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

Abstract

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.

Electron microscopy and morphometric analyses of microtubules in two differently sized types of axons in the protocerebral tract of a crustacean

“…However, as reported previously (Corrêa et al, 2004), we found NF proteins in regions corresponding to the MT side-arms in the crustacean protocerebral tract (PCT).…”

“…Other numbers of protofilaments are possible: for example, certain MTs in the neurons of nematode worms contain 11 or 15 protofilaments (Karabinos et al, 2001). In crustaceans, there is at least one remarkable difference in the structure of MTs compared to vertebrates: the side-arms departing from the vertebrate MTs are constituted mainly by tau and MT-associated proteins (MAPs) (Hirokawa, 1986); whereas in crustaceans, besides MAP 2 (Bloom et al, 1984(Bloom et al, , 1985Matus et al, 1981), NF proteins also constitute the MT side-arms (Corrêa et al, 2004).…”

“…In decapod crustaceans, the PCT is the nerve pathway that connects the terminal medulla and hemiellipsoid body to the anterior medial protocerebral structures (Corrêa et al, 2004(Corrêa et al, , 2005Sandeman et al, 1992). This tract has been used in our laboratory to study glial-axon relationships and specific issues related to the cell biology of the crustacean axoplasm (Allodi et al, 1999;Corrêa et al, 2004Corrêa et al, , 2005.…”

New Insights on the Olfactory Lobe of Decapod Crustaceans

“…One series of sections was immediately dewaxed, hydrated, and stained with Mallory trichromic, hematoxylin and eosin or 5% toluidine blue for routine histological analyses. Series of sections destined for the immunohistochemical reactions were also dewaxed, hydrated, and then washed in PBS with 0.3% Triton X-100, incubated with 5% normal goat serum, and afterwards with the primary antibody overnight at 4 ° C. The primary antibodies used were: rabbit anti-serotonin diluted at 1: 100 (Sigma), based on Harzsch and Waloszek [2000], who used this label in order to enhance the visualization of functional neurites in decapod crustaceans, showing active neural routes; mouse anti-neurofilament medium (N-160), to reveal axon bundles [Corrêa et al, 2004] and rabbit anti-neurofilament high (N-200; Sigma), diluted at 1: 100; rabbit anti-neuronal nitric oxide synthase (NOS; Sigma) diluted 1: 100, for revealing projection neurons [Benton et al, 2007]; and rabbit anti-synapsin (synorf 1; Hybridoma bank), diluted at 1: 50, because it labels synaptic contacts; therefore the distribution this marker reveals directly relates to the synaptic and probably to the functional organization [Vilpoux et al, 2006;Harzsch and Mül-ler, 2007].…”

Purification of Mitochondrial Proteins HSP60 and ATP Synthase from Ascidian Eggs: Implications for Antibody Specificity

“…NN18, also called NF-160 or NF-M, reacts with the medium molecular weight (160 kDa) neurofilament subunit and labels exclusively neurofilaments in vertebrates as well as crab [30]–[33]. In the ascidian, it was found that NN18 recognizes a 58 kDa protein and strongly labels myoplasm in eggs from numerous species [13], [34]–[37].…”