Assembly of vimentin in vitro and its implications concerning the structure of intermediate filaments.
Assembly of vimentin in vitro and its implications concerning the structure of intermediate filaments.
复制标题
波形蛋白的体外组装及其对中间丝结构的影响。
DOI：
10.1016/0022-2836(85)90007-5
复制
发表时间：
1985
影响因子：
5.6
通讯作者：
Richard M. Robson
中科院分区：
文献类型：
--
作者：
W. Ip;Michael K. Hartzer;Y. Pang;Richard M. Robson
经10 mm-三羟甲基氨基甲烷醋酸盐（pH 8.5）透析后，在尿素存在下纯化的波形蛋白以四聚体2至3 nm × 48 nm棒状物（称为原丝）的形式存在。这些积木又变成中间丝（10至12纳米直径），当他们对生理离子强度和pH值的溶液透析。通过改变离子条件下发生聚合，我们已经确定了两类组装中间体的结构提供线索，如何可以构建一个中间丝。第一类的结构，看到组装时发生在10至20毫米盐在pH值8.5，强烈表明，长丝组装的初始步骤之一是协会的原丝成对的半单位轴向交错。增加组装缓冲液的离子强度导致在pH 8.5下约50 mm-盐下出现短的全宽中间细丝。在存在额外的原丝的情况下，当离子强度和pH进一步调节到生理水平时，这些短的丝伸长到许多微米。组装中间体的电子显微镜图像表明，含有波形蛋白的中间丝由8个原丝组成，组装使得相邻原丝之间存在约22 nm的轴向交错。我们认为，这种半单位的原丝交错是中间丝结构和组装的一个基本特征，并且它可以解释到目前为止在所有中间丝中观察到的20至22 nm轴向重复。
After dialysis against 10 mm-Tris-acetate (pH 8.5), vimentin that has been purified in the presence of urea is present in the form of tetrameric 2 to 3 nm × 48 nm rods known as protofilaments. These building blocks in turn polymerize into intermediate filaments (10 to 12 nm diameter) when they are dialyzed against a solution of physiological ionic strength and pH. By varying the ionic conditions under which polymerization takes place, we have identified two classes of assembly intermediates whose structures provide clues as to how an intermediate filament may be constructed. The structure of the first class, seen when assembly takes place at 10 to 20 mm-salt at pH 8.5, strongly suggests that one of the initial steps of filament assembly is the association of protofilaments into pairs with a half-unit axial stagger. Increasing the ionic strength of the assembly buffer leads to the emergence of short, full-width intermediate filaments at approximately 50 mm-salt at pH 8.5. In the presence of additional protofilaments, these short filaments elongate to many micrometers when the ionic strength and pH are further adjusted to physiological levels. The electron microscope images of the assembly intermediates suggest that vimentin-containing intermediate filaments are made up of eight protofilaments, assembled such that there is an approximately 22 nm axial stagger between neighboring protofilaments. We propose that this half-unit staggering of protofilaments is a fundamental feature of intermediate filament structure and assembly, and that it could account for the 20 to 22 nm axial repeat seen in all intermediate filaments examined so far.