Steven A C, Hainfeld J F, Trus B L, Wall J S, Steinert P M
J Biol Chem. 1983 Jul 10;258(13):8323-9.
We have studied the distribution of mass in several types of intermediate filaments (IF) assembled in vitro, by analyzing scanning transmission electron micrographs (STEM) of unstained specimens imaged in dark-field mode. Bovine epidermal keratin IF, which are obligate heteropolymers, were thus characterized and compared with facultative heteropolymers of vimentin and desmin and with earlier observations of homopolymer vimentin IF. The major components of each type of IF have linear densities of 37 kilodaltons/nm. Minor polymorphic variants are also present in each case, with linear densities of approximately 25 and 48 kilodaltons/nm, respectively. In view of the known subunit masses, these results are consistent with the proposition that at least three IF types (bovine epidermal keratin, vimentin, and desmin) are structurally homologous. For each type of IF studied, measurements taken near the ends of long IF or from short IF tend to have lower densities, characteristic of the least dense polymorphic variant. This form may represent a precursor "minimal core" structure in the sense of a minimal aggregate of protofilaments sufficiently stable to permit elongation. In the course of in vitro assembly, this form would be maturable to the normal IF structure at 37 kilodaltons/nm by the accretion of further protein. We also find the projected widths of these IF as measured from the STEM images to be significantly greater than the corresponding diameters determined in previous studies by conventional electron microscopy. Whereas the conventional techniques have routinely yielded diameters of 7-11 nm for IF contrasted by heavy metal staining, the STEM images of unstained IF provide estimates of 15 +/- 1 nm for the outer diameter of the major density class for each IF type studied and average values of 13.8 and 16.1 nm for the less dense and more dense variants, respectively.
我们通过分析暗场模式下成像的未染色标本的扫描透射电子显微照片（STEM），研究了体外组装的几种中间丝（IF）类型中的质量分布。牛表皮角蛋白IF是专性杂聚物，因此对其进行了表征，并与波形蛋白和结蛋白的兼性杂聚物以及先前对同聚物波形蛋白IF的观察结果进行了比较。每种IF类型的主要成分的线性密度为37千道尔顿/纳米。每种情况下也存在次要的多态变体，其线性密度分别约为25和48千道尔顿/纳米。鉴于已知的亚基质量，这些结果与至少三种IF类型（牛表皮角蛋白、波形蛋白和结蛋白）在结构上同源的观点一致。对于所研究的每种IF类型，在长IF末端附近或短IF上进行的测量往往具有较低的密度，这是最低密度多态变体的特征。这种形式可能代表一种前体“最小核心”结构，即原丝的最小聚集体，其稳定性足以允许伸长。在体外组装过程中，这种形式可通过进一步蛋白质的积累成熟为正常的37千道尔顿/纳米的IF结构。我们还发现，从STEM图像测量的这些IF的投影宽度明显大于先前通过传统电子显微镜研究确定的相应直径。虽然传统技术常规得出经重金属染色对比的IF直径为7-11纳米，但未染色IF的STEM图像显示，所研究的每种IF类型的主要密度类别的外径估计为15±1纳米，较低密度和较高密度变体的平均值分别为13.8和16.1纳米。
