Direct observation of steady-state microtubule dynamics.
Direct observation of steady-state microtubule dynamics.
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DOI：
10.1083/jcb.102.3.1007
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发表时间：
1986-03
期刊：
影响因子：
--
通讯作者：
Kirschner M
中科院分区：
文献类型：
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作者：
Kristofferson D;Mitchison T;Kirschner M
不同类型的不寻常的动态行为已被报道的稳态微管。虽然几乎所有早期的报告依赖于动力学测量的本体聚合，我们已经直接可视化的稳定状态下，通过使用微管蛋白衍生生物素添加到单个微管的亚基。生物素化的微管蛋白既用作聚合的内部“种子”，又用作组装到由纯化的微管蛋白组成的微管末端上的标记物。通过双标记免疫荧光将生物素化片段与未修饰的微管蛋白区分开。在两种缓冲液条件下，在稳态下随时间监测微管长度、数量浓度和片段长度。结果表明，在这些条件下的微管稳态是大多数缓慢生长的微管和少数快速解聚的微管之间的平衡，如“动态不稳定性”模型所描述的（Mitchison T.，和M. Kirschner，1984，Nature（Lond.）.，312：232-242）。微管没有显示出螺旋铣削的证据;相反，大多数微管在稳态时显示出两端的进行性生长。虽然溶剂条件显着影响生长速率，定性的行为是不变的。
Different types of unusual dynamic behavior have been reported for steady-state microtubules. While almost all earlier reports relied on kinetic measurements of bulk polymerization, we have directly visualized the steady-state addition of subunits to individual microtubules through the use of tubulin derivitized with biotin. Biotinylated tubulin was used both as an internal "seed" for polymerization and as a marker for assembly onto the ends of microtubules composed of purified tubulin. Biotinylated segments were distinguished from unmodified tubulin by double-label immunofluorescence. Microtubule lengths, number concentrations, and segment lengths have been monitored with time at steady state under two buffer conditions. The results indicate that the microtubule steady state under these conditions is a balance between a majority of slowly growing microtubules and a minority of rapidly depolymerizing ones as described by the "dynamic instability" model (Mitchison T., and M. Kirschner, 1984, Nature (Lond.)., 312:232-242). Microtubules show no evidence of treadmilling; instead most show progressive growth off both ends at steady state. Although solvent conditions markedly influence the growth rates, qualitatively the behavior is unchanged.