Pearson Chad G, Gardner Melissa K, Paliulis Leocadia V, Salmon E D, Odde David J, Bloom Kerry
Department of Molecular, Cellular, and Developmental Biology, University of Colorado at Boulder, Boulder, CO 80309-0347, USA.
Mol Biol Cell. 2006 Sep;17(9):4069-79. doi: 10.1091/mbc.e06-04-0312. Epub 2006 Jun 28.
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.
一种用于出芽酵母有丝分裂纺锤体的计算模型预测，着丝粒附着微管（kMT）的正端聚合和解聚动力学产生了微管蛋白周转的空间梯度。然而，酵母中的kMT通常比光学显微镜的分辨率极限短得多，使得可视化这种梯度变得困难。为了克服这一限制，我们将光漂白后荧光重新分布（FRAP）的数字成像与模型卷积方法相结合，以将纳米级分辨率的计算机模拟与微观数据进行比较。我们以大约65纳米的空间间隔测量了酵母纺锤体中微管动力学的梯度。微管蛋白周转在着丝粒附近最大，在纺锤体极附近最小。一种正端动力学降低的β-微管蛋白突变体在较慢的时间尺度上保持微管蛋白周转的空间梯度，使平均着丝粒微管长度增加约14%，并降低着丝粒处的张力。β-微管蛋白突变体细胞的染色体丢失频率增加，这表明染色体分离的准确性与强大的kMT正端动力学有关。
