Identification of a neurofilament-like protein in the protocerebral tract of the crab Ucides cordatus.

Neurofilaments (NFs) have not been observed in crustaceans using conventional electron microscopy, and intermediate filaments have never been described in crustaceans and other arthropods by immunocytochemistry. Since polypeptides, labeled by the NN18-clone antibody, were revealed on microtubule side-arms of crayfish, we have tested, in this study, whether proteins similar to mammalian NFs are present in the protocerebral tract (PCT) of the crab Ucides cordatus. We used immunohistochemistry for light microscopy with monoclonal antibodies against three different NF subunits, high (NF-H), medium (NF-M), and light (NF-L). Labeling was observed with the NN18-clone, which recognizes NF-M. In order to confirm the results obtained with the immunohistochemical reactions, Western blotting, using the three primary antibodies, was performed and the presence of NF-M was confirmed. The NN18-clone monoclonal antibody recognized a protein of approximately 160 kDa, similar to the mammalian NF-M protein, but NF-L and NF-H were not recognized. Conventional transmission electron microscopy was used to observe the ultrastructural components of the axons and immunoelectron microscopy was used to show the distribution of the NF-M-like polypeptides along cytoskeletal elements of the PCT. Our results agree with previous studies on crustacean NF proteins that have reported negative immunoreactions against NF-H and NF-L subunits and positive immunoreactions against the mammalian NF-M subunit. However, the protein previously referred to as P600 and recognized by the NN18-clone, has a very high molecular weight, thus, being different from mammalian NF-M subunit and from the protein revealed now in our study.

Binding of an antibody against a noncompact myelin protein to presumptive glial cells in the visual system of the crab Ucides cordatus.

Glial cells, in both vertebrate and invertebrate nervous systems, provide an essential environment for developmental, supportive, and physiological functions. However, information on glial cells themselves and on glial cell markers, with the exception of those of Drosophila and other insects, is not abundant in invertebrate organisms. A common ultrastructural feature of invertebrate nervous systems is that layers of glial cell cytoplasm-rich processes ensheath axons and neuronal and glial somata. In the present study, we have examined the binding of a monoclonal antibody to 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) in the compound eye and optic lobe of the crab Ucides cordatus using both light and electron microscopy. CNPase is a noncompact myelin protein that is a phenotypic marker of oligodendroglial and Schwann cells, is apparently involved in the ensheathment step prior to myelin compaction, and is also expressed by the potentially myelinating olfactory ensheathing glia. CNPase has raised much interest, first by virtue of its unusual enzymatic activity and more recently by its membrane-skeletal features and possible involvement in migration or expansion of membranes. We have found CNPase-like immunoreactivity in most cells of the compound eye basement membrane and both in optic cartridges of the synaptic layer and cells of the outer sublayer of the lamina ganglionaris. The results suggest that in the crab visual system some, but not all, glial cells, including some adaxonal glia, may express the noncompact myelin protein CNPase or a related protein.