- Saccharomyces cerevisiaes Cin8, a member of the kinesin-5 family of motors, performs important functions in mitotic spindle dynamics such as spindle assembly and anaphase spindle elongation. Resent work has shown that Cin8 is a bidirectional motor and moves in vitro towards the minus-end of microtubules (MTs) and changes directionality as a function on ionic strength conditions and MT binding geometry (Gerson-Gurwitz et al., 2011). Previous work from our laboratory had also indicated that Cin8 is differentially phosphorylated during late anaphase at three cyclin-dependent kinase 1 (Cdk1) specific sites located in its motor domain. In vivo, this phosphorylation causes Cin8 detachment from the spindles, reduces spindle elongation rate and aids in maintaining proper spindle morphology (Avunie-Masala et al., 2011).Here, we examined the in vitro motile properties of Cin8 by a single-molecule fluorescence motility assay. To study the effect of phosphorylation, we examined the activity of phosphorylation-deficient and phosphorylation-mimic mutant of Cin8. The analysis was done in whole cell extracts as well as on purified Cin8 samples. We found that addition of negative charge in the phospho-mimic mutant weakens the MT-motor interaction and alters the motile properties of Cin8. These results indicate that phosphorylation of Cin8 in the catalytic domain, regulates its motor function.