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MBC in Press, published online ahead of print June 28, 2006
Mol. Biol. Cell 10.1091/mbc.E06-04-0312

A more recent version of this article appeared on September 1, 2006
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Submitted on April 17, 2006
Revised on June 9, 2006
Accepted on June 20, 2006

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

Chad G. Pearson,*{dagger} Melissa K. Gardner,{dagger}{ddagger} Leocadia V. Paliulis,{sect} E. D. Salmon,{sect} David J. Odde,{ddagger} and Kerry Bloom{sect}

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

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 {beta}-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% percent, and decreases tension at kinetochores. The {beta}-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.

{dagger}These authors contributed equally to this work.

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




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