Head-to-tail polymerization of microtubules in vitro. Electron microscope analysis of seeded assembly.
Head-to-tail polymerization of microtubules in vitro. Electron microscope analysis of seeded assembly.
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DOI：
10.1083/jcb.84.1.141
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发表时间：
1980-01
期刊：
影响因子：
--
通讯作者：
Borisy GG
中科院分区：
文献类型：
--
作者：
Bergen LG;Borisy GG
微管是极性结构，这种极性体现在其有偏向的定向生长上。按照先前确立的惯例（G. G. 博里西，1978年，《分子生物学杂志》124卷：565 - 570页），我们将微管的正（+）端和负（ - ）端定义为在结构取向分别等同于鞭毛外双联体A亚纤维的远端和近端的两端。以鞭毛轴丝为晶种，猪脑微管蛋白为亚基，测定了两端的伸长速率。由于鞭毛晶种的两端在形态上可区分，因此可以分别分析每一端的伸长情况。通过绘制在不同亚基蛋白浓度下的伸长速率曲线，我们确定了正端和负端的结合与解离速率常数。在我们30℃的实验条件下，正端和负端的结合常数分别为7.2×10⁶ M⁻¹ s⁻¹和2.25×10⁶ M⁻¹ s⁻¹，解离常数分别为17 s⁻¹和7 s⁻¹。根据这些数值以及韦格纳方程（1976年，《分子生物学杂志》108卷：139 - 150页），我们确定微管的正端为其头部，并计算了“s”，即头 - 尾聚合参数。发现其值小得令人惊讶（s = 0.07 ± 0.02）。鉴于s值较小，对基于头 - 尾聚合的有丝分裂模型的有效性进行了讨论（马戈利斯等人，1978年，《自然（伦敦）》272卷：450 - 452页）。
Microtubules are polar structures, and this polarity is reflected in their biased directional growth. Following a convention established previously (G. G. Borisy, 1978, J. Mol. Biol. 124:565--570), we define the plus (+) and minus (-) ends of a microtubule as those equivalent in structural orientation to the distal and proximal ends, respectively, of the A subfiber of flagellar outer doublets. Rates of elongation were obtained for both ends using flagellar axonemes as seeds and porcine brain microtubule protein as subunits. Since the two ends of a flagellar seed are distinguishable morphologically, elongation of each end may be analyzed separately. By plotting rates of elongation at various concentrations of subunit protein, we have determined the association and dissociation rate constants for the plus and minus ends. Under our conditions at 30 degrees C, the association constants were 7.2 X 10(6) M-1 s-1 and 2.25 X 10(6) M-1 s-1 for the plus and minus ends, respectively, and the dissociation constants were 17 s-1 and 7 s-1. From these values and Wegner's equations (1976, J. Mol. Biol. 108:139--150), we identified the plus end of the microtubule as its head and calculated "s," the head-to-tail polymerization parameter. Surprisingly small values (s = 0.07 +/- 0.02) were found. The validity of models of mitosis based upon head-to-tail polymerization (Margolis et al., 1978, Nature (Lond.) 272:450--452) are discussed in light of a small value for s.