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Chromosoma

, Volume 119, Issue 4, pp 371–379 | Cite as

Re-evaluating the role of Tao1 in the spindle checkpoint

  • Frederick G. Westhorpe
  • Maria A. Diez
  • Mark D. J. Gurden
  • Anthony Tighe
  • Stephen S. TaylorEmail author
Research Article

Abstract

The spindle checkpoint restrains anaphase onset and mitotic exit until all chromosomes are stably attached to the mitotic spindle via their kinetochores. The Tao1 protein kinase was recently reported as a novel spindle checkpoint component. When an siRNA was used to repress Tao1, the essential spindle checkpoint component Mad2 failed to localise to kinetochores, and cells rapidly exited mitosis. Tao1 was also shown to interact with BubR1, another essential checkpoint component, and be rapidly degraded after mitosis, a feature typical of many mitotic regulators. Here, we identify four different siRNAs that repress Tao1 protein levels as efficiently as the previously reported siRNA. However, these siRNAs do not override the spindle checkpoint. We also present data indicating that Tao1 does not interact with BubR1 and that it is not rapidly degraded after mitosis. We show that the previously reported siRNA not only represses Tao1 but also dramatically reduces Mad2 protein levels. Crucially, expression of exogenous Mad2, but not Tao1, rescued the spindle checkpoint phenotype induced by this siRNA. Thus, the key functional data implicating Tao1 in the spindle checkpoint can be explained by an off-target siRNA phenomenon that results in Mad2 inhibition. Taken together, our data do not support the notion that Tao1 is a component of the spindle checkpoint.

Keywords

Nocodazole Spindle Checkpoint Mitotic Exit Mitotic Entry Mad2 Protein 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

Acknowledgements

F.G.W. and M.D.J.G. are funded by studentships from the Wellcome Trust. M.A.D., A.T. and S.S.T. are funded by Cancer Research UK.

Supplementary material

412_2010_261_Fig1_ESM.gif (66 kb)
Fig. S1

Quantification of Tao1 RNAi. (a) Immunoblot of HeLa cells treated with the indicated siRNAs probed with antibodies against Tao1, Mad2 and Bub3. Note that this immunoblot is an extension of the immunoblot in Fig. 5c, now showing the individual Tao1-si-oligos from Tao1-Sp. (b) Quantitative immunoblotting showing the relative Tao1 and Mad2 protein levels after the indicated RNAi treatment. Note that the Tao1 SMARTpool (Tao1-Sp) and the four individual siRNAs repress Tao1 as effectively as si3NCB but have little effect on Mad2. (c) Immunoblot of HeLa cells treated with si3NCB at the indicated concentrations probed with antibodies against Tao1, Mad2 and Bub3. Tao1-Sp and control-treated HeLa cells (both 200 nM) are used as controls. (d) Quantitative immunoblotting showing the relative Tao1 and Mad2 protein levels after the indicated RNAi treatment. The values in (b) and (d) show the mean ± SEM and are derived from three independent experiments. (GIF 66 kb)

412_2010_261_Fig1_ESM.tif (3.1 mb)
High resolution image (TIFF 3217 kb)

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Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Frederick G. Westhorpe
    • 1
  • Maria A. Diez
    • 1
  • Mark D. J. Gurden
    • 1
  • Anthony Tighe
    • 1
  • Stephen S. Taylor
    • 1
    Email author
  1. 1.Faculty of Life Sciences, Michael Smith Building, Oxford RoadUniversity of ManchesterManchesterUK

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