Figure 9. Cofilin-1 promotes disassembly of non-contractile dorsal stress fibers.

(A) Endogenous cofilin-1 localizes to dorsal stress fibers (red arrowheads) but it is absent from contractile arcs (yellow arrowheads) as shown by phalloidin and anti-cofilin-1 staining of a U2OS cell treated with Triton-X 100 prior to PFA fixation. Bar, 10 μm. (B) Line intensity profiles show incorporation of cofilin-1 into dorsal stress fibers (dsf) but not to the contractile ventral stress fibers (vsf). Cofilin (green); Actin (red). (C) Depletion of cofilin-1 leads to an appearance of abnormally long dorsal stress fibers and defects in the fusion of transverse arcs. Bar, 10 μm. (D) Quantification of the lengths of dorsal stress fibers (μm) in control and cofilin-1-depleted cells. Mean lengths (+/- SEM) of 50 dorsal stress fibers from control and cofilin-1 RNAi cells are displayed in the graph.

DOI: http://dx.doi.org/10.7554/eLife.06126.023

Figure 9—figure supplement 1. Cofilin-1 localizes to dorsal stress fibers and affects their turnover.

(A) Flag-tagged cofilin-1 localizes to non-contractile dorsal stress fibers, whereas it is absent from contractile ventral stress fibers and transverse arcs. Cofilin-1 (red) was visualized by anti-flag antibody and F-actin (green) by phalloidin staining. Bar, 10 μm. (B) Magnifications of a flag-tagged cofilin-1 expressing cell. The regions selected for the line intensity profiles from dorsal and ventral stress fibers are indicated by red and orange lines, respectively. (C) Line intensity profiles show incorporation of cofilin-1 into dorsal stress fibers (dsf) but not to the contractile ventral stress fibers (vsf). (D) Expression of constitutively active Ser239Ala;Thr278Ala mutant VASP leads to formation of curly actomyosin structures. Cofilin-1 localizes to these structures. (E) Cofilin-1-depleted cells show abnormally long and thick dorsal stress fibers as well as impaired fusion of transverse arcs into thick contractile ventral stress fibers. Stress fibers were visualized with phalloidin staining. Bar, 10 μm. (F) Time lapse frames from a cofilin-1-depleted U2OS cell, transfected with GFP-actin. Depletion of cofilin-1 leads to formation of abnormally stable dorsal stress fibers, which appear to prevent arc fusion, but continue to grow between adjacent arcs. Red arrowhead indicates a dorsal stress fiber and yellow brackets indicate two arcs that are not able to fuse. Bar, 5 μm.

DOI: http://dx.doi.org/10.7554/eLife.06126.024