Effect of Phosphorylation on Motile Properties of the Mitotic Kinesin-5 Cin8

Abstract

The mitotic spindle is a microtubule-based bipolar structure which undergoes a well-defined set of morphological changes while mediating the segregation of duplicated chromosomes. It has been established that spindle morphogenesis is governed, in part, by the activity of molecular motors from the kinesin-5 family of bipolar motors with two pairs of catalytic domains located at the opposite sides of the active complex. Kinesin-5 motors are believed to perform their functions by crosslinking and sliding apart antiparallel microtubules originating from opposite spindle poles. Saccharomyces cerevisiaes cells express two kinesin-5 homologues; Cin8 and Kip1 that overlap in essential mitotic functions such as spindle assembly and anaphase spindle elongation.Previous work from our laboratory indicated that Cin8 is differentially phosphorylated during mid-late anaphase at three cyclin-dependent kinase 1 (Cdk1) specific sites located in its motor domain. Phosphorylation of Cin8 at anaphase, causes its detachment from the spindles, reduces spindle elongation rate and aids in maintaining spindle morphology (Avunie-Masala et al., 2011). To understand how phosphorylation regulates the functions of Cin8, we examined its motile properties by a single-molecule fluorescence motility assay, in which movements of single Cin8-3GFP molecules were observing on fixed microtubules. Since Cin8 was shown to be phosphorylated only during anaphase, we first examined its motile properties in crude extracts of yeast cells arrested in the different points of the cells-cycle. We also compared the motile properties of phosphorylation-deficient and phosphorylation-mimic mutants of Cin8 to the motile properties of the wild type Cin8. We found differences in characteristics of Cin8 motility under the different conditions. Results will be presented.