Phosphoregulation of Cdc8-tropomyosin is important for actin cable stability and cytokinesis. (A) Schematic representation of the phosphorylation site on tropomyosin cdc8. (B) Graphical representation of the coiled-coil heptad repeat organization of Cdc8. Serine (S125) residue is positioned at f on the outside of the coiled-coil heptad. (C) Liquid chromatography-MS/MS spectra of one representative phosphopeptide carrying S125^p. (D) Recognition of Cdc8 S125^P, but not Cdc8 S125A, by an antibody against RXXS^P. Cell lysates from cdc8 and cdc8-S125A cells were immunoprecipitated (IP) with Cdc8 antibodies and immune complexes or cell lysates (whole-cell extract; WCE) were immunoblotted using antibodies against Cdc8, RxxS^p, and actin (loading control). (E) Assessment of colony formation by cdc8 mutants. 10-fold serial dilutions of cdc8⁺, cdc8-S125A, and cdc8-S125E inoculated on YES plates were incubated at indicated temperatures for 3 d. (F) Exponentially growing cdc8⁺, cdc8-S125A, and cdc8-S125E at 24°C were stained for actin structures with CF-633-phalloidin. Also shown are the corresponding segmentation of filaments (green) and patches (pink). Scale bar represents 3 µm. (G) Quantification of F by calculating the ratio of filament to patch area normalized to the average found in WT cells; n = [19, 30, 31] fields of view; box depicts median and 25% to 75% range, whiskers depict maximum and minimum. ***, P < 0.0001; **, P < 0.008.