Neurofilaments (NFs) are cytoskeletal proteins that are localized within nerve cells, which form long oriented bundles running the length of axons. While abnormal aggregations of these proteins have been implicated in several neurological disorders including Parkinson's disease and ALS, interfilament interactions in both the normal and diseased states are not well understood. In vivo, NFs are supramolecular structures composed of three subunit proteins of low (NF-L), medium (NF-M), and high molecular (NF-H) weight that assemble into a 10 nm diameter rod with radiating sidearms, forming a bottle-brush conformation. In this study we alter the subunit composition and probe the resulting networks with polarized microscopy and synchrotron small angle x-ray scattering (SAXS), in order to isolate the role of each subunit in interfilament interactions.

By reassembling NFs in vitro from varying ratios of the subunit proteins, purified from bovine spinal cord, we form filaments with controlled subunit compositions. The resulting filaments, at a high volume fraction, are nematic liquid crystalline gels with a well defined spacing, determined with SAXS. Upon dilution the difference between the subunits is realized with NF-M grafted filaments being dominated by attractive interactions and remaining aligned, while those flanked with NF-H sidearms repel and become isotropic gels. Interplay between these forces is seen in the ternary system composed of all three subunit proteins (NF-LMH).

The polyampholytic subunits have a charge distribution that varies along the length of the sidearm, which forms the brush layer, and the distribution is different for each subunit. The interfilament interactions are highly dependent on environmental conditions including salt concentration, pH, and osmotic pressure. Increasing ionic strength induces attractive interactions and a stabilization of the nematic phase in filaments that were repulsive at lower monovalent salt concentration. The interfilament spacing decreases with ionic strength and the effect is more dramatic in NF-H grafted filaments. Attractive forces are also increased at lower pH. Introducing an inert polymer to the solution surrounding the NF gel shows that the elastic response of the network to osmotic pressure is dependent on the subunit composition.