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Cellular and Molecular Bioengineering

, Volume 11, Issue 1, pp 25–36 | Cite as

Kinesin-5 Mediated Chromosome Congression in Insect Spindles

  • Emily Tubman
  • Yungui He
  • Thomas S. Hays
  • David J. Odde
Article

Abstract

Introduction

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.

Methods

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.

Results

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.

Conclusions

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.

Keywords

Kinesin-5 Microtubules Mitosis 

Abbreviations

kMT

Kinetochore microtubule

iMT

Interpolar microtubule

RNAi

RNA interference

dsRNA

Double stranded RNA

NEB

Nuclear envelope breakdown

AO

Anaphase onset

PEF

Polar ejection force

Notes

Acknowledgments

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.

Author Contributions

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.

Ethical Standards

No human studies were carried out by the authors for this article. No animal studies were carried out by the authors for this article.

Supplementary material

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© Biomedical Engineering Society 2017

Authors and Affiliations

  1. 1.Department of Biomedical EngineeringUniversity of MinnesotaMinneapolisUSA
  2. 2.Department of Genetics, Cell Biology, and DevelopmentUniversity of MinnesotaMinneapolisUSA

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