Walker R A, O'Brien E T, Pryer N K, Soboeiro M F, Voter W A, Erickson H P, Salmon E D
Department of Biology, University of North Carolina, Chapel Hill 27599-3280.
J Cell Biol. 1988 Oct;107(4):1437-48. doi: 10.1083/jcb.107.4.1437.
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.
我们开发了视频显微镜方法，以33毫秒的间隔观察单个微管的组装和解聚过程。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，并分析了微管正负两端在7至15.5微摩尔微管蛋白浓度下的动态行为。伸长和快速缩短是明显不同的阶段。在每一端，伸长阶段的特征是二级缔合和大量的一级解离反应。正端和负端的缔合速率常数分别为8.9和4.3微摩尔-1秒-1；相应的解离速率常数分别为44和23秒-1。对于两端，微管蛋白解离速率在5微摩尔时等于微管蛋白缔合速率。两端的快速缩短速率相似（正端=733秒-1；负端=915秒-1），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，两端的灾难频率降低，负端的救援频率显著增加。这导致在较高微管蛋白浓度下，两端的快速缩短阶段减少，负端的快速缩短阶段更短。在每个浓度下，正端的灾难频率略高，负端的救援频率更高。我们的数据表明，由纯微管蛋白组装的微管在两倍的微管蛋白浓度范围内经历动态不稳定性，并且微管正负两端的动态不稳定性可能有显著差异。我们的分析表明，这种差异可能导致踏车行为，并为作为动态不稳定性度量的长度重新分布的有效性设定了一般限制。我们的结果与伸长过程中存在GTP帽一致，但与现有的GTP帽模型不一致。
