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Vol. 15, Issue 4, 1776-1784, April 2004
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* Woods Hole Marine Biological Laboratory, Woods Hole, Massachusetts 02543;
Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115; and
Department of Biology, University of North Carolina, Chapel Hill, North Carolina 27599
Submitted November 18, 2003;
Revised January 9, 2004;
Accepted January 10, 2004
Monitoring Editor: Frank Solomon
Microtubule dynamics underlie spindle assembly, yet we do not know how the spindle environment affects these dynamics. We developed methods for measuring two key parameters of microtubule plus-end dynamic instability in Xenopus egg extract spindles. To measure plus-end polymerization rates and localize growing plus ends, we used fluorescence confocal imaging of EB1. This revealed plus-end polymerization throughout the spindle at 
11 µm/min, similar to astral microtubules, suggesting polymerization velocity is not regionally regulated by the spindle. The ratio of EB1 to microtubule fluorescence revealed an enrichment of polymerizing ends near the spindle middle, indicating enhanced nucleation or rescue there. We measured depolymerization rates by creating a front of synchronized depolymerization in spindles severed with microneedles. This front could be tracked by polarization and fluorescence microscopy as it advanced from each cut edge toward the associated pole. Both imaging modalities revealed rapid depolymerization (30 µm/min) superimposed on a subset of microtubules stable to depolymerization. Larger spindle fragments contained a higher percentage of stable microtubules, which we believe were oriented with their minus ends facing the cut. Depolymerization was blocked by the potent microtubule stabilizing agent hexylene glycol, but was unaffected by 
-MCAK antibody and AMPPNP, which block catastrophe and kinesin motility, respectively. These measurements move us closer to understanding the complete life history of a spindle microtubule.
Abbreviations used: EM, electron microscopy; FSM, fluorescence speckle microscopy; MCAK, mitotic centromere associated kinesin (also called XKCM1 Xenopus kinesin central motor domain 1).
Online version of this article contains videos. Online version is available at www.molbiolcell.org.
Corresponding author. E-mail address: jennifer_tirnauer{at}hms.harvard.edu.
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