Zhai Y, Kronebusch P J, Borisy G G
Laboratory of Molecular Biology, University of Wisconsin-Madison 53706, USA.
J Cell Biol. 1995 Nov;131(3):721-34. doi: 10.1083/jcb.131.3.721.
We have quantitatively studied the dynamic behavior of kinetochore fiber microtubules (kMTs); both turnover and poleward transport (flux) in metaphase and anaphase mammalian cells by fluorescence photoactivation. Tubulin derivatized with photoactivatable fluorescein was microinjected into prometaphase LLC-PK and PtK1 cells and allowed to incorporate to steady-state. A fluorescent bar was generated across the MTs in a half-spindle of the mitotic cells using laser irradiation and the kinetics of fluorescence redistribution were determined in terms of a double exponential decay process. The movement of the activated zone was also measured along with chromosome movement and spindle elongation. To investigate the possible regulation of MT transport at the metaphase-anaphase transition, we performed double photoactivation analyses on the same spindles as the cell advanced from metaphase to anaphase. We determined values for the turnover of kMTs (t1/2 = 7.1 +/- 2.4 min at 30 degrees C) and demonstrated that the turnover of kMTs in metaphase is approximately an order of magnitude slower than that for non-kMTs. In anaphase, kMTs become dramatically more stable as evidenced by a fivefold increase in the fluorescence redistribution half-time (t1/2 = 37.5 +/- 8.5 min at 30 degrees C). Our results also indicate that MT transport slows abruptly at anaphase onset to one-half the metaphase value. In early anaphase, MT depolymerization at the kinetochore accounted, on average, for 84% of the rate of chromosome movement toward the pole whereas the relative contribution of MT transport and depolymerization at the pole contributed 16%. These properties reflect a dramatic shift in the dynamic behavior of kMTs at the metaphase-anaphase transition. A release-capture model is presented in which the stability of kMTs is increased at the onset of anaphase through a reduction in the probability of MT release from the kinetochore. The reduction in MT transport at the metaphase-anaphase transition suggests that motor activity and/or subunit dynamics at the centrosome are subject to modulation at this key cell cycle point.
我们通过荧光光活化定量研究了中期和后期哺乳动物细胞中动粒纤维微管（kMTs）的动态行为，包括周转和向极运输（通量）。将用可光活化荧光素衍生化的微管蛋白显微注射到前中期的LLC-PK和PtK1细胞中，并使其掺入至稳态。使用激光照射在有丝分裂细胞的半纺锤体中的微管上产生一条荧光条，并根据双指数衰减过程确定荧光重新分布的动力学。还测量了活化区的移动以及染色体移动和纺锤体伸长。为了研究中期 - 后期转变时微管运输的可能调节，我们在细胞从中期进入后期时，对同一纺锤体进行了双光活化分析。我们确定了kMTs的周转值（30℃时t1/2 = 7.1 +/- 2.4分钟），并证明中期kMTs的周转比非kMTs慢约一个数量级。在后期，kMTs变得明显更稳定，荧光重新分布半衰期增加了五倍（30℃时t1/2 = 37.5 +/- 8.5分钟）证明了这一点。我们的结果还表明，微管运输在后期开始时突然减慢至中期值的一半。在后期早期，动粒处的微管解聚平均占染色体向极移动速率的84％，而极处微管运输和解聚的相对贡献为16％。这些特性反映了中期 - 后期转变时kMTs动态行为的巨大变化。提出了一种释放 - 捕获模型，其中后期开始时kMTs的稳定性通过降低微管从动粒释放的概率而增加。中期 - 后期转变时微管运输的减少表明，中心体处的运动活性和/或亚基动力学在这个关键的细胞周期点受到调节。
