Biochemical and Biophysical Research Communications
Depletion of CPAP by RNAi disrupts centrosome integrity and induces multipolar spindles
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70% CPAP proteins were depleted in siRNA-transfected cells (unpublished data).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).
The details of
Acknowledgments
This work was supported by the National Science Council project and a grant from the National Health Research Institute, Taipei, Taiwan.
References (15)
- et al.
Mitotic spindle assembly and chromosome segregation: refocusing on microtubule dynamics
Mol. Cell
(2004) - et al.
Mutations in aurora prevent centrosome separation leading to the formation of monopolar spindles
Cell
(1995) - et al.
A novel mechanism for activation of the protein kinase Aurora A
Curr. Biol.
(2003) - et al.
hTPX2 is required for normal spindle morphology and centrosome integrity during vertebrate cell division
Curr. Biol.
(2002) - et al.
A complex of NuMA and cytoplasmic dynein is essential for mitotic spindle assembly
Cell
(1996) - et al.
A Ran signalling pathway mediated by the mitotic kinase Aurora A in spindle assembly
Nat. Cell Biol.
(2003) - et al.
The ch-TOG/XMAP215 protein is essential for spindle pole organization in human somatic cells
Genes Dev.
(2003)
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