9723288 Cohlberg Research will be conducted on the pathway and mechanism of assembly of mammalian neurofilaments (NFs) from their constituent proteins and the role of phosphorylation in regulating the assembly process and the properties of the filaments. The goals of the research are to identify and quantitatively characterize the interactions which determine the specificity of interaction of neurofilament proteins and are responsible for filament stabilization, and to gain information about the parameters which characterize the dynamic properties of NFs. In large caliber neurons of the central nervous system, NFs are composed of three "NF triplet" proteins, termed NF-H (high), NF-M (middle), and NF-L (low) according to their molecular weights. In certain neurons, other proteins, including alpha-internexin, are present in NF. Neurofilament (NF) proteins contain alpha-helical rod domains flanked by nonhelical head and tail domains. They aggregate to form coiled coil dimers, which then aggregate further to form NF-L/NF-M and NF-L/NF-H heterotetramers, which in turn serve as intermediates in the assembly of filaments. It is not certain whether the coiled coil dimers are heterodimers or homodimers. Disulfide cross-linking will be used as a probe to detect the formation of heterodimers from homodimers, and the kinetics of monomer exchange and its dependence on solvent conditions will be characterized. The stabilities of various heterodimers containing both full-length proteins and their rod domains will be investigated by sedimentation and cross-linking studies, and the roles of various proteins domains in the association of dimers to tetramers will be characterized. The roles of the various domains in the formation of heterotetramer assembly intermediates will be investigated by the use of nondenaturing polyacrylamide gel electrophoresis. The principal question to be answered is whether specific interactions among rod domains are fully responsible for th e specificity of protein association at this level (LM and MH complexes are formed, but not MH) or whether the head and tail domains also play a role. Furthermore, the roles of the various protein domains in determining the specificity with which proteins associate to form filaments will be examined by filament reconstitution experiments and analyzed by filament pelleting experiments and by examination of reconstituted filaments by electron microscopy, including immunogold labeling studies. Finally, NF proteins will be modified by the covalent attachment of fluorophores, and the assembly and disassembly of filaments and the exchange of proteins between filaments will be followed by fluorescence energy transfer measurements. All of these studies will include experiments on alpha- internexin as well as the NF triplet proteins. A key attribute of eukaryotic cells is the presence of filamentous structures inside the cells, collectively termed the cytoskeleton, which provide a structural framework for the cytoplasm and its functions. The three major types of cytoskeletal "elements" in cells, distinguished by their molecular composition and structural features, are the actin- containing microfilaments, the 10-nm diameter intermediate filaments, and microtubules. Neurofilaments (NF) are those members of the family of intermediate filaments (IFs) that form part of the cytoskeleton in neurons (cells of the nervous system). They are found primarily running longitudinally down the long cellular projections known as axons, along with microtubules, and they are thought to be responsible for promoting the radial growth (thickening) of axons, establishing axonal diameter, and maintaining the structural integrity of the axon and its resistance to compressive forces. In large caliber neurons of the central nervous system, NFs are composed of three "NF triplet" proteins, distinguished by their molecular weights (heavy-, middle-, and low-weight). In certain neurons, add itional proteins, e.g., alpha-internexin, are present in NFs. The proteins are synthesized in the cell body and move down the axon in a process known as slow axonal transport, eventually leaving the moving phase and becoming incorporated into the stationary filament network of the axon. A number of diseases of motor neurons involve abnormal NFs, and overexpression of either the light or heavy weight NF in transgenic mice leads to pathology very similar to amyotrophic lateral sclerosis (ALS, or "Lou Gherig's Disease"). Very little is known about how these NF proteins assemble to form functional filaments. This research will provide vital information on the chemical and physical properties of NF protein components and their interactions with each other, which will help us better understand filament assembly and transport in living neuronal cells. ***
