Kinesin-5 Mediated Chromosome Congression in Insect Spindles
The microtubule motor protein kinesin-5 is well known to establish the bipolar spindle by outward sliding of antiparallel interpolar microtubules. In yeast, kinesin-5 also facilitates chromosome alignment “congression” at the spindle equator by preferentially depolymerizing long kinetochore microtubules (kMTs). The motor protein kinesin-8 has also been linked to chromosome congression. Therefore, we sought to determine whether kinesin-5 or kinesin-8 facilitates chromosome congression in insect spindles.
RNAi of the kinesin-5 Klp61F and kinesin-8 Klp67A were performed separately in Drosophila melanogaster S2 cells to test for inhibited chromosome congression. Klp61F RNAi, Klp67A RNAi, and control metaphase mitotic spindles expressing fluorescent tubulin and fluorescent Cid were imaged, and their fluorescence distributions were compared.
RNAi of Klp61F with a weak Klp61F knockdown resulted in longer kMTs and less congressed kinetochores compared to control over a range of conditions, consistent with kinesin-5 length-dependent depolymerase activity. RNAi of the kinesin-8 Klp67A revealed that kMTs relative to the spindle lengths were not longer compared to control, but rather that the spindles were longer, indicating that Klp67A acts preferentially as a length-dependent depolymerase on interpolar microtubules without significantly affecting kMT length and chromosome congression.
This study demonstrates that in addition to establishing the bipolar spindle, kinesin-5 regulates kMT length to facilitate chromosome congression in insect spindles. It expands on previous yeast studies, and it expands the role of kinesin-5 to include kMT assembly regulation in eukaryotic mitosis.
KeywordsKinesin-5 Microtubules Mitosis
Double stranded RNA
Nuclear envelope breakdown
Polar ejection force
We thank Professor Lawrence Goldstein for providing us with rat-anti-Klp61F antibody. Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award No. R01GM071522 and R01GM076177 to D.J.O. and Award RO1GM044757 to T.S.H. E.T. was a recipient of a University of Minnesota Interdisciplinary Doctoral Fellowship through the Institute for Advanced Study.
E.T. conducted RNAi experiments, collected images, wrote analysis algorithms, ran statistical tests, analyzed and interpreted results, prepared figures, and wrote paper. E.T. and Y.H. designed primers, prepared dsRNA, and ran Western Blot. Y.H. contributed to intellectual ideas. T.H. and D.O., co-principal investigators, oversaw the project and contributed to intellectual ideas.
Conflicts of interest
Emily Tubman, Yungui He, Thomas S. Hays, and David J. Odde declare that they have no conflicts of interest.
No human studies were carried out by the authors for this article. No animal studies were carried out by the authors for this article.
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