Molecular anatomical analysis of neurofilament

本研究获得了关于神经丝（NF）组织的四个主要新结果。1. 用速冻深蚀刻云母电子显微镜观察从牛脊髓中纯化出来的NF以不同的NF亚基组合重组，发现NF- h是最大的NF亚基，是形成核心细丝之间交叉桥的最重要成分。2. NF-H是磷酸化程度最高的亚基，在轴突中被大量磷酸化，但在树突或核周中磷酸化程度较低。此外，轴突NFs在结构组织上与树突或核周NFs有很大不同。根据这些事实和结果1，我们用各种电子显微镜技术检查了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交叉桥结构。因此，我们提供了NF-H羧基末端尾部区域磷酸化依赖性结构变化的证据。3. 为了阐明为什么NF-H在轴突而在其他隔室中严重磷酸化，我们用免疫金标记检查了大鼠中枢神经元。我们在树突中发现，当NFs单独出现时，它们的NF-H不被磷酸化，而当NFs捆绑在一起时，NF-H被磷酸化。结果表明NF- h的磷酸化是形成交叉桥的必要条件，而非磷酸化是NF与其他蛋白相互作用的必要条件。4. 从以上结果可以看出，根据NF-H磷酸化程度的不同，交叉桥在结构上是可变的。为了在体内证实这一观点，我们比较了铝中毒后核周或树突中积累的NFs与轴突中积累的NFs。我们的研究表明，与轴突NFs相比，累积NFs的核心细丝具有更少的规则排列，更少的交叉桥和更低的NF-H磷酸化程度。总之，我们的数据表明NF-H磷酸化对于de的形成是必要的

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