Weaver D J, Viancour T A
Department of Biological Sciences, University of Maryland, Baltimore 21228.
Brain Res. 1991 Mar 22;544(1):49-58. doi: 10.1016/0006-8993(91)90884-x.
We have evaluated the possibility that proteins similar to mammalian neurofilament proteins (NFPs) are present in crustacean neurons. A panel of monoclonal antibodies (mAbs), raised against mammalian NFP, was used to identify candidate proteins. The degree to which these proteins are similar to mammalian NFPs was further evaluated using the following criteria: tissue specificity, recognition by the neurofilament-specific Bodian silver strain, recognition by the intermediate filament-specific Pruss mAb, and insolubility following detergent-extraction. Three candidate polypeptides were identified by mAb screening: a very high molecular weight polypeptide (Mr greater than 300 kDa), a 40 kDa polypeptide, and a group of 4 bands at Mr = 66-84 kDa. Although all of these polypeptides were recognized by one or more anti-NFP mAb, not one of them was found exclusively in neuronal tissue, not one was stained by the NFP-specific Bodian method, and all were soluble under conditions in which mammalian NFPs are insoluble. As a result of this thorough evaluation, we conclude that crayfish neurons do not contain neurofilament-like proteins. Although not closely related to mammalian neurofilaments, the very high molecular weight crayfish polypeptide which was strongly labeled by a commercially available anti-NF-M mAb (clone NN18) during the mAb screening, may be a novel cytoskeletal protein. The evidence for this conclusion comes from immunocytochemical labeling experiments. Indirect immunofluorescence labeling of this protein differentially labeled axons, such that labeling intensity of specific axons was proportional to the relative concentration of cytoskeletal organelles in those axons. Labeling of neuronal cell bodies delineated a fibrous network throughout the cytoplasm, and intensely labeled microtubule-rich regions of cytoplasm which are characteristic of larger neuronal somata. Immunogold labeling and electron microscopic analysis of the distribution of this protein revealed that the NN18-clone antibody bound to an antigen located on microtubule side-arms.
我们评估了甲壳类动物神经元中是否存在与哺乳动物神经丝蛋白（NFPs）相似的蛋白质。使用一组针对哺乳动物NFP产生的单克隆抗体（mAb）来鉴定候选蛋白质。利用以下标准进一步评估这些蛋白质与哺乳动物NFPs的相似程度：组织特异性、神经丝特异性博迪安银染法的识别、中间丝特异性普鲁士mAb的识别以及去污剂提取后的不溶性。通过mAb筛选鉴定出三种候选多肽：一种非常高分子量的多肽（Mr大于300 kDa）、一种40 kDa的多肽以及一组Mr = 66 - 84 kDa的四条带。尽管所有这些多肽都被一种或多种抗NFP mAb识别，但它们都没有仅在神经组织中被发现，没有一种能用NFP特异性博迪安法染色，并且在哺乳动物NFPs不溶的条件下它们都是可溶的。经过这种全面评估，我们得出结论：小龙虾神经元不含有神经丝样蛋白。在mAb筛选过程中被市售抗NF - M mAb（克隆NN18）强烈标记的非常高分子量的小龙虾多肽，虽然与哺乳动物神经丝没有密切关系，但可能是一种新型细胞骨架蛋白。这一结论的证据来自免疫细胞化学标记实验。这种蛋白质的间接免疫荧光标记对轴突进行了差异性标记，使得特定轴突的标记强度与这些轴突中细胞骨架细胞器的相对浓度成正比。神经元细胞体的标记勾勒出整个细胞质中的纤维网络，并强烈标记了富含微管的细胞质区域，这些区域是较大神经元胞体的特征。对这种蛋白质分布的免疫金标记和电子显微镜分析表明，NN18克隆抗体与位于微管侧臂上的抗原结合。
