Zhou B B, Kirschner M W
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Cell Motil Cytoskeleton. 1999;43(1):43-51. doi: 10.1002/(SICI)1097-0169(1999)43:1<43::AID-CM5>3.0.CO;2-A.
Previous work has shown that catastrophe frequency is the predominant dynamic parameter of microtubules that changes dramatically during the cell cycle. As an alternative to videomicroscopy assays, we have developed a biochemical assay to measure directly the average catastrophe rate of a population of microtubules. In this assay, the growing plus end of the microtubules, polymerized off seeds, are labeled with a brief pulse of alpha-32P-GTP, followed by a cold GTP chase. The rate of loss of 32P label in microtubules measured by this method is equal to the catastrophe frequency at microtubule plus ends measured by videomicroscopy of individual microtubules. Addition of mitotic extract from Xenopus eggs increases the catastrophe rate of purified tubulin by almost 100-fold, while interphase extract alters the catastrophe rate by about 20-fold as compared to pure tubulin. Most of the catastrophe-promoting activities in both mitotic and interphase extracts is found in particulate fractions. High-speed centrifugation of extracts appears to eliminate the components required for increasing microtubule catastrophe, but does not eliminate the cell cycle difference in microtubule dynamics. This assay provides a new approach to quantitate microtubule catastrophe rates. It will be of particular interest to search for catastrophe factors associated with intracellular membranes or other insoluble components.
先前的研究表明，灾难频率是微管的主要动态参数，在细胞周期中会发生显著变化。作为视频显微镜检测的替代方法，我们开发了一种生化检测方法，以直接测量一群微管的平均灾难率。在该检测中，从种子上聚合生长的微管的正端，用短暂的α-32P-GTP脉冲标记，随后进行冷GTP追踪。通过这种方法测量的微管中32P标记的丢失率，等于通过单个微管的视频显微镜测量的微管正端的灾难频率。添加非洲爪蟾卵的有丝分裂提取物可使纯化微管蛋白的灾难率增加近100倍，而间期提取物与纯微管蛋白相比，可使灾难率改变约20倍。有丝分裂和间期提取物中大多数促进灾难的活性都存在于颗粒部分。提取物的高速离心似乎消除了增加微管灾难所需的成分，但并未消除微管动力学中的细胞周期差异。该检测提供了一种定量微管灾难率的新方法。寻找与细胞内膜或其他不溶性成分相关的灾难因子将特别有趣。
