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Abstract
The metaphase-spindle steady-state length occurs as spindle microtubules ;flux', incorporating new subunits at their plus ends, while simultaneously losing subunits from their minus ends. Orbit/Mast/CLASP is required for tubulin subunit addition at kinetochores, and several kinesins regulate spindle morphology and/or flux by serving as microtubule depolymerases. Here, we use RNA interference in S2 cells to examine the relationship between Orbit and the four predicted kinesin-type depolymerases encoded by the Drosophila genome (Klp10A, Klp59C, Klp59D and Klp67A). Single depletion of Orbit results in monopolar spindles, mitotic arrest and a subsequent increase in apoptotic cells. These phenotypes are rescued by co-depleting Klp10A but none of the other three depolymerases. Spindle bipolarity is restored by preventing the spindle collapse seen in cells that lack Orbit, leading to functional spindles that are similar to controls in shape and length. We conclude that Klp10A exclusively antagonises Orbit in the regulation of bipolar spindle formation and maintenance.
Highlights
In mitotic Drosophila cells the replicated centrosomes usually separate from one another around the intact prophase nuclear envelope
The recent finding that Orbit is required for the incorporation of tubulin subunits at the kinetochore for MT elongation and flux (Maiato et al, 2005) might offer an explanation for the spindle collapse seen in Orbit-deficient cells
Knockdown of Klp10A but not of the other kinesin-type depolymerases moderates the amount of apoptosis in Orbit-deficient cells To determine which, if any, of the kinesin-8 and/or kinesin-13 family members might counteract the activity of Orbit, we performed double RNA interference (RNAi) depletion experiments for orbit combined with each of the four identified Drosophila kinesin-like protein (Klp) depolymerases in S2 cells
Summary
In mitotic Drosophila cells the replicated centrosomes usually separate from one another around the intact prophase nuclear envelope As this envelope becomes fenestrated at prometaphase onset, dynamic astral microtubules (MTs) nucleated from each of the opposing centrosomes invade the nuclear space where their plus-ends become attached to the sister kinetochores of each chromosome. Mutations in orbit or RNA interference (RNAi) knockdown do not affect prophase centrosome separation but cause spindle collapse at flux-like velocities only after kMT formation during prometaphase (Maiato et al, 2002; Maiato et al, 2005) The kinetochores in such cells fail to remain associated with MT plus-ends and the chromosomes become buried in the centre of a monoaster causing a mitotic arrest (Maiato et al, 2002). The recent finding that Orbit is required for the incorporation of tubulin subunits at the kinetochore for MT elongation and flux (Maiato et al, 2005) might offer an explanation for the spindle collapse seen in Orbit-deficient cells
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