Measuring nanometer scale gradients in spindle microtubule dynamics using model convolution microscopy.
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 approximately 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 approximately 14%, 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.
Pearson
Chad G
CG
Department of Molecular, Cellular, and Developmental Biology, University of Colorado at Boulder, Boulder, CO 80309-0347, USA.
Gardner
Melissa K
MK
Paliulis
Leocadia V
LV
Salmon
E D
ED
Odde
David J
DJ
Bloom
Kerry
K
D020090:Chromosome Segregation / Q000235:genetics; D015825:Chromosomes, Fungal / Q000235:genetics; D003198:Computer Simulation; D049452:Green Fluorescent Proteins / Q000378:metabolism; D018386:Kinetochores / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D008853:Microscopy / Q000379:methods*; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008870:Microtubules / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D036103:Nanotechnology / Q000379:methods*; D010957:Plasmids / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D008941:Spindle Apparatus / Q000737:chemistry* / Q000378:metabolism*; D014404:Tubulin / Q000235:genetics / Q000378:metabolism; D000097573:Red Fluorescent Protein
D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D008869:Microtubule-Associated Proteins; D050505:Mutant Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014404:Tubulin; D000097573:Red Fluorescent Protein; C505600:Spc29 protein, S cerevisiae