Novel Kinesin Regulators of Gamma-TuRC

Abstract

Mitotic spindle assembly is a critical control point in eukaryotic chromosome segregation. In fission yeast conserved mitotic kinesin-like proteins (Klps) Kinesin-14 and Kinesin-5 are emerging as novel regulators of the γ-tubulin ring complex (γ-TuRC) MTOC. While these Klps typically participate in microtubule roles for spindle assembly, alternatively in fission yeast they localize to γ-TuRC and when co-deleted a bipolar spindle forms. Our analysis by genetics, biochemistry, cell biology and nanotechnology provides new knowledge on mitotic mechanisms in S. pombe. Previous work from our lab and others uncovered a novel functional relationship between Kinesin-14 Pkl1 and all proteins of the γ-tubulin small complex (γ-TuSC). We demonstrated that Pkl1 binds γ-TuRC through Motor and Tail interactions to directly regulate its function. A Pkl1 Tail peptide is sufficient in vitro and disrupts γ-TuRC structure by removal of γ-tubulin subunits (Cell Cycle 2013), an action that is reversible. New data from our lab indicates that the essential Kinesin-5 Cut7 is actually dispensable in the absence of Pkl1 with which it shares an important antagonistic MTOC regulatory relationship. Cut-7 associates with FPLC purified γ-TuRC and binds to γ-tubulin similarly as Pkl1. We demonstrate in vivo that Pkl1 blocks microtubule nucleation from γ-TuRC and additionally influences spindle microtubule numbers and microtubule overlap. Kinetochore-based microtubule motors Klp5 and Klp6 may facilitate spindle organization, since triple mutants of Pkl1, Cut7 and either Kinesin-6 Klp9 or Kinesin-14 Klp2 are viable. Previously we showed in vivo that human Kinesin-14 HSET, but not Drosophila Ncd, functionally replaces Pkl1 and more recently that human γ-TuSC proteins GCP2 and GCP3 replace fission yeast Alp4 and Alp6 (JCS 2013). Our research highlights conserved in vivo functions of γ-TuSC and provides a new model of spindle assembly in S. pombe.