Microtubule plus-end dynamics in Xenopus egg extract spindles

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 local...

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Bibliographic Details
Published in:Molecular biology of the cell 2004-04, Vol.15 (4), p.1776-1784
Main Authors: Tirnauer, Jennifer S, Salmon, E D, Mitchison, Timothy J
Format: Article
Language:English
Subjects:
Adenylyl Imidodiphosphate - pharmacology
Animals
Glycols - chemistry
Glycols - pharmacology
Image Processing, Computer-Assisted
Kinesin - metabolism
Metaphase
Microscopy, Confocal
Microscopy, Fluorescence
Microtubules - enzymology
Microtubules - ultrastructure
Ovum - metabolism
Spindle Apparatus
Xenopus
Xenopus laevis
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Summary: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 approximately 11 microm/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 ( approximately 30 microm/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 alpha-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.
ISSN:1059-1524
1939-4586
1059-1524
DOI:10.1091/mbc.E03-11-0824