Mitotic Functions and Motile Properties of the S. cerevisiae Kinesin-5 motors

Mitotic chromosome segregation is mediated by mitotic spindle, a highly dynamic microtubule-based structure, which undergoes a distinct set of morphological changes in each mitotic cycle. Major factors that contribute to spindle morphogenesis are microtubule (MT) plus-end dynamics and function of molecular motors from the Kinesin-5 family. Kinesin-5 family members are conserved, homotetrameric motors with two catalytic domains located on opposite sides of the active complex. This special architecture enables these motors to crosslink and slide anti-parallel MTs originating from opposite spindle poles and thereby perform their essential functions in mitotic spindle morphogenesis. It was recently shown that Kinesin-5 motors affect anaphase spindle symmetry and midzone organization (1). S. cerevisiae cells express two Kinesin-5 homologues, Cin8p and Kip1p that overlap in function during spindle assembly, metaphase and anaphase B and at least one of them need to be expressed for viability. So far, the extent of redundancy between these two Kinesin-5 proteins and their motile properties in vitro have not been thoroughly investigated. In the present study, we use high temporal and spatial resolution imaging and FRAP to characterize interpolar MT (iMT) plus-end dynamics during spindle morphogenesis in S. cerevisiae cells expressing tubulin-GFP(2). This approach allowed us to study the role of the major midzone organizing protein Ase1(2) in controlling iMT plus-end dynamics and to compare between the effects of Cin8 and Kip1 on these dynamics during anaphase. In addition, in order to understand in vivo functions of the Kinesin-5 Kip1, we characterized its motile properties in single-molecule fluorescence motility assay. Results from this assay
will be presented