9316365 Cohlberg Research will be conducted on the pathway and mechanisms of assembly of mammalian neurofilaments from their constituent proteins and the role of phosphorylation in regulating the assembly process and the properties of the filaments. The goals are to: 1, gain information about the arrangement of the three proteins in neurofilaments (NF), their structural roles, and the pathway by which they assemble into filaments and by which they become incorporated in preexisting filaments; 2, investigate the role of phosphorylation in regulating assembly and in molecular conformation of the proteins; and 3, gain information on the protein kinase enzymes which regulate neurofilament phosphorylation. The first phase of the project will center on the role of certain mixed tetramer assembly intermediates, one containing both NF-L and NF-M and another containing NF-L and NF-H. The presence of these species is detected by polyacrylamide "native gel" electrophoresis. The arrangement of polypeptide chains in the complex will be investigated by electron microscopy of rotary shadowed protein preparations and by experiments examining the ability of chemically cross-linked proteins to form the tetrameric complexes. Proteins produced by recombinant DNA techniques will be used to determine the nature of the specific chemical interactions which determine the rules by which the three proteins combine. NF proteins contain three domains -- a central rod domain which forms the body of the filament, a head domain important in the assembly of the rods, and tail domains which project from the filament. By cutting the DNA from the genes for the proteins into fragments encoding the individual domains and then pasting the fragments together in various combinations, and then expressing the resultant DNAs in bacteria, once can swap domains and produce a modular protein with domains derived from different members of the NF triplet. Determining the ability of these modular proteins to combine with each other should help to determine the roles of different domains in the specific interactions which determine the arrangement of the three NF proteins in filaments. The effect of phosphorylation of the head domains of NF-M and NF-H on assembly will be determined by adding phosphates with the use of enzymes known to phosphorylate these domains and then conducting centrifugation experiments to examine the ability of the phosphorylated proteins to form pelletable filaments. The effect of tail domain phosphorylation on protein conformation will be investigated by using kinase and phosphatase enzymes to prepare NF in different states of phosphorylation and then to measure their circular dichroism and infrared spectra. Protein kinases which catalyze NF phosphorylation will be identified from neuronal extracts. The action of the enzyme, glycogen synthase kinase 3, shown in previous work to phosphorylate NF in vitro, will be further characterized. %%% Neurofilaments (NF) are those members of the family of "intermediate filaments" which form part of the cytoskeleton in neurons of the central nervous system. They are found primarily running longitudinally down the axon along with microtubules, and they are thought to be responsible for promoting the radial growth of axons, establishing axonal diameter, and maintaining the structural integrity of the axon and its resistance to compressive forces. The are composed of three "NF triplet" proteins, termed NF-H (high), NF-M (middle), and NF-L (low) according to their molecular weights. These proteins are synthesized in the cell body and move down the axon in a process termed "slow axonal transport," eventually leaving the moving phase and becoming incorporated into the stationary filament network. As they move down the axon, phosphate groups are added, primarily to the carboxyl-terminal "tail" domains which project from the filaments. A number of diseases of motor neurons involve abnormal NF, and overexpression of either NF-L or NF-H in mice leads to pathology very similar to amyotrophic lateral sclerosis ("Lou Gherig's disease"). In addition, in this and other neuropathologies, abnormally phosphorylated NF accumulate in different parts of the neuron. In addition to the obvious relevance to health and the normal function of CNS neurons, these biochemical studies of NF can be expected to lead to insights into "general principles" of cytoskeletal assembly in other intermediate filament systems. The mechanism of self-assembly of biological polymers is of particular interest in the area of biomolecular materials, materials science, and materials and nanofabrication engineering, since the underlying principles, and even the biological polymers themselves, have potential for adaptation and commercial exploitation as "smart" materials for a variety of uses. ***
