Spindle microtubule dynamics in sea urchin embryos: analysis using a fluorescein-labeled tubulin and measurements of fluorescence redistribution after laser photobleaching.
Spindle microtubule dynamics in sea urchin embryos: analysis using a fluorescein-labeled tubulin and measurements of fluorescence redistribution after laser photobleaching.
复制标题
DOI：
10.1083/jcb.99.6.2165
复制
发表时间：
1984-12
期刊：
影响因子：
--
通讯作者：
McIntosh JR
中科院分区：
文献类型：
--
作者：
Salmon ED;Leslie RJ;Saxton WM;Karow ML;McIntosh JR
微管蛋白被纳入纺锤体微管与二聚体微管蛋白在细胞质中的交换率已在海胆卵中通过研究光漂白后的荧光再分布（FRAP）进行了测量。用二氯三嗪基氨基荧光素（DTAF）标记牛脑微管蛋白。将DTAF-微管蛋白注射到杂色鱼虱的受精卵中，并使其与内源性微管蛋白库平衡。荧光纺锤体形成的同时，纺锤体被视为在控制鸡蛋，和注射胚胎进行了许多周期的分裂计划，这表明DTAF-微管蛋白是一个很好的类似物微管蛋白在体内。氩激光的微束已被用来漂白荧光纺锤体的部分，和FRAP已被记录与敏感的摄像机。激光漂白不影响纺锤体结构，如偏振光学所见，也不影响纺锤体功能，如通过有丝分裂的进展速度所见，即使一个纺锤体在单个细胞周期中被漂白多次。视频图像分析已被用来测量FRAP的速率，并获得荧光再分布过程的低分辨率视图。纺锤体FRAP的半衰期约为19秒，即使整个半纺锤体被漂白。非动粒纺锤体和星状微管中微管蛋白的完全交换似乎在稳态下60-80 s内发生。这个速率太快，不能用微管蛋白的简单微管末端依赖性交换来解释。有效的微管微研磨将足够快，但是利用当前技术，我们没有看到在恢复期间漂白点移动的证据，这是我们基于Margolis和Wilson的模型所期望的（Nature（Lond.），1981，293：705）--荧光均匀地恢复。微管可以解聚和重新聚合迅速和异步整个纺锤体和星形，但FRAP数据是最兼容的微管蛋白亚基的快速交换所有沿着整个长度的非动粒纺锤体和星形微管。
The rate of exchange of tubulin that is incorporated into spindle microtubules with dimeric tubulin in the cytoplasm has been measured in sea urchin eggs by studying fluorescence redistribution after photobleaching (FRAP). Dichlorotriazinyl amino fluorescein (DTAF) has been used to label bovine brain tubulin. DTAF-tubulin has been injected into fertilized eggs of Lytechinus variegatus and allowed to equilibrate with the endogenous tubulin pool. Fluorescent spindles formed at the same time that spindles were seen in control eggs, and the injected embryos proceeded through many cycles of division on schedule, suggesting that DTAF-tubulin is a good analogue of tubulin in vivo. A microbeam of argon laser light has been used to bleach parts of the fluorescent spindles, and FRAP has been recorded with a sensitive video camera. Laser bleaching did not affect spindle structure, as seen with polarization optics, nor spindle function, as seen by rate of progress through mitosis, even when one spindle was bleached several times in a single cell cycle. Video image analysis has been used to measure the rate of FRAP and to obtain a low resolution view of the fluorescence redistribution process. The half-time for spindle FRAP is approximately 19 s, even when an entire half-spindle is bleached. Complete exchange of tubulin in nonkinetochore spindle and astral microtubules appeared to occur within 60-80 s at steady state. This rate is too fast to be explained by a simple microtubule end-dependent exchange of tubulin. Efficient microtubule treadmilling would be fast enough, but with current techniques we saw no evidence for movement of the bleached spot during recovery, which we would expect on the basis of Margolis and Wilson's model (Nature (Lond.)., 1981, 293:705)-- fluorescence recovers uniformly. Microtubules may be depolymerizing and repolymerizing rapidly and asynchronously throughout the spindle and asters, but the FRAP data are most compatible with a rapid exchange of tubulin subunits all along the entire lengths of nonkinetochore spindle and astral microtubules.