Neurofilament sidearms modulate parallel and crossed-filament orientations inducing nematic to isotropic and re-entrant birefringent hydrogels

Abstract

Neurofilaments are intermediate filaments assembled from the subunits neurofilament-low, neurofilament-medium and neurofilament-high. In axons, parallel neurofilaments form a nematic liquid-crystal hydrogel with network structure arising from interactions between the neurofilaments' C-terminal sidearms. Here we report, using small-angle X-ray-scattering, polarized-microscopy and rheometry, that with decreasing ionic strength, neurofilament-lowhigh, neurofilament-low-medium and neurofilament-low-medium-high hydrogels transition from the nematic hydrogel to an isotropic hydrogel (with random, crossed-filament orientation) and to an unexpected new re-entrant liquid-crystal hydrogel with parallel filaments-the bluish-opaque hydrogel-with notable mechanical and water retention properties reminiscent of crosslinked hydrogels. Significantly, the isotropic gel phase stability is sidearm-dependent: neurofilament-low-high hydrogels exhibit a wide ionic strength range, neurofilament-low-medium hydrogels a narrow ionic strength range, whereas neurofilamentlow hydrogels lack the isotropic gel phase. This suggests a dominant regulatory role for neurofilament-high sidearms in filament reorientation plasticity, facilitating organelle transport in axons. Neurofilament-inspired biomimetic hydrogels should therefore exhibit remarkable structure-dependent moduli and slow and fast water-release properties.

Neurofilament networks: Salt-responsive hydrogels with sidearm-dependent phase behavior

“…This distinction in network substructure is also reflected in phasedependent mechanical and water retention properties. The N G and I G phases had elastic plateau modulus (G 0 ) ≈ 100 -200 Pa, comparable to other physical hydrogels [31], whereas the B G phase had significantly larger G 0 ≈ 3000 -4000 Pa (similar to chemically cross-linked hydrogels), presumably due to a much larger number and/or strength of crosslinks per unit volume [27,32]. In addition, the water retention propensity (reported as a percent of the normalized gel weight after 24 hrs of drying) of the B G (≈ 60%) is remarkably higher than that of the N G (≈ 40%) I G (less than 10%) phase.…”

“…The shape and water-retention properties of each of the hydrogels, coupled with POM images and scattering data, helped construct probable models for the relative strength of interfilament interactions and the average arrangement of constituent filaments, depicted in (iv.). Figure adapted from [27].…”

“…Previous small-angle X-ray scattering (SAXS) work on in vitro reconstituted NF networks demonstrate the direct role subunits play in tailoring the interfilament interactions, and thus the network properties [27][28][29][30]. At nearphysiological ionic strengths (86 mM monovalent salt, pH 6.8), and in the absence of external osmotic pressure, heteropolymers of neurofilament-lowmedium (NF-LM) at various mole ratios of NF-M form compact gels in the liquid-ordered nematic gel (N G ) phase.…”

“…With decreasing salt concentration (C S ) from physiological conditions, intended to mimic charge variation achieved in vivo via phosphorylation or dephosphorylation, hydrogels in the N G phase melt into the I G phase, and back into a liquid-ordered phase, the bluish-opaque gel (B G ) phase [27]. The regulatory role of the sidearms is most evident for the I G phase.…”

“…While homopolymer NF-Low hydrogels lack the I G phase entirely, NF-H in 70:30 wt% NF-L:NF-H (70:30 wt% NF-LH) hydrogels stabilizes the I G phase over a much wider salt range (∆C S ≈ 70 mM) than NF-M (∆C S ≈ 10 mM) in 60:40 wt% NF-L:NF-M (60:40 wt% NF-LM) hydrogels. In addition, both sidearms play a concerted role in the ternary 7:3:2 NF-LMH system: NF-H determines the C S transition from B G to I G , and NF-M plays a larger role in stabilizing the N G phase, and thus the I G to N G transition [27]. This distinction in network substructure is also reflected in phasedependent mechanical and water retention properties.…”