Salmon E D, Leslie R J, Saxton W M, Karow M L, McIntosh J R
J Cell Biol. 1984 Dec;99(6):2165-74. doi: 10.1083/jcb.99.6.2165.
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.
通过研究光漂白后的荧光再分布（FRAP），已在海胆卵中测量了整合到纺锤体微管中的微管蛋白与细胞质中二聚体微管蛋白的交换速率。二氯三嗪基氨基荧光素（DTAF）已用于标记牛脑微管蛋白。将DTAF - 微管蛋白注入多色紫球海胆的受精卵中，并使其与内源性微管蛋白库平衡。荧光纺锤体与对照卵中观察到的纺锤体同时形成，并且注射后的胚胎按计划进行了许多次分裂循环，这表明DTAF - 微管蛋白在体内是微管蛋白的良好类似物。使用氩激光微束漂白荧光纺锤体的部分区域，并用灵敏的摄像机记录FRAP。如通过偏振光学观察到的，激光漂白不影响纺锤体结构，也不影响纺锤体功能，如通过有丝分裂进程速率观察到的，即使在单个细胞周期中一个纺锤体被漂白多次也是如此。视频图像分析已用于测量FRAP速率并获得荧光再分布过程的低分辨率视图。即使整个半纺锤体被漂白，纺锤体FRAP的半衰期约为19秒。在稳态下，非动粒纺锤体和星体微管中的微管蛋白似乎在60 - 80秒内完全交换。这个速率太快，无法用简单的依赖微管末端的微管蛋白交换来解释。有效的微管踏车运动速度足够快，但根据目前的技术，我们在恢复过程中没有看到漂白斑点移动的证据，而根据马戈利斯和威尔逊的模型（《自然》（伦敦），1981年，293:705）我们应该会看到这种情况——荧光均匀恢复。微管可能在整个纺锤体和星体中快速且异步地解聚和重新聚合，但FRAP数据与沿着非动粒纺锤体和星体微管的整个长度快速交换微管蛋白亚基最为相符。
