Cross-Species Analysis of Kinesin-14s: Human HSET Functions in Fission Yeast to Regulate Spindle Bipolarity

Abstract

The discovery of Kinesin-like proteins in 1990, five years after conventional Kinesin, has blossomed into the identification of hundreds of Klps that are classified into fourteen families and one orphan family. Not all Klp families are conserved between eukaryotes, however the Kinesin-14 Klps are ubiquitous and key regulators of bipolar spindle assembly and microtubule dynamics and organization. Kinesin-14 Klps associate with microtubules through ATP-dependent binding of their motor domains, but an additional ATP-independent microtubule binding site in the stalk domain is proposed to allow bundling of microtubules. Kinesin-14 members represent some of the most extensively studied Klps, however limited analysis has been done to examine conserved domains or mechanisms. Kinesin-14s share a domain structure in the stalk that consists of an N-terminal region followed by a region of coiled coil. They can vary drastically in size and share minimal sequence similarities. Features of a second microtubule-binding domain are unknown as well as regulatory elements. Most recently this lab has determined that Kinesin-14/γ-tubulin interactions at the γ-TuSC MTOC can regulate bipolarity. Other genetic and biochemical analysis indicates that Kinesin-14 interactions with other MTOC proteins also occur. We are using deletion analysis, site-directed mutagenesis and chimeric Kinesin-14 proteins with human HSET and Drosophila Ncd to relate structural and sequence domains in Kinesin-14s to their functional importance. Twenty-five Kinesin-14 constructs have been generated and are helping to define the minimal Kinesin-14 and to separate domains for bipolar spindle assembly versus later mitotic roles. Length requirements of the coiled-coil domain are also being examined as a critical component in models that emphasize microtubule cross-linking by Kinesin-14s. This work provides insights into Kinesin-14 functional needs and unique tools for assessing Kinesin-14 interactions with tubulin and non-tubulin proteins in conserved molecular mechanisms.