Lamprey neurofilaments contain a previously unreported 50-kDa protein

Abstract

We have previously hypothesized that regeneration of axons after spinal cord injury in the lamprey may involve assembly and transport of neurofilaments (NFs) into the growing tip. A single NF, NF-180, has been cloned in this laboratory and until now was thought to be the only NF subunit in lamprey nervous system. However, homopolymerization of NF-180 has not been observed either in experiments on transfected cells or in self-assembly tests in vitro. Forty-three monoclonal antibodies designated as LCM series were generated previously against cytoskeletal proteins of the lamprey nervous system. Seven LCMs were NF specific, and five were keratin specific, as demonstrated by immunohistochemistry. In the present study, one antibody, LCM40, selectively labeled axons in immunohistochemical sections and recognized a single 50-kDa protein in Western blots. Other neuron-specific LCMs and anti-NF antibodies, e.g., LCM39, recognized a known NF subunit, NF-180. Two-dimensional (2-D) gel electrophoresis was employed to separate otherwise indistinguishable individual cytoskeletal proteins. Western blot analysis with an antibody (IFA) that selectively labels all known intermediate filaments indicated that this 50-kDa protein is an intermediate filament (IF). The new protein was incorporated into IF polymers in vitro. Immunoelectron microscopy confirmed that neuronal IFs contain this novel protein. These results suggest that the 50-kDa protein is a previously unrecognized neuronal IF subunit in the lamprey.