse arcs (indicated by red arrowheads). Two separate video frame series are shown in the panels. In the images, dorsal stress fibers and arcs are oriented vertically and horizontally, respectively. Stress fibers were visualized by expression of GFP-actin. Bar, 1 μm. (D) Live imaging of YFP-Tm4 and CFP-α-actinin-1 expressing U2OS cell reveals that homotypic coalescence of adjacent Tm4 and α-actinin foci occurs during arc fusion, thus allowing to retain the periodic pattern of transverse arcs. Yellow brackets indicate fusing arcs and yellow arrowheads highlight pairs of fusing α-actinin-1 foci. Bar, 1 μm.
Figure 1—figure supplement 1. Fusion of transverse arcs.
