Neurofilaments (NFs), the class of intermediate filaments characteristic of neuronal cells, are one of three structural protein groups that collectively form the cytoskeletal network. The active structure that NFs assume in the cell, and that permits them to fulfill their physiological role, is a space-filling expanded hydrogel held together by interfilament associations. These lateral inter- filament associations are predominantly electrostatic and established by the polyampholytic nature of the sidearms, the unstructured C-termini of the three constituent subunits: NF-Low (NF-L), NF-Medium (NF-M), and NF-High (NF-H).

Using synchrotron x-ray scattering experiments and rheology, we examine the strength and range of these regulatory electrostatic associations by varying the salinity of the in vitro buffer. Furthermore, motivated by variable in vivo subunit expression in axons versus dendrites that results in variable network packing, reassembled (in vitro ) binary system NF-hydrogels have revealed the different contributions of individual subunits to interfilament interactions and to network interfilament spacings. Upon varying the monovalent salt concentration of the in vitro buffer for binary (and ternary) NF systems, we discover the presence of three distinct salt-induced phases, with a re-entrant isotropic phase (at intermediate salts ≈ 40mM) that persists between two different liquid crystal nematic phases.

Hydrogels in each of these phases exhibit characteristic physical features and mechanical properties, which served as the inspiration for the tri-phase classification system, and that ultimately stem from the defining nature and strength of the interfilament interactions and thus the hydrogels' water retention capabilities. Within the lowest and non-physiologically relevant salt range (≈ 5mM), the nematic hydrogels are very elastic and brittle, observed for the first time with filamentous protein hydrogels, and display a bluish tint macroscopically as well as enhanced small angle scattering---the BG hydrogels. An increase in sample salt concentration "melts" the nematic order, resulting in an isotropic phase at intermediate salts---the IG. The third phase---NG---appears with a further increase in salt concentration, and is also characterized by internal filament order. Interestingly, NF- L homopolymer hydrogels do not abide by the tri-phase classification system in that an isotropic phase is never observed for these gels.

In BG, the sidearms are highly charged, stretched, and drive counterion condensation from the buffer. BG hydrogels also have a phenomenal ability to hold their macroscopic shape and are non-sticky with reference to hydrophilic surfaces: self-associations have saturated all the sidearms making them unavailable for non-self interactions. Furthermore, it is this higher energetic barrier (from stronger bond formation) that discourages constituent rearrangement, as would occur as a result of water loss for instance, which could be another reason for the low rate of water loss. Upon melting into IG (≈ 40mM), the network looses internal order (filament orientation) in an attempt to minimize repulsion, an aspect that is paralleled by the marked increase in the viscous component of the hydrogel, and clears up. The loss of filament orientation, as well as the ensuing loss of extensive sidearm overlap, means that a larger percentage of the sidearms is free from association: this explains the tendency for IG hydrogels to spread along and "wet" a hydrophilic glass surface. Furthermore, loss of a non-continuous mesh and the increased flexibility in filament rearrangement (since they are minimally bound by interactions) hastens subsequent gel dehydration.

By comparing and contrasting the experimental findings for hydrogels of varied subunit compositions in all three hydrogel phases---BG , IG, and NG---we address the relationship between interfilament association strength and the nature of the resultant network. As such, we can present an image of network substructure and an understanding of how it translates to the observed macroscopic properties. (Abstract shortened by UMI.).