Distinct regions of the kinesin-5 C-terminal tail are essential for mitotic spindle midzone localization and sliding force

Abstract

Kinesin-5 motor proteins play essential roles during mitosis in most organisms. Their tetrameric structure and plus-end-directed motility allow them to bind to and move along antiparallel microtubules, thereby pushing spindle poles apart to assemble a bipolar spindle. Recent work has shown that the C-terminal tail is particularly important to kinesin-5 function: the tail affects motor domain structure, ATP hydrolysis, motility, clustering, and sliding force measured for purified motors, as well as motility, clustering, and spindle assembly in cells. Because previous work has focused on presence or absence of the entire tail, the functionally important regions of the tail remain to be identified. We have therefore characterized a series of kinesin-5/Cut7 tail truncation alleles in fission yeast. Partial truncation causes mitotic defects and temperature-sensitive growth, while further truncation that removes the conserved BimC motif is lethal. We compared the sliding force generated bycuts7mutants using a kinesin-14 mutant background in which some microtubules detach from the spindle poles and are pushed into the nuclear envelope. These Cut7 driven protrusions decreased as more of the tail was truncated, and the most severe truncations produced no observable protrusions. Our observations suggest that the BimC motif and adjacent C-terminal amino acids are essential for sliding force, while the residues N-terminal to the BimC motif are more important for midzone localization. Spindle microtubule protrusions may be a useful tool to study sliding force generated by kinesin-5 motors.