Molecular anatomical analysis of neurofilament

在本研究中获得了四个主要的新结果，关于神经丝（NF）的组织。1.从牛脊髓中分离纯化的NF以不同的NF亚基组合存在，用快速冷冻深蚀云母电镜观察，发现NF中最大的NF亚基NF-H是形成核心丝之间横桥的最重要成分。2. NF-H是磷酸化程度最高的亚基，在轴突中磷酸化程度很高，但在树突或核周体中磷酸化程度很低。此外，轴突神经纤维在结构组织上与树突或核周神经纤维有相当大的不同。根据这些事实和结果1，我们用各种电子显微镜技术研究了NF-H磷酸化状态如何影响体外NF结构，发现去磷酸化显著改变了横桥结构。因此，我们提供的证据磷酸化依赖的结构变化的NF-H羧基末端尾部结构域。3.为了阐明为什么NF-H是严重磷酸化的轴突，但不是在其他车厢，我们研究了大鼠中枢神经元免疫金标记。我们发现在树突中，当NFs单独出现时，它们的NF-H没有磷酸化，而当NFs成束时，NF-H变得磷酸化。结果表明NF-H的磷酸化是形成横桥所必需的，而去磷酸化是NF与其他蛋白质相互作用所必需的。4.从所有上述结果，横桥被认为是根据NF-H磷酸化程度在结构上可变的。为了在体内证实这一观点，我们比较了铝中毒后在核周体或树突中积累的神经纤维与轴突神经纤维。我们的研究表明，核心丝有较少的规则排列，更少的crossbridge和较低程度的NF-H磷酸化积累的神经纤维比轴突的神经纤维。总之，我们的数据表明，NF-H磷酸化是形成脱钙蛋白所必需的。

Four main new results concerning neurofilament (NF)organizations were obtained in the present study. 1. When NFs reassembeld in various combinations of NF subunits after purified from bovine spinal cord were examined by quick-freeze deep-etch mica electron microscopy, NF-H, the largest NF subunit, was found to be the most essential component for forming cross-bridges between core filaments. 2. NF-H, the most phosphorylated subunit, is heavily phosphorylated in the axon but poorly phosphorylated in the dendrite or perikaryon. Further, axonal NFs are considerably different in structural organization from the dendritic or perikaryal NFs. From these facts and the result 1, we examined with various electron microscopic techniques how NF-H phosphorylation state influences the NF structure in vitro, and found that crossbridge structures were significantly altered by dephosphorylation. Thus, we provide the evidence of phosphorylation-de-pendent structural change in the NF-H carboxy-terminal tail domain. 3. To clarify why NF-H is heavily phosphorylated in the axon but not in the other compartments, we examined rat central neurons with immunogold labeling. We found in dendrites that when NFs appeared singly their NF-H was not phosphorylated while when NFs were bundled NF-H became phosphorylated. The result suggests that phosphorylation of NF-H is necessary for forming crossbridges and unphosphorylation is necessary for NF interaction with other proteins. 4. From all above results, crossbridges are considered to be structurally changeable according to the NF-H phosphorylation degree. To confirm this idea in vivo, we compared NFs accumulated in perikaryon or dendrite following aluminum intoxication with axonal NFs. Our examination showed that core filaments have less regular alignment, fewer crossbridges and lower degree of NF-H phosphorylation in accumulated NFs than in axonal NFs. In conclusion, our data indicate that NF-H phosphorylation is necessary for the formation of de