Dynamic instability of individual microtubules analyzed by video light microscopy: rate constants and transition frequencies.
Dynamic instability of individual microtubules analyzed by video light microscopy: rate constants and transition frequencies.
复制标题
通过视频光学显微镜分析的单个微管的动态不稳定性：速率常数和过渡频率。
DOI：
10.1083/jcb.107.4.1437
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发表时间：
1988-10
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;O'Brien, E T;Pryer, N K;Soboeiro, M F;Voter, W A;Erickson, H P;Salmon, E D
我们开发了视频显微镜方法，可以以 33 毫秒的间隔可视化单个微管的组装和拆卸。将不含微管相关蛋白的猪脑微管蛋白在 37 摄氏度下组装到轴丝片段上，并分析微管正负端的动态行为，以了解 7 至 15.5 µM 之间的微管蛋白浓度。伸长和快速缩短是明显不同的阶段。在每一端，延伸阶段的特征在于二级缔合反应和大量的一级解离反应。正端和负端的结合速率常数分别为 8.9 和 4.3 microM-1 s-1；相应的解离速率常数分别为44和23 s-1。对于两端，微管蛋白解离速率等于 5 microM 的微管蛋白结合速率。两端的快速缩短率相似（正 = 733 s-1；负 = 915 s-1），并且不随微管蛋白浓度变化。阶段之间的转变是突然且随机的。随着微管蛋白浓度的增加，两端的灾难频率下降，负端的救援频率急剧增加。这导致两端的微管蛋白浓度较高时快速缩短相较少，负端的快速缩短相较短。在每个浓度下，正端发生灾难的频率稍高，负端发生救援的频率稍高。我们的数据表明，由纯微管蛋白组装而成的微管在微管蛋白浓度的两倍范围内经历动态不稳定性，并且微管正端和负端的动态不稳定性可能显着不同。我们的分析表明，这种差异可能会产生跑步现象，并对长度重新分布作为动态不稳定性衡量标准的有效性建立了一般限制。我们的结果与伸长过程中 GTP 帽的存在一致，但与现有的 GTP 帽模型不一致。
We have developed video microscopy methods to visualize the assembly and disassembly of individual microtubules at 33-ms intervals. Porcine brain tubulin, free of microtubule-associated proteins, was assembled onto axoneme fragments at 37 degrees C, and the dynamic behavior of the plus and minus ends of microtubules was analyzed for tubulin concentrations between 7 and 15.5 microM. Elongation and rapid shortening were distinctly different phases. At each end, the elongation phase was characterized by a second order association and a substantial first order dissociation reaction. Association rate constants were 8.9 and 4.3 microM-1 s-1 for the plus and minus ends, respectively; and the corresponding dissociation rate constants were 44 and 23 s-1. For both ends, the rate of tubulin dissociation equaled the rate of tubulin association at 5 microM. The rate of rapid shortening was similar at the two ends (plus = 733 s-1; minus = 915 s-1), and did not vary with tubulin concentration. Transitions between phases were abrupt and stochastic. As the tubulin concentration was increased, catastrophe frequency decreased at both ends, and rescue frequency increased dramatically at the minus end. This resulted in fewer rapid shortening phases at higher tubulin concentrations for both ends and shorter rapid shortening phases at the minus end. At each concentration, the frequency of catastrophe was slightly greater at the plus end, and the frequency of rescue was greater at the minus end. Our data demonstrate that microtubules assembled from pure tubulin undergo dynamic instability over a twofold range of tubulin concentrations, and that the dynamic instability of the plus and minus ends of microtubules can be significantly different. Our analysis indicates that this difference could produce treadmilling, and establishes general limits on the effectiveness of length redistribution as a measure of dynamic instability. Our results are consistent with the existence of a GTP cap during elongation, but are not consistent with existing GTP cap models.