Figure 2: Sac6 phosphorylation regulates actin cable dynamics and is important for normal cell growth.

(a) GST-Bni1 FH1-COOH-functionalized beads were added to cytoplasmic extracts from hydroxyurea (HU)-arrested yeast cells of indicated genotypes expressing Abp140-3 × GFP. (b) Protein concentration for cytoplasmic extracts used for actin reconstitution in a is compared using Pgk1 by immunoblotting. (c) Growth test by serial dilution of WT, cap2Δ, sac6Δ, cap2Δ SAC6, cap2Δ sac6(T103A) double mutant on YPD plates. Plates were incubated at indicated temperature for 24 h. (d) Fluorescent pattern of actin filaments in SAC6 and sac6(T103A) showed by maximum intensity Z-projection. Cells were grown at 39 °C for 30 min before imaging. Star indicates sac6(T103A). (e) Actin cable movement speed in HU-arrested cells treated with or without CK666 (n=100). A concentration of 50 μM CK666 treatment was applied for 30 min at room temperature. Actin filament elongation rate was measured by monitoring the ends of the elongating cables. (f) Depolymerization of actin cables by Lat A treatment. Real-time imaging of actin cables at the cell cortex in SAC6 and sac6(T103A) cells. Cells were pretreated with 50 μM CK666 or DMSO for 30 min at room temperature before adding 0.4 μM Lat A. Images were acquired at 2-min intervals after adding Lat A. Images at time point 0 and 8 min are shown. Scale bars, 5 μm. Error bars are in s.d. One-way analysis of variance was used. **P<0.0001. DMSO, dimethylsulphoxide; NS, not significant.