Dilution-induced disassembly of microtubules: relation to dynamic instability and the GTP cap.
Dilution-induced disassembly of microtubules: relation to dynamic instability and the GTP cap.
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稀释诱导的微管解体：与动态不稳定性和 GTP 帽的关系。
DOI：
10.1002/cm.970180106
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发表时间：
1991
影响因子：
--
通讯作者：
Erickson,HP
中科院分区：
文献类型：
--
作者：
Voter,WA;O'Brien,ET;Erickson,HP
在最初用于研究动态不稳定性的缓冲液（100 mM PIPES、2 mM EGTA、1 mM镁、0.2 mM GTP）中由纯化的微管蛋白组装微管，然后在相同的缓冲液中稀释以研究分解速率。在15倍稀释后，微管聚合物在15秒内线性降低至起始值的约20%。我们确定了稀释前微管的长度分布，并准备了计算机模拟的聚合物损失为不同的假设率的拆卸。我们的实验数据与60 μ m/min的微管解聚率一致。这是通过单个微管的光学显微镜测定的快速缩短期微管解聚的总速率（步行者等人：Journal of Cell Biology 107：1437 - 1448，1988）。因此，我们的结论是，微管开始迅速缩短在两端稀释。此外，由于我们可以检测到稀释和快速分解的开始之间没有滞后，从伸长到快速缩短的过渡显然发生在稀释后1秒内。假设这种转变（灾难）涉及GTP帽的损失，并且帽的损失是通过稀释后GTP-微管蛋白亚基的连续解离来实现的，我们可以根据动力学数据和步行者等人的模型解释来估计帽的最大尺寸。帽在正端和负端分别可能短于40和20个亚基。
Microtubules were assembled from purified tubulin in the buffer originally used to study dynamic instability (100 mM PIPES, 2 mM EGTA, 1 mM magnesium, 0.2 mM GTP) and then diluted in the same buffer to study the rate of disassembly. Following a 15-fold dilution, microtubule polymer decreased linearly to about 20% of the starting value in 15 sec. We determined the length distribution of microtubules before dilution, and prepared computer simulations of polymer loss for different assumed rates of disassembly. Our experimental data were consistent with a disassembly rate per microtubules of 60 μm/min. This is the total rate of depolymerization for microtubules in the rapid shortening phase, as determined by light microscopy of individual microtubules (Walker et al.: Journal of Cell Biology 107: 1437–1448, 1988). We conclude, therefore, that microtubules began rapid shortening at both ends upon dilution. Moreover, since we could detect no lag between dilution and the onset of rapid disassembly, the transition from elongation to rapid shortening apparently occurred within 1 sec following dilution. Assuming that this transition (catastrophe) involves the loss of the GTP cap, and that cap loss is achieved by the sequential dissociation of GTP-tubulin subunits following dilution, we can estimate the maximum size of the cap based on the kinetic data and model interpretation of Walker et al. The cap is probably shorter than 40 and 20 subunits at the plus and minus ends, respectively.