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Published online 25 February 2003. doi:10.1083/jcb.200208143
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© The Rockefeller University Press, 0021-9525/2003/3/671 $5.00
The Journal of Cell Biology, Volume 160, Number 5, 671-683

Article

 Minus-end capture of preformed kinetochore fibers contributes to spindle morphogenesis

Alexey Khodjakov1,2, Lily Copenagle4, Michael B. Gordon3, Duane A. Compton3 and Tarun M. Kapoor2,4

1 Division of Molecular Medicine, Wadsworth Center, New York State Department of Health, Albany, NY 12201
2 Marine Biology Laboratory, Woods Hole, MA 02543
3 Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755
4 Laboratory of Chemistry and Cell Biology, The Rockefeller University, New York, NY 10021

Address correspondence to Tarun M. Kapoor, 1230 York Ave., The Rockefeller University, New York, NY 10021. Tel.: (212) 327-8176. Fax: (212) 327-8177. E-mail: kapoor{at}rockefeller.edu

Near-simultaneous three-dimensional fluorescence/differential interference contrast microscopy was used to follow the behavior of microtubules and chromosomes in living {alpha}-tubulin/GFP-expressing cells after inhibition of the mitotic kinesin Eg5 with monastrol. Kinetochore fibers (K-fibers) were frequently observed forming in association with chromosomes both during monastrol treatment and after monastrol removal. Surprisingly, these K-fibers were oriented away from, and not directly connected to, centrosomes and incorporated into the spindle by the sliding of their distal ends toward centrosomes via a NuMA-dependent mechanism. Similar preformed K-fibers were also observed during spindle formation in untreated cells. In addition, upon monastrol removal, centrosomes established a transient chromosome-free bipolar array whose orientation specified the axis along which chromosomes segregated. We propose that the capture and incorporation of preformed K-fibers complements the microtubule plus-end capture mechanism and contributes to spindle formation in vertebrates.

Key Words: mitosis; mitotic spindle; microtubules; spindle pole; chromosome positioning


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