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Poly(ADP-ribose) is required for spindle assembly and structure

Nature volume 432, pages 645–649 (2004)Cite this article

Abstract

The mitotic spindle is typically thought of as an array of microtubules, microtubule-associated proteins and motors that self-organizes to align and segregate chromosomes1. The major spindle components consist of proteins and DNA, the primary structural elements of the spindle1. Other macromolecules including RNA and lipids also associate with spindles, but their spindle function, if any, is unknown. Poly(ADP-ribose) (PAR) is a large, branched, negatively charged polymeric macromolecule whose polymerization onto acceptor proteins is catalysed by a family of poly(ADP-ribose) polymerases (PARPs)2. Several PARPs localize to the spindle in vertebrate cells, suggesting that PARPs and/or PAR have a role in spindle function2. Here we show that PAR is enriched in the spindle and is required for spindle function—PAR hydrolysis or perturbation leads to rapid disruption of spindle structure, and hydrolysis during spindle assembly blocks the formation of bipolar spindles. PAR exhibits localization dynamics that differ from known spindle proteins and are consistent with a low rate of turnover in the spindle. Thus, PAR is a non-proteinaceous, non-chromosomal component of the spindle required for bipolar spindle assembly and function.

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Figure 1: PAR is enriched in the spindle.
Figure 2: PAR is required for bipolar spindle assembly and function.
Figure 3: PAR exchange between the spindle and cytoplasm is slow.

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Acknowledgements

We thank A. Pollington, B. Ward, J. Tirnauer and B. Brieher for critical reading of the manuscript; A. Straight for CENPE antibodies; A. Groen and D. Miyamoto for TPX2, NuMA and Eg5 antibodies; Z. Perlman for assistance with data analysis; D. L. Coyle for technical support; and the Nikon Imaging Facility at Harvard Medical School for use of microscopes. This work was supported by NIH grants to T.M.J. and M.K.J.

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Authors and Affiliations

  1. Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, 02115, USA

    Paul Chang & Timothy J. Mitchison

  2. Department of Pharmacology and Toxicology, College of Pharmacy, and Arizona Cancer Center, University of Arizona, Tucson, Arizona, 85721, USA

    Myron K. Jacobson

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  1. Paul Chang
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Correspondence to Paul Chang.

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The authors declare that they have no competing financial interests.

Supplementary information

Supplementary Figure S1

PAR Localization to the kinetochore. Xenopus extract spindles were incubated with Alexa 488-labeled PAR antibody, and X-rhodamine-labeled CENPE antibody (CENP) and, visualized using spinning disk confocal microscopy every 30 s for 20 min. PAR co-localized with CENPE at every time point. In Merge, PAR is green and CENPE, red. (JPG 36 kb)

Supplementary Movie M1

Real time spinning disk confocal microscopy of Xenopus egg extract spindles treated with 100 µg/ml PARG. Images were obtained every 30s for 15 min. (MP4 156 kb)

Supplementary Movie M2

Real time spinning disk confocal microscopy of Xenopus egg extract spindles treated with 500 µg/ml anti PAR antibody. Images were obtained every 30s for 15 min. (MP4 186 kb)

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Chang, P., Jacobson, M. & Mitchison, T. Poly(ADP-ribose) is required for spindle assembly and structure. Nature 432, 645–649 (2004). https://doi.org/10.1038/nature03061

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