Supplementary Figure 5: Long “single-ended” stress-fibers replace radial fibers in GFP-ABDdel-α-actinin-1 expressing cells.

(a) Schematic diagram illustrating the molecular organization of α-actinin-1 and its mutants. The α-actinin-1 monomer has an N-terminal actin-binding domain (ABD) composed of two calponin-homology (CH) domains, a central rod consisting of four spectrin repeats (R1-R4), and a C-terminal calmodulin (CaM)-like domain. α-actinin-1 monomers form an anti-parallel dimer with the ABD domain present at both ends. The ABDdel-α-actinin-1 mutant lacks the ABD domain⁴¹. Spectrin repeats 1 and 2 are responsible for integrin binding. The SR12 mutant consists only of these spectrin repeats (R1 and R2)⁴¹. (b) Cells expressing GFP-ABDdel-α-actinin-1 were extracted prior to fixation to reduce background due to the presence of excess soluble GFP-ABDdel-α-actinin-1. Truncated GFP-ABDdel-α-actinin-1 mutant protein localized to long actin fibers (purple). Unlike radial fibers, these fibers also contained myosin-IIA (blue). However, unlike ventral stress-fibers, they were associated with only one focal adhesion (labeled by vinculin, red). Thus, we classified this type of actin fibers as “single-ended” stress-fibers. Scale bar, 10 μm. (c) Histogram showing the distribution of “single-ended” actin fiber tilt angle (°) in a cell expressing GFP-ABDdel-α-actinin-1. 30 actin fibers of a representative cell were analyzed. See also Supplementary Video 12. Note that unlike radial fibers in cells overexpressing the full-length α-actinin-1, the “single-ended” stress-fibers in cells expressing GFP-ABDdel-α-actinin-1 tilt in the same direction as radial fibers in control cells (see Fig. 6c).