Depletion of CPAP by RNAi disrupts centrosome integrity and induces multipolar spindles

doi:10.1016/j.bbrc.2005.11.074

Biochemical and Biophysical Research Communications

Volume 339, Issue 3, 20 January 2006, Pages 742-747

https://doi.org/10.1016/j.bbrc.2005.11.074 Get rights and content

Abstract

We previously identified a novel microtubule-destabilizing motif in CPAP that can disassemble microtubules. To examine further the CPAP function in human cells, we used siRNA to knockdown its expression. Our results showed that CPAP depletion arrested cells in mitosis and induced apoptosis. Interestingly, more than 40% of these mitotic cells had multiple spindle poles. Furthermore, inhibition of the kinesin Eg5 in CPAP-depleted cells resulted in monopolar spindles, indicating that Eg5 function is required for multipolar spindle formation in the absence of CPAP. Together, our results reveal a structural role for CPAP to maintain centrosome integrity and normal spindle morphology during cell division.

Section snippets

Materials and methods

Synthesis of SMARTpool siRNA duplexes. The SMARTpool oligonucleotides that contain four selected CPAP siRNA duplexes were synthesized with symmetric 3′-UU overhangs (Dharmacon RNA Technologies, Lafayette, CO) and double-stranded RNA annealed according to the manufacturer’s instructions. These four CPAP siRNA duplexes were sp1 (GGACTGACCTTGAAGAGAA, position 2395–2413); sp2 (CCAAACAACTTCATTCATT, position 1963–1981); sp3 (AGAATTAGCTCGAATAGAA, position 2861–2879); and sp4 (AAACAGACCTTTGAAGATT,

Results

The depletion efficiency of CPAP was first analyzed by SMARTpool CPAP siRNA duplexes, which contain four selected siRNA duplexes. Immunoblot analyses showed that >70% CPAP proteins were depleted in siRNA-transfected cells (unpublished data). Each selected siRNA duplex in the SMARTpool was then tested for the depletion efficiency of CPAP. The siRNA duplex (position 2395–2413) that revealed high knockdown efficiency was selected for the target sequence to construct a plasmid-based pSuper-GFP

Discussion

It has been shown that the establishment and maintenance of spindle bipolarity require a coordinated action of a number of proteins, such as the plus-end motor Eg5, the minus-end motor dynein, TPX2, NuMA, and the Aurora kinases [1]. Here, we report for the first time that CPAP, a microtubule destabilizer, may also be required for centrosome integrity and normal spindle morphology, since depletion of CPAP disrupts centrosome integrity and induces multipolarity (Fig. 2, Fig. 3).

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Acknowledgments

This work was supported by the National Science Council project and a grant from the National Health Research Institute, Taipei, Taiwan.

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Depletion of CPAP by RNAi disrupts centrosome integrity and induces multipolar spindles

Abstract

Section snippets

Materials and methods

Results

Discussion

Acknowledgments

Mol. Cell

Cell

Curr. Biol.

Curr. Biol.

Cell

A Ran signalling pathway mediated by the mitotic kinase Aurora A in spindle assembly

Nat. Cell Biol.

The ch-TOG/XMAP215 protein is essential for spindle pole organization in human somatic cells

Genes Dev.