Ofer Shapira 1 Alina Goldstein 1 Jawdat Al-Bassam 2 Larisa Gheber 1
1Department of Chemistry and the Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev, Beer-Sheva, Israel
2Department of Molecular Cell Biology, UC Davis, Davis, California, USA

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.

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