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A more recent version of this article appeared on December 1, 2002
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Submitted on April 29, 2002
Revised on August 6, 2002
Accepted on August 20, 2002
1 Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115
2 Department of Biology, C.B. 3280, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
3 Department of Biology, C.B. 3280, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599; and Marine Biological Laboratory, Woods Hole, Massachusetts 02543
4 Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115; and Marine Biological Laboratory, Woods Hole, Massachusetts 02543
* Corresponding author. E-mail address: jennifer_tirnauer{at}hms.harvard.edu.
Microtubule polymerization dynamics at kinetochores are closely coupled to chromosome movements, but their regulation there is poorly understood. The microtubule plus end tracking protein EB1 is required both for regulation of microtubule dynamics and for maintenance of a euploid genome. To address the role of EB1 in aneuploidy, we visualized the dynamic localization of EB1 in mitotic PtK1 cells. Fluorescently labeled EB1 protein localized to the polymerizing ends of astral and spindle microtubules, and could be used to track their polymerization over time. EB1 also associated with a subset of attached kinetochores in late prometaphase and metaphase, and rarely in anaphase. Localization occurred in a narrow crescent, concave toward the centromere, consistent with targeting to the microtubule plus end - kinetochore interface. EB1 did not localize to kinetochores lacking attached kinetochore microtubules in prophase or early prometaphase, or in cells treated with Nocodazole. By time-lapse, EB1 specifically targeted to kinetochores moving anti-poleward, coupled to microtubule plus end polymerization, and was not present during plus end depolymerization. Its localization was independent of spindle bipolarity, the spindle checkpoint, and dynein/dynactin function. EB1 is thus first protein whose targeting reflects microtubule polymerization as well as kinetochore directionality. In this way EB1 differs from other microtubule plus end tracking proteins, which show enhanced kinetochore binding in the absence of microtubules. Our results suggest EB1 may have a role in regulating kinetochore microtubule polymerization and/or attachment.
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