STRUCTURAL POLARITY AND DIRECTIONAL GROWTH OF MICROTUBULES OF CHLAMYDOMONAS FLAGELLA
STRUCTURAL POLARITY AND DIRECTIONAL GROWTH OF MICROTUBULES OF CHLAMYDOMONAS FLAGELLA
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DOI：
10.1016/0022-2836(74)90381-7
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发表时间：
1974-01-01
影响因子：
5.6
通讯作者：
BORISY, GG
中科院分区：
文献类型：
--
作者：
ALLEN, C;BORISY, GG
关于微管组装的一个基本问题是生长的极性，即亚基是否可以添加到正在生长的微管的两端，或者生长是否通过亚基添加到唯一的一端进行。为了探讨这一问题，在体外系统中，对分离的鞭毛轴丝添加微管亚单位进行了实验。用非离子洗涤剂短暂处理从衣藻上分离出鞭毛，用差速离心法分离出鞭毛，然后与猪脑组织的高速粗提物或纯化的微管蛋白(通过重复温度依赖的组装和拆卸获得的微管蛋白)孵育。阴性染色标本的电子显微镜显示，90%以上的轴突的一端增加了多达11个长微管。秋水仙碱(100μm)、氯化钙(2.5 mM)和低温(0℃)均可阻止和逆转微管组装，但对轴突长度无影响。在粗提物中，微管形成在轴丝中央对的两个成员上，但仅在外二联体的A管上形成。机械剪切产生的鞭毛碎片也与微管亚单位孵育。单管仅在外部双片段的一端形成；在中心对片段的一个或两个成员上的单管的出现主要是单向的。对磨损的轴丝的结构分析和侧臂附着的不对称性使轴丝碎片的绝对极性得以确定，并揭示了轴丝微管的远端通过添加亚单位进行组装。使用纯化的脑微管蛋白，也发生了有限范围的近端添加和在B管上的生长。近端加成的程度随蛋白质浓度和温度的升高而增加。我们得出结论，鞭毛微管有一种内在的极性，反映在它们的侧臂附着和生长的方向性上。
A basic question concerning microtubule assembly is the polarity of growth, namely, whether subunits can add to either end of a growing microtubule or whether growth proceeds by subunit addition to only one end. To approach this question in anin vitrosystem, experiments were carried out on the addition of microtubule subunits to isolated flagellar axonemes. Flagella were detached fromChlamydomonasby brief treatment with non-ionic detergent, isolated by differential centrifugation, and incubated with crude high-speed extracts of porcine brain tissue or with purified tubulin (obtained by repetitive temperature-dependent assembly and disassembly). Electron microscopy of negatively stained samples showed as many as 11 long microtubules added at one end of more than 90% of the axonemes. Colchicine (100 μm), CaCl2(2.5 mm), and low temperature (0 °C) both prevented and reversed microtubule assembly but had no effect on axonemal length. In crude extracts microtubules formed on both members of the axonemal central pair but on only the A-tubule of the outer doublets. Flagellar fragments, produced by mechanical shearing, were also incubated with microtubule subunit. Single tubules formed at only one end of outer doublet fragments; the appearance of single tubules on one or both members of central pair fragments was predominantly unidirectional. Structural analysis of frayed axonemes and the asymmetry of side-arm attachments permitted the absolute polarity of the axonemal fragments to be determined and revealed that assembly proceeded by addition of subunits to the distal ends of the axonemal microtubules. Using purified brain tubulin, a limited extent of proximal addition and growth on the B-tubule also occurred. The extent of proximal addition increased with increasing protein concentration and temperature. We conclude that the microtubules of flagella have an intrinsic polarity reflected in their side-arm attachments and in their directionality of growth.