- The homoterameric bipolar kinesin-5 motors perform essential functions in mitotic spindle dynamics by crosslinking and sliding apart antiparallel microtubules. S. cerevisiae cells express two kinesin-5s Cin8 and Kip1, which overlap in function. We have recently demonstrated that Cin8 and Kip1 are minus-end directed on the single-molecule level and can switch directionality under a number of conditions (Duselder et al., 2015; Fridman et al., 2013; Gerson-Gurwitz et al., 2011). The mechanism of this directionality switch and its physiological significance remain unclear. We have also demonstrated that Cin8 is differentially phosphorylated during late anaphase at three cyclin-dependent kinase 1 (Cdk1) sites located in its motor domain. This phosphorylation regulates Cin8 activity during anaphase (Avunie-Masala et al., 2011), but its mechanism remains unclear.Here we examined the in vitro motile properties and in vivo functions of Cin8 by TIRF microscopy and live-cell imaging. We found that addition of negative charge in a phospho-mimic Cin8 mutant weakens the MT-motor interaction and regulates the motile properties and directionality of Cin8. We also found that of the three Cdk1 sites in the catalytic domain of Cin8, the S277 site contributes the most to regulation of Cin8 localization and function during anaphase. Finally, we found that in vitro under high ionic strength conditions, Cin8 not only moves to- but also clusters at the minus-end of the MTs. This clustering causes Cin8 to reverse its directionality from fast minus- to slow plus-end directed motility. Clustering of Cin8 at the minus-end of the MTs serves as a primary site for capturing and antiparallel sliding of MTs. Based on these results, we propose a revised model for activity of Cin8 during mitosis and propose a physiological role for its minus-end directionality.