2 DISTINCT FUNCTIONS OF THE CARBOXYL-TERMINAL TAIL DOMAIN OF NF-M UPON NEUROFILAMENT ASSEMBLY - CROSS-BRIDGE FORMATION AND LONGITUDINAL ELONGATION OF FILAMENTS

Neurofilaments are the major cytoskeletal elements in the axon that ta ke highly ordered structures composed of parallel arrays of 10-nm fila ments linked to each other with frequent cross-bridges, and they are b elieved to maintain a highly polarized neuronal cell shape. Here we re port the function of rat NF-M in this characteristic neurofilament ass embly. Transfection experiments were done in an insect Sf9 cell line l acking endogenous intermediate filaments. NF-L and NF-M coassemble to form bundles of 10-nm filaments packed in a parallel manner with frequ ent cross-bridges resembling the neurofilament domains in the axon whe n expressed together in Sf9 cells. Considering the fact that the expre ssion of either NF-L or NF-M alone in these cells results in neither f ormation of any ordered network of 10-nm filaments nor crossbridge str uctures, NF-M plays a crucial role in this parallel filament assembly. In the case of NF-H the carboxyl-tail domain has been shown to consti tute the cross-bridge structures. The similarity in molecular architec ture between NF-M and NF-H suggests that the carboxyl-terminal tail do main of NF-M also constitutes cross-bridges. To examine this and to fu rther investigate the function of the carboxyl-terminal tail domain of NF-M, we made various deletion mutants that lacked part of their tail domains, and we;expressed these with NF-L. From this deletion mutant analysis, we conclude that the carboxyl-terminal tail domain of NF-M h as two distinct functions. First, it is the structural component of cr oss-bridges, and these crossbridges serve to control the spacing betwe en core filaments. Second, the portion of the carboxyl-terminal tail d omain of NF-M that is directly involved in crossbridge formation affec ts the core filament assembly by helping them to elongate longitudinal ly so that they become straight.