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Vol. 18, Issue 1, 282-294, January 2007
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*Medical Science Program and Department of Biology, Indiana University, Bloomington, IN 47405; and Department of Biological Sciences, Wright State University, Dayton, OH 45435
Submitted August 17, 2006;
Revised September 27, 2006;
Accepted October 30, 2006
Monitoring Editor: Stephen Doxsey
Spindle assembly and accurate chromosome segregation require the proper regulation of microtubule dynamics. MCAK, a Kinesin-13, catalytically depolymerizes microtubules, regulates physiological microtubule dynamics, and is the major catastrophe factor in egg extracts. Purified GFP-tagged MCAK domain mutants were assayed to address how the different MCAK domains contribute to in vitro microtubule depolymerization activity and physiological spindle assembly activity in egg extracts. Our biochemical results demonstrate that both the neck and the C-terminal domain are necessary for robust in vitro microtubule depolymerization activity. In particular, the neck is essential for microtubule end binding, and the C-terminal domain is essential for tight microtubule binding in the presence of excess tubulin heterodimer. Our physiological results illustrate that the N-terminal domain is essential for regulating microtubule dynamics, stimulating spindle bipolarity, and kinetochore targeting; whereas the C-terminal domain is necessary for robust microtubule depolymerization activity, limiting spindle bipolarity, and enhancing kinetochore targeting. Unexpectedly, robust MCAK microtubule (MT) depolymerization activity is not needed for sperm-induced spindle assembly. However, high activity is necessary for proper physiological MT dynamics as assayed by Ran-induced aster assembly. We propose that MCAK activity is spatially controlled by an interplay between the N- and C-terminal domains during spindle assembly.
The online version of this contains supplemental material at MBC Online (http://www.molbiolcell.org). This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E06-08-0724)) on November 8, 2006.
Address correspondence to: Claire E. Walczak (cwalczak{at}indiana.edu)
Abbreviations used: MT, microtubule; GMPCPP, guanylyl-(a,b)-methylene-diphosphonate; GFP, green fluorescent protein; GM, GFP-MCAK; SEM, standard error of the mean.
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