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Vol. 17, Issue 9, 4069-4079, September 2006

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Measuring Nanometer Scale Gradients in Spindle Microtubule Dynamics Using Model Convolution Microscopy

Chad G. Pearson^*,, Melissa K. Gardner^,, Leocadia V. Paliulis, E. D. Salmon, David J. Odde, and Kerry Bloom

*Department of Molecular, Cellular, and Developmental Biology, University of Colorado at Boulder, Boulder, CO 80309-0347; Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN 55455; and Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3280

Submitted April 17, 2006; Revised June 9, 2006; Accepted June 20, 2006
Monitoring Editor: Orna Cohen-Fix

A computational model for the budding yeast mitotic spindle predicts a spatial gradient in tubulin turnover that is produced by kinetochore-attached microtubule (kMT) plus-end polymerization and depolymerization dynamics. However, kMTs in yeast are often much shorter than the resolution limit of the light microscope, making visualization of this gradient difficult. To overcome this limitation, we combined digital imaging of fluorescence redistribution after photobleaching (FRAP) with model convolution methods to compare computer simulations at nanometer scale resolution to microscopic data. We measured a gradient in microtubule dynamics in yeast spindles at 65-nm spatial intervals. Tubulin turnover is greatest near kinetochores and lowest near the spindle poles. A -tubulin mutant with decreased plus-end dynamics preserves the spatial gradient in tubulin turnover at a slower time scale, increases average kinetochore microtubule length 14%, and decreases tension at kinetochores. The -tubulin mutant cells have an increased frequency of chromosome loss, suggesting that the accuracy of chromosome segregation is linked to robust kMT plus-end dynamics.

This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E06-04-0312) on June 28, 2006.

These authors contributed equally to this work.

Address correspondence to: Kerry Bloom (kbloom{at}email.unc.edu)