Rapid rate of tubulin dissociation from microtubules in the mitotic spindle in vivo measured by blocking polymerization with colchicine.
Rapid rate of tubulin dissociation from microtubules in the mitotic spindle in vivo measured by blocking polymerization with colchicine.
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DOI：
10.1083/jcb.99.3.1066
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发表时间：
1984-09
期刊：
影响因子：
--
通讯作者：
Hays T
中科院分区：
文献类型：
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作者：
Salmon ED;McKeel M;Hays T
在分裂中期，组装成纺锤体微管的微管蛋白的量相对恒定;微管蛋白结合的速率等于解离的速率。为了测量固有的解离速率，我们显微注射高浓度的秋水仙碱，或其衍生物秋水仙胺，海胆胚胎中期绑定的自由微管蛋白，从而迅速阻断聚合。通过双折射延迟（BR）的变化从校准的视频信号测量微管解体的速率。注射秋水仙碱或秋水仙胺后，在0.1-3.0 mM的最终细胞内浓度的初始延迟后，BR迅速下降，同时在整个中央纺锤体和紫菀。在中心半纺锤体中测得的BR在约20 s的特征周期内呈指数下降至其初始值的10%;在此浓度范围内，BR衰减的速率常数k = 0.11 +/- 0.023 s-1和相应的半衰期t1/2约为6.5 +/- 1.1 s。低于0.1 mM秋水仙碱或秋水仙胺，BR降低的速率是浓度依赖性的。电子显微镜照片显示，BR的快速下降对应于nonkinetochore微管的消失; kinetochore纤维微管差异稳定。作为对照，光秋水仙碱，它不结合微管蛋白具有高亲和力，被证明是没有影响的纺锤体BR在细胞内浓度为0.5 mM。如果秋水仙碱和秋水仙胺块只聚合，然后从nonkinetochore纺锤体微管的微管蛋白解离的初始速率是在每秒180-992二聚体的范围内。这个速率范围是基于k = 11%的初始聚合物每秒和从电子显微照片估计的半纺锤体微管的平均长度为1- 5.5微米。更慢的速率微管协会预测从末端依赖性微管组装以前在体外测量的特性时，关联速率常数校正为较低的速率微管在胚胎细胞质中的扩散。这种差异的各种可能性进行了讨论。
At metaphase, the amount of tubulin assembled into spindle microtubules is relatively constant; the rate of tubulin association equals the rate of dissociation. To measure the intrinsic rate of dissociation, we microinjected high concentrations of colchicine, or its derivative colcemid, into sea urchin embryos at metaphase to bind the free tubulin, thereby rapidly blocking polymerization. The rate of microtubule disassembly was measured from a calibrated video signal by the change in birefringent retardation (BR). After an initial delay after injection of colchicine or colcemid at final intracellular concentrations of 0.1-3.0 mM, BR decreased rapidly and simultaneously throughout the central spindle and aster. Measured BR in the central half-spindle decreased exponentially to 10% of its initial value within a characteristic period of approximately 20 s; the rate constant, k = 0.11 +/- 0.023 s-1, and the corresponding half-time, t 1/2, of BR decay was approximately 6.5 +/- 1.1 s in this concentration range. Below 0.1 mM colchicine or colcemid, the rate at which BR decreased was concentration dependent. Electron micrographs showed that the rapid decrease in BR corresponded to the disappearance of nonkinetochore microtubules; kinetochore fiber microtubules were differentially stable. As a control, lumicolchicine, which does not bind to tubulin with high affinity, was shown to have no effect on spindle BR at intracellular concentrations of 0.5 mM. If colchicine and colcemid block only polymerization, then the initial rate of tubulin dissociation from nonkinetochore spindle microtubules is in the range of 180-992 dimers per second. This range of rates is based on k = 11% of the initial polymer per second and an estimate from electron micrographs that the average length of a half-spindle microtubule is 1- 5.5 micron. Much slower rates of tubulin association are predicted from the characteristics of end-dependent microtubule assembly measured previously in vitro when the association rate constant is corrected for the lower rate of tubulin diffusion in the embryo cytoplasm. Various possibilities for this discrepancy are discussed.