Phosphorylation of S125 in Cdc8 reduces affinity to F-actin. (A) Coomassie blue–stained gels of actin-tropomyosin cosedimentation assay using high-speed centrifugation. SDS-PAGE gel of supernatant. Each lane represents a pelleting experiment with a different concentration of ASCdc8 or ASCdc8-S125E. (B) Pellet fractions of ASCdc8 and ASCdc8-S125E mutants, respectively. Each lane represents a pelleting experiment with a different concentration of ASCdc8 or ASCdc8-S125E. (C) Image sequence showing the decoration of 125 nM F-actin-Alexa Fluor 488 (magenta) with ASCdc8-I76C-Atto-565 (top) or ASCdc8-I76C-S125E-Atto-565 (bottom) at indicated concentrations of Cdc8. Scale bar represents 5 µm. (D) Corresponding plot of relative F-actin decoration by ASCdc8-I76C (black) and ASCdc8-I76C-S125E (red), respectively, and fitted Hill functions (lines); WT, n = [20, 33, 46, 31, 25, 37, 6] filaments; S125E, n = [11, 9, 25, 20, 14, 27, 8] filaments. (E) Images showing decoration of 125 nM F-actin-Alexa Fluor 488 (magenta) with 400 nM ASCdc8-I76C-Atto-565 alone (left) or 5 min after addition of Pom1-WT (center) or Pom1-KD (right). Scale bar represents 5 µm. (F) Corresponding box plot of relative F-actin decoration by ASCdc8-I76C at indicated conditions; n = [119, 93, 56] fields of view. (G) Images showing decoration of 125 nM F-actin-Alexa Fluor 488 (magenta) with 400 nM ASCdc8-I76C-S125A-Atto-565 alone (left) or 5 min after addition of Pom1-WT (center) or Pom1-KD (right). Scale bar represents 5 µm. (H) Corresponding box plot of relative F-actin decoration by ASCdc8-I76C at indicated conditions; n = [40, 113, 18] fields of view. Error bars represent SD.