The crayfish neuronal cytoskeleton: an investigation of proteins having neurofilament-like immunoreactivity

Abstract

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 stain, 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 (M r > 300kDa), a 40 kDa polypeptide, and a group of 4 bands atM r = 66−84kDa. 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.