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Microtubule plus-end dynamics in Xenopus egg extract spindles
Jennifer S Tirnauer1, E D Salmon, Timothy J Mitchison
1Woods Hole Marine Biological Laboratory, Woods Hole, Massachusetts 02543, USA. jennifer_tirnauer@hms.harvard.edu
Molecular Biology of the Cell
|February 10, 2004
Summary
Microtubule dynamics in Xenopus egg extract spindles were measured. Polymerization rates were consistent throughout, but depolymerization showed rapid rates with a subset of stable microtubules.
Area of Science:
- Cell Biology
- Biophysics
Background:
- Microtubule dynamics are crucial for spindle assembly.
- The influence of the spindle environment on microtubule dynamics remains unclear.
Purpose of the Study:
- To measure microtubule plus-end polymerization and depolymerization rates within Xenopus egg extract spindles.
- To investigate how the spindle environment affects microtubule dynamic instability.
Main Methods:
- Utilized fluorescence confocal imaging of EB1 to track growing microtubule plus-ends and measure polymerization rates.
- Employed microneedle-induced depolymerization fronts, tracked by polarization and fluorescence microscopy, to measure depolymerization rates.
- Assessed the effect of microtubule-stabilizing agents (hexylene glycol) and inhibitors (alpha-MCAK antibody, AMPPNP) on depolymerization.
Main Results:
- Microtubule plus-end polymerization occurred at ~11 microm/min throughout the spindle, similar to astral microtubules.
- An enrichment of polymerizing ends was observed near the spindle middle, suggesting increased nucleation or rescue.
- Depolymerization proceeded at ~30 microm/min, with a subset of microtubules exhibiting stability, particularly in larger fragments.
- Depolymerization was inhibited by hexylene glycol but not by alpha-MCAK antibody or AMPPNP.
Conclusions:
- Microtubule polymerization velocity is not regionally regulated within the spindle.
- The spindle middle may be a site of enhanced microtubule nucleation or rescue.
- Microtubule depolymerization is rapid but involves a stable subpopulation, potentially influenced by microtubule orientation.
- Hexylene glycol stabilizes microtubules against depolymerization, while MCAK and kinesin activity do not appear to be the primary drivers of depolymerization in this context.