FIGURE 2.

Cytoplasmic actin-cofilin rod assembly/disassembly dynamics. (A) Fluorescence recovery after photobleaching (FRAP) of cytoplasmic rods. Formation of cytoplasmic rods was induced by addition of 10 mM sodium azide for 60 min. Rods were selectively photobleached by drawing a ROI to apply high laser power for 10 s to bleach the GFP. The GFP diffusion into the ROI area (fluorescence recovery) was recorded for 300 s. See also Supplementary Video S4. Scale bar, 10 µm. (B) Fluorescence recovery of photobleached rods. Bleaching curves of three different cytoplasmic rods of sodium azide-treated cells (blue, pink, purple lines) and the untreated control cell are plotted (red). (C) The histogram depicts the relative proportions of mobile and immobile fractions of cells without rods (control) and with cytoplasmic rods. The average of fluorescence recovery was about 20%, indicating that rods are rather stable structures with low exchange inside the rod bundles. (D) Actin-cofilin rods are rapidly disintegrating structures. After induction of cytoplasmic rods by addition of 10 mM sodium azide, the percentage of cells with cytoplasmic rods decreases rapidly when the sodium azide is washed out and replaced by standard medium. Three experiments were performed and for each experiment at least 50 cells were evaluated. Data are presented as mean ± SD (red bars). Statistical significance was calculated by unpaired Welch´s t-test. p ≤ 0.05 was considered significant and (***p < 0.0001). (E) Visualization of actin-cofilin rod disassembly stages. Within 30 min after wash-out of the sodium azide, actin-cofilin rods are almost completely disintegrated. The disassembly takes place via shorter bundles and punctiform actin-cofilin aggregates. Scale bars, 10 µm.