Biochemical and immunocytochemical characterization and distribution of phosphorylated and nonphosphorylated subunits of neurofilaments in squid giant axon and stellate ganglion

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Abstract
Monoclonal antibodies to squid neurofilament (aNFP) and intermediate filament (aIFA) proteins were used as probes for the biochemical and immunocytochemical analyses of neurofilament structure and distribution in the squid giant axon and stellate ganglion. On Western blots the aNFP antibody stained exclusively the 220 kDa and high-molecular-weight (HMW) components of neurofilaments in the giant axon, whereas the aIFA antibody primarily labeled the 60 kDa protein in the giant axon and the 60 and 65 kDa proteins in the stellate ganglion. Dephosphorylation of axoplasmic proteins by alkaline phosphatase resulted in a decrease in the molecular weights of both the 220 kDa and HMW neurofilament proteins and a concomitant loss of reactivity with the aNFP antibody on Western blots. This indicated that the aNFP antibody is specific for a phosphorylated epitope in the neurofilament. Increased dephosphorylation of the 220 kDa protein led to an enhanced immunostaining of the resultant 190 kDa polypeptide by the aIFA antibody, suggesting that the phosphorylated epitope may mask the conserved epitope recognized by aIFA. Light and electron microscopic immunocytochemical studies show intense labeling by the aNFP antibody in the giant axon. In contrast, the aIFA antibody labeled the glial cells around the giant axon intensely, while labeling of the giant axon itself was considerably less than that with the aNFP antibody. Since the 60 kDa protein in axoplasm is intensely stained by the aIFA antibody on Western blots, the relatively low amounts of labeling seen on semithin and thin sections of the giant axon by this antibody may be due to the masking of the 60 kDa protein by in situ fixed axoplasmic proteins. However, the aIFA antibody intensely labeled glial cells within the stellate ganglion and "islands" of filaments and nuclear membranes within ganglion cells. No reactivity for either antibody was seen in synapses. The aNFP antibody specifically labeled "beadlike" portions and cross-bridges on the axonal neurofilaments, suggesting that these components consist of the 220 kDa and HMW proteins. In contrast, the aIFA antibody labeled relatively smooth filaments in ganglion and glial cells. These data suggest that the 65 kDa protein represents the squid glial filament protein and that the 60 kDa protein found in axoplasm represents the low-molecular weight subunit in the axonal neurofilament. The latter appears to be formed and/or organized in "islands" of filaments within ganglion cells. These results support the view that the formation, maturation, and degradation of neurofilaments are regionally segregated within the neuron.