Gamma Tubulin Phospho Regulation: Insights into Spindle Assembly

Abstract

Kinesin-5 proteins are microtubule associated motors, which are highly conserved from yeast to human cells. They share high homology in their catalytic motor domain sequence, fulfill similar essential mitotic roles in spindle assembly and anaphase B spindle elongation and, until recently (Roostalu et al., Science, 2011), were all thought to move towards plus ends of microtubules. Mechanisms that regulate Kinesin-5 function, specifically during anaphase B, are not well understood. S. cerevisiae cells express two Kinesin-5 homologues, Cin8 and Kip1, which overlap in function. Here we have examined in vitro and in vivo functions and regulation of Cin8 during anaphase B. We followed Cin8 localization and carried out single molecule fluorescence motility assays to study Cin8 motile properties. We found that in vitro, Cin8 molecules are able to switch directionality along a single microtubule as a function of ionic strength conditions and that during anaphase B, Cin8 moves not only towards the plus, but also towards the minus ends of spindle microtubules. Compared to kinesin-5 homologues of higher eukaryotes, S. cerevisiae Cin8 carries a uniquely large insert in loop 8 in its motor domain. To probe the role of the large loop 8 in the directionality switch of Cin8, we studied a construct in which this segment was replaced with the seven amino acids of loop 8 in the related S. cerevisiae kinesin-5 Kip1 (Cin8D99) (Hoyt et al.,J Cell Biol, 1992). We examined the anaphase B localization and in vitro motile properties of the Cin8D99 variant. Using combined in vitro and in vivo approaches, we were able to characterize the role of loop 8 in controlling Cin8 motility and function during S. cerevisiae anaphase.