The single neurofilament subunit of lamprey may need another element for filament assembly

Regenerating axon tips in transected lamprey spinal cord contain dense accumulations of neurofilaments (NFs), suggesting that NFs may play a role in the mechanism of axonal regeneration. Compared with heteropolymeric assemblies of NF triplet proteins in mammals, NF in lampreys has been thought to contain only a single subunit (NF180). This would imply that NF180 self-assembles, which would be important for manipulating its expression in studies of axonal regeneration. In order to study the possible role of NF in process outgrowth and to determine whether NF180 can self-assemble, its gene was transfected into mammalian and fish cell lines that either contain or lack vimentin. In transfected NIH3T3 cells, NF180 was poorly phosphorylated and its expression did not alter the length or number of cell processes. Nor did it appear to form typical intermediate filaments, suggesting that it may not self-assemble. NF180 also did not form typical filaments in SW13cl cells that either possessed or lacked vimentin, nor in transfected fish cells that were cultured at 18 degrees C. In vitro, NF180 could not self-assemble but interacted with NF-L to interrupt its self-assembly. When cotransfected with rat NF-L into SW13c1.2vim(-) cells, NF180 did form thick, rod-like filamentous structures on immunofluorescence. More typical NFs were observed when NF180 was cotransfected with both NF-L and NF-M. Thus, NF180 cannot self-assemble but appears to require one or more additional elements for incorporation into NFs.