This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The proposed experiments aim to elucidate the nature of structures and interactions in reconstituted assemblies of filamentous neurofilaments (NFs) derived from neurons of the bovine central nervous system. NFs form networks in neurons, which are believed to play a key role both in the structural stability of neuronal processes (axons and dendrites) and as scaffolds for microtubules dispersed throughout the cytoskeleton of the processes. Neurofilaments consist of three homopolymers NF-L, NF-M, and NF-H, assembled in varying composition to form the mature NF filament with protruding C-terminus sidearms. However, the nature of the interfilament interactions and the precise role of each homopolymer within the network remain unclear. In mature neurons the composition of the three NF fractions is regulated over a narrow range with deviations in composition resulting in the disruption of the NF-network (hypothesized to be due to incorrect sidearm interactions) and motor neuron diseases (e.g. amyotrophic lateral sclerosis, Lou Gehrig?s disease). Thus, it is important to understand the role of distinct sidearms in imparting stability to the network through sidearm-sidearm interactions. Our recent synchrotron SAXS data suggests qualitatively different behavior for interfilament interactions with sidearms consisting of either NF-M or NF-H. Using the synchrotron SAXS-osmotic pressure technique, a series of direct force measurements are proposed, which when combined with complementary phase behavior studies, should result in a comprehensive understanding of the nature of the interactions between neurofilaments. The studies will be conducted in NF gels as a function of sidearm densities in binary (NF-LH, NF-LM), and ternary (NF-LMH) mixtures mimicking in-vivo conditions. Aside from further enhancing our knowledge of charged biopolymer networks, the experiments should enhance the understanding of structures and interactions within the axonal cytoskeleton.