STRUCTURE OF THE PERIPHERAL DOMAINS OF NEUROFILAMENTS REVEALED BY LOW-ANGLE ROTARY SHADOWING
STRUCTURE OF THE PERIPHERAL DOMAINS OF NEUROFILAMENTS REVEALED BY LOW-ANGLE ROTARY SHADOWING
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DOI：
10.1016/0022-2836(88)90459-7
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发表时间：
1988-07-20
影响因子：
5.6
通讯作者：
HIROKAWA, N
中科院分区：
文献类型：
--
作者：
HISANAGA, S;HIROKAWA, N
用旋转阴影电镜观察神经丝周围区的结构。用Sepharose CL-4 B凝胶过滤法从牛脊髓中分离神经纤维，并通过低角度旋转阴影法进行检查。外围域表现为从中间丝芯突出的薄的、柔性的丝状结构，沿其整个长度沿着具有恒定的密度。突起的平均长度约为85 nm，宽度约为4 nm。 这些突出物出现在22 nm间距的规则分布的位点，这似乎对应于α-β的典型重复。核心丝的富含螺旋的棒状结构域。测得突起的密度为4.1 ± 0.01。0.6)每22 nm。我们使用纯化的NF多肽进行重建实验，以确认该突起确实是NF外周结构域。在6 M-尿素存在下，通过DE-52和Mono-Q阴离子交换色谱法纯化NF三联体的各个组分，即NF-L、NF-M和NF-H，并以各种组合组装成细丝。重新组装的细丝比孤立的NF更细长，并表现出明显的22 nm轴向周期性。虽然在单独由NF-L组装的细丝中没有观察到突出的突起，但含有NF-L加上NF-M或NF-H的重建细丝显示出许多突起。从NF-L和NF-H重建的细丝中的突起的平均长度为约63 nm。从NF-L和NF-M重建的纤维的投影长度约为55 nm。突起长度的差异可能反映了NF-M和NF-H之间羧基端尾结构域长度的差异。结果被解释为显示核因子的羧基末端尾部结构域以规则的模式从核丝投射，这与四聚体亚基的半交错组织一致。
The structure of the peripheral domains of neurofilaments (NFs) was revealed by rotary shadowng electron microscopy. NFs were isolated from bovine spinal cords by Sepharose CL-4B gel filtration and examined by low angle rotary shadowing. The peripheral domains appeared as thin, flexible, filamentous structures projecting from the intermediate filament core, with a constant density along their entire length. The average length of the projections was approximately 85 nm and the width about 4 nm. These projections appeared from regularly distributed sites, at 22 nm spacing, which seemed to correspond to the typical repeat of the .alpha.-helix-rich rod domain of the core filament. The density of the projections was found to be 4.1 (.+-. 0.6) per 22 nm. We performed reconstitution experiments using purified NF polypeptides to confirm that the projection was indeed the NF peripheral domain. Individual components of the NF triplet, i.e. NF-L, NF-M and NF-H, were purified by DE-52 and Mono-Q anion exchange chromatographies in the presence of 6 M-urea and were assembled in various combinations into filaments. Reassembled filaments were somewhat more slender than the isolated NFs and exhibited a distinct 22 nm axial periodicity. While prominent projections were not observed in the filaments assembled from NF-L alone, reconstructed filaments containing NF-L plus either NF-M or NF-H revealed many projections. The average length of the projections in the filaments reconstructed from NF-L and NF-H was about 63 nm. The projections of reconstructed filaments from NF-L and NF-M were about 55 nm in length. The difference in the lengths of the projections might reflect the difference in the length of the carboxy-teminal tail domain between NF-M and NF-H. The results are interpreted to show that the carboxy-terminal tail domains of NFs project in a regular pattern from the core filament, which is consistent with a half-staggered organization of the tetrameric subunits.