The homoterameric bipolar kinesin-5 motor proteins perform essential functions in mitotic spindle dynamics by crosslinking and sliding apart antiparallel microtubules (MTs). Plus-end-directed motility of kinesin-5 motors is required for sliding apart of interpolar spindle MTs and providing the outwardly-directed force to separate the spindle pole bodies during spindle assembly and anaphase spindle elongation dowering mitosis. However in previous work at our lab we demonstrated that the kinesin-5 from Saccharomyces cerevisiae (baking yeast), Cin8 is minusend-directed on the single molecule level and can switch directionality under a number of conditions (Duselder et al., 2015; Gerson-Gurwitz et al., 2011). Recently, two additional yeast kinesin-5 motors were reported to be bi-directional, the Saccharomyces cerevisiae kinesin-5 Kip1 (Fridman et al., 2013) and the Schizosaccharomyces pombe Cut7 (Edamatsu, 2014). These findings indicate that minus-end-directed motility in kinesins-5 motors may be more common than previously believed, and that the directionality switch of yeast kinesin-5 motors may be physiologically important for unique functions required during yeast mitosis. In previous work we have also demonstrated that Cin8 is differentially phosphorylated during mitosis at three cyclin-dependent kinase 1 (Cdk1) sites located in its motor domain. This phosphorylation regulates Cin8 localization during anaphase (Avunie Masala et al., 2011), but its mechanism remains unclear.
Here we examined the in vitro motile properties and in vivo localization of Cin8 by TIRF microscopy and live cell imaging. We found that addition of negative charge in a phosphomimic Cin8 mutant weakens the MT-motor interaction and regulates the motile properties and directionality of Cin8. We also 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 closed mitosis in yeast and propose a possible physiological role for its minus-end directed motility.
