Dilution of individual microtubules observed in real time in vitro: evidence that cap size is small and independent of elongation rate.
Dilution of individual microtubules observed in real time in vitro: evidence that cap size is small and independent of elongation rate.
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实时观察到的单个微管的稀释：帽子大小很小且与伸长率无关。
DOI：
10.1083/jcb.114.1.73
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发表时间：
1991-07
影响因子：
7.8
通讯作者：
Salmon, E D
中科院分区：
文献类型：
--
作者：
Walker, R A;Pryer, N K;Salmon, E D
虽然微管动力学不稳定性的机制被认为涉及微管蛋白结合的GTP的水解，但GTP水解的机制和微管稳定性的基础是有争议的。使用单个微管的视频显微镜和稀释方案来检查稳定帽的大小和寿命。在37 ℃下，在微型流动池中将纯化的猪脑微管蛋白（7-23 μ M）组装到分离的海胆轴丝片段的两端上，以得到10倍的延伸率变化。微管生长区域中的微管蛋白浓度可以被迅速稀释（在开始稀释的3秒内稀释84%）。在用不含微管蛋白的缓冲液灌注后，微管在稀释至初始浓度的16%后平均在4-6秒内经历灾难（从伸长到快速缩短的转化），与伸长和长度的预稀释速率无关。基于突变频率外推至零微管蛋白浓度，对于正端和负端，无限稀释后稳定帽的估计寿命小于3-4秒，比在稳态下观察到的约200秒短得多（步行者，R.一、E. T.奥布莱恩，N. K. Pryer，M.索博什湾A.选民，H。P. Erickson和E. D.三文鱼1988. 107：1437-1448）。我们的结论是，在延长过程中，正负两端稳定的短区域（约200二聚体或更少）和稳定帽的大小是独立的10倍的变化，延长率。这些结果排除了动态不稳定性模型，该模型预测了广泛的“建立”稳定帽和支持模型，该模型将帽限制在伸长尖端。我们建议，细胞可以利用这样的装配机制，通过使用“灾难因子”，可以促进频繁的灾难，即使在高伸长率的短暂结合微管末端和短暂抑制GTP-微管蛋白协会。
Although the mechanism of microtubule dynamic instability is thought to involve the hydrolysis of tubulin-bound GTP, the mechanism of GTP hydrolysis and the basis of microtubule stability are controversial. Video microscopy of individual microtubules and dilution protocols were used to examine the size and lifetime of the stabilizing cap. Purified porcine brain tubulin (7-23 microM) was assembled at 37 degrees C onto both ends of isolated sea urchin axoneme fragments in a miniature flow cell to give a 10-fold variation in elongation rate. The tubulin concentration in the region of microtubule growth could be diluted rapidly (by 84% within 3 s of the onset of dilution). Upon perfusion with buffer containing no tubulin, microtubules experienced a catastrophe (conversion from elongation to rapid shortening) within 4-6 s on average after dilution to 16% of the initial concentration, independent of the predilution rate of elongation and length. Based on extrapolation of catastrophe frequency to zero tubulin concentration, the estimated lifetime of the stable cap after infinite dilution was less than 3-4 s for plus and minus ends, much shorter than the approximately 200 s observed at steady state (Walker, R. A., E. T. O'Brien, N. K. Pryer, M. Soboeiro, W. A. Voter, H. P. Erickson, and E. D. Salmon. 1988. J. Cell Biol. 107:1437-1448.). We conclude that during elongation, both plus and minus ends are stabilized by a short region (approximately 200 dimers or less) and that the size of the stable cap is independent of 10-fold variation in elongation rate. These results eliminate models of dynamic instability which predict extensive "build- up" stabilizing caps and support models which constrain the cap to the elongating tip. We propose that the cell may take advantage of such an assembly mechanism by using "catastrophe factors" that can promote frequent catastrophe even at high elongation rates by transiently binding to microtubule ends and briefly inhibiting GTP-tubulin association.