Abstract
ABSTRACTKinesin-13 motors are unusual in that they do not walk along microtubules, but instead diffuse to the ends, where they remove tubulin dimers, regulating microtubule dynamics. Here we show that Drosophila kinesin-13 klp10A regulates oocyte meiosis I spindle length and is haplo-insufficient – KLP10A, reduced by RNAi or a loss-of-function P element insertion mutant, results in elongated and mispositioned oocyte spindles, and abnormal cortical microtubule asters and aggregates. KLP10A knockdown by RNAi does not significantly affect microtubule growth rates in oocyte spindles, but, unexpectedly, EB1 binding and unbinding are slowed, suggesting a previously unobserved role for kinesin-13 in mediating EB1 binding interactions with microtubules. Kinesin-13 may regulate spindle length both by disassembling subunits from microtubule ends and facilitating EB1 binding to plus ends. We also observe an increased number of paused microtubules in klp10A RNAi knockdown spindles, consistent with a reduced frequency of microtubule catastrophes. Overall, our findings indicate that reduced kinesin-13 decreases microtubule disassembly rates and affects EB1 interactions with microtubules, rather than altering microtubule growth rates, causing spindles to elongate and abnormal cortical microtubule asters and aggregates to form.
Highlights
The kinesin motor proteins bind to microtubules and hydrolyze ATP, producing force in the cell to transport cargo along microtubules or slide microtubules relative to one another
We examined live oocytes from wild-type and klp10A RNAi/atubGal4 knockdown (RNAi) knockdown females, and klp10A P mut line #50/+ heterozygous females to determine the effects of reduced KLP10A levels on oocyte spindles
Ceq, which is a measure of the fraction of bound protein at equilibrium, was the same or only slightly different for the RNAi knockdown and wild type. These results indicate that klp10A RNAi knockdown slows EB1 binding and dissociation in the spindle, consistent with the hypothesis that klp10A RNAi knockdown reduces the frequency of microtubule catastrophes
Summary
The kinesin motor proteins bind to microtubules and hydrolyze ATP, producing force in the cell to transport cargo along microtubules or slide microtubules relative to one another. Many kinesin motor proteins play essential roles in spindle assembly and elongation in dividing cells, driving spindle and chromosome motility during mitosis and meiosis (Mitchison and Salmon, 2001; Mogilner and Craig, 2010). The motors crosslink and slide microtubules, producing force to assemble spindles, and position chromosomes for movement to opposite poles.
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