Direct Measurement of Inter-filament Forces in Neurofilament Networks: Synchrotron X-ray Diffraction Study under Osmotic Pressure

Abstract

Neurofilaments (NFs) are the major protein constituents in neuronal processes (axons and dendrites) that impart mechanical stability and act as structural scaffolds. The filaments assemble from 3 different subunit proteins (NF-L, NF-M, NF-H) to form a 10 nm diameter flexible polymer with radiating unstructured sidearms. Recent work, showed that at high protein concentration, the NFs form a nematic hydrogel network with a well-defined interfilament spacing as can be measured by synchrotron small angle x-ray scattering (SAXS) [1]. The x-ray/phase behavior study showed the role the different subunit protein compositions play in the interfilament interaction. In order to directly elucidate the interfilament forces responsible for the mechanical properties of NFs hydrogel, we conducted a SAXS-osmotic pressure study, which yielded pressure-distance curves at different subunit compositions and monovalent salts. We show that filaments composed with NF-L and NF-M strongly attract each other through their polyampholyte sidearms, in particularly at high monovalent salt. However, filaments comprised of NF-L and NF-H, at high NF-H grafting density, show a distinctly different pressure-distance dependency, with much larger interfilament spacing and weaker salt dependence. Supported by DOE DE-FG-02-06ER46314, NIH GM-59288, NSF DMR-0503347, and the Human Frontier Science Program organization.[1] J.B. Jones, C.R. Safinya, Biophys. J. 95, 823 (2008).