The mechanism of assembly of Acanthamoeba myosin-II minifilaments: minifilaments assemble by three successive dimerization steps.
The mechanism of assembly of Acanthamoeba myosin-II minifilaments: minifilaments assemble by three successive dimerization steps.
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棘阿米巴肌球蛋白-II 微丝的组装机制：微丝通过三个连续的二聚化步骤组装。
DOI：
10.1083/jcb.109.4.1537
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发表时间：
1989-10
影响因子：
7.8
通讯作者：
Pollard, T D
中科院分区：
文献类型：
--
作者：
Sinard, J H;Stafford, W F;Pollard, T D
我们使用 90 度光散射、分析超速离心和电子显微镜来推断棘阿米巴肌球蛋白-II 微丝（每个由八个分子组成）是通过由三个连续二聚步骤组成的新颖机制组装的，而不是通过向核添加单体或平行二聚体。高于 200 mM KCl，棘阿米巴肌球蛋白-II 是单体。在低离子强度（低于 100 mM KCl）下，肌球蛋白-II 聚合成双极微丝。在 100 至 200 mM KCl 之间，光散射与肌球蛋白浓度的关系图均外推至原点，但斜率随着 KCl 的增加而减小。这表明形成了尺寸介于单体和全长微丝之间的结构，并且组装这些结构的临界浓度非常低。分析超速离心已证实在这些盐浓度下存在中间结构，并且它们彼此处于快速平衡。我们相信这些结构代表组装中间体，并使用平衡分析超速离心和电子显微镜来识别它们。聚合从反平行二聚体的形成开始，两个尾部重叠约 15 nm。然后两个反平行二聚体与 15 nm 交错结合形成反平行四聚体。最后，两个四聚体与 30 nm 交错结合形成完整的微丝。在离子强度非常低的情况下，组装机制的最后一步很大程度上是相反的，并且反向平行四聚体是主要种类。碱性 pH 值也可以诱导微丝分解，产生与盐诱导分解相同的组装中间体。
We used 90 degrees light scattering, analytical ultracentrifugation, and electron microscopy to deduce that Acanthamoeba myosin-II minifilaments, composed of eight molecules each, assemble by a novel mechanism consisting of three successive dimerization steps rather than by the addition of monomers or parallel dimers to a nucleus. Above 200 mM KCl, Acanthamoeba myosin-II is monomeric. At low ionic strength (less than 100 mM KCl), myosin-II polymerizes into bipolar minifilaments. Between 100 and 200 mM KCl, plots of light scattering vs. myosin concentration all extrapolate to the origin but have slopes which decrease with increasing KCl. This indicates that structures intermediate in size between monomers and full length minifilaments are formed, and that the critical concentrations for assembly of these structures is very low. Analytical ultracentrifugation has confirmed that intermediate structures exist at these salt concentrations, and that they are in rapid equilibrium with each other. We believe these structures represent assembly intermediates and have used equilibrium analytical ultracentrifugation and electron microscopy to identify them. Polymerization begins with the formation of antiparallel dimers, with the two tails overlapping by approximately 15 nm. Two antiparallel dimers then associated with a 15-nm stagger to form an antiparallel tetramer. Finally, two tetramers associate with a 30-nm stagger to form the completed minifilament. At very low ionic strengths, the last step in the assembly mechanism is largely reversed and antiparallel tetramers are the predominant species. Alkaline pH, which can also induce minifilament disassembly, produces the same assembly intermediates as are found for salt induced disassembly.