Cdc8 phosphorylation on S125 facilitates Adf1-mediated F-actin disassembly in vitro and actin cable turnover in vivo. (A) Images showing 125 nM F-actin-Alexa Fluor 488 (magenta) with 400 nM ASCdc8-I76C-Atto-565 (left) or 1,200 nM ASCdc8-I76C-S125E-Atto-565 (right) before (top) or after (bottom) addition of 100 nM Adf1/Cofilin (bottom). Scale bar represents 5 µm. (B) Corresponding box plot of average F-actin contour with ASCdc8-I76C (purple) and ASCdc8-I76C-S125E (red), respectively, before (hollow) and after (filled) Adf1 addition; WT, n = [74, 51] fields of view; S125E, n = [167, 221] fields of view. (C) Images showing 125 nM F-actin-Alexa Fluor 488 (magenta) with 400 nM ASCdc8-I76C-Atto-565 (left) or 1,200 nM ASCdc8-I76C-S125E-Atto-565 (right) before (top) or after (bottom) addition of 1,000 nM Swinholide-A (bottom). Scale bar represents 5 µm. (D) Corresponding box plot of average F-actin contour with ASCdc8-I76C (purple) and ASCdc8-I76C-S125E (red), respectively, before (hollow) and after (filled) Swinholide-A addition; WT, n = [41, 94] fields of view; S125E, n= [34, 76]. (E) cdc8⁺, cdc8-S125A and cdc8-S125E were treated with Lat-A (2.5 µM), and samples were taken every 2 min followed by 4% PFA fixation. Permeabilized cells were stained for actin structures with CF-633 (phalloidin). Scale bar represents 3 µm. (F) Quantification of E. Graph shows the fraction of cells with clearly detectable actin cables (n > 120 cells each from three independent experiments). (G) Quantification of E. Graph shows the fraction of cells with clearly detectable actin rings (n > 120 cells each from three independent experiments). Error bars represent SD. ns, not significant; ***, P < 0.0001; *, P < 0.017.