Abstract
Bipolar mitotic spindles play a critical part in accurate chromosome segregation. During late mitosis, spindle microtubules undergo drastic elongation in a process called anaphase B. Two kinesin motors, Kinesin-5 and Kinesin-6, are thought to generate outward forces to drive spindle elongation, and the microtubule crosslinker Ase1/PRC1 maintains structural integrity of antiparallel microtubules. However, how these three proteins orchestrate this process remains unknown. Here we explore the functional interplay among fission yeast Kinesin-5/Cut7, Kinesin-6/Klp9 and Ase1. Using total internal reflection fluorescence microscopy, we show that Klp9 forms homotetramers and that Klp9 is a processive plus end-directed motor. klp9Δase1Δ is synthetically lethal. Surprisingly, this lethality is not ascribable to the defective motor activity of Klp9; instead, it is dependent upon a nuclear localisation signal and coiled coil domains within the non-motor region. We isolated a cut7 mutant (cut7-122) that displays temperature sensitivity only in the absence of Klp9. Interestingly, cut7-122 alone is impaired in spindle elongation during anaphase B, and furthermore, cut7-122klp9Δ double mutants exhibit additive defects. We propose that Klp9 plays dual roles during anaphase B; one is motor-dependent that collaborates with Cut7 in force generation, while the other is motor-independent that ensures structural integrity of spindle microtubules together with Ase1.
Highlights
Bipolar mitotic spindles play a critical part in accurate chromosome segregation
Using total internal reflection fluorescence microscopy, we show that Klp[9] forms homotetramers and that Klp[9] is a processive plus end-directed motor. klp9Δase1Δ is synthetically lethal
We propose that Klp[9] plays dual roles during anaphase B; one is motor-dependent that collaborates with Cut[7] in force generation, while the other is motor-independent that ensures structural integrity of spindle microtubules together with Ase[1]
Summary
Bipolar mitotic spindles play a critical part in accurate chromosome segregation. During late mitosis, spindle microtubules undergo drastic elongation in a process called anaphase B. Kinesin-5 and Kinesin-6, are thought to generate outward forces to drive spindle elongation, and the microtubule crosslinker Ase1/PRC1 maintains structural integrity of antiparallel microtubules How these three proteins orchestrate this process remains unknown. We propose that Klp[9] plays dual roles during anaphase B; one is motor-dependent that collaborates with Cut[7] in force generation, while the other is motor-independent that ensures structural integrity of spindle microtubules together with Ase[1]. In fission yeast, the sole member of Kinesin-6, Klp[9], appears to play multiple roles on its own[26,37,45] Despite these similarities and adaptations, some evolutionary diversifications exist; for instance, MKLP1/CHO1/Kif[23], but not Klp[9], is a structural component of the Centralspindlin complex[46]
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