Interplay between liquid crystalline and isotropic gels in self-assembled neurofilament networks

Abstract

Neurofilaments (NFs) are a major constituent of nerve cell axons that assemble from three subunit proteins of low (NF-L), medium (NF-M), and high (NF-H) molecular weight into a 10 nm diameter rod with radiating sidearms to form a bottle-brush-like structure. Here, we reassemble NFs in vitro from varying weight ratios of the subunit proteins, purified from bovine spinal cord, to form homopolymers of NF-L or filaments composed of NF-L and NF-M (NF-LM), NF-L and NF-H (NF-LH), or all three subunits (NF-LMH). At high protein concentrations, NFs align to form a nematic liquid crystalline gel with a well-defined spacing determined with synchrotron small angle x-ray scattering. Near physiological conditions (86 mM monovalent salt and pH 6.8), NF-LM networks with a high NF-M grafting density favor nematic ordering whereas filaments composed of NF-LH transition to an isotropic gel at low protein concentrations as a function of increasing mole fraction of NF-H subunits. The interfilament distance decreases with NF-M grafting density, opposite the trend seen with NF-LH networks. This suggests a competition between the more attractive NF-M sidearms, forming a compact aligned nematic gel, and the repulsive NF-H sidearms, favoring a more expansive isotropic gel, at 86 mM monovalent salt. These interactions are highly salt dependent and the nematic gel phase is stabilized with increasing monovalent salt.

FIGURE 1

Cartoon showing how the three subunit proteins, NF-L, NF-M, and NF-H (a) form dimers that associate into a mature filament with radiating sidearms (b, redrawn from Fuchs and Cleveland (4) and modified to show the different NF subunits). The dimers are half staggered and antiparallel in orientation, resulting in a 22 nm spacing between protruding tails along the length of the filaments. NF-L is the only subunit that can homopolymerize, whereas NF-M and NF-H can only form a dimer with NF-L. In a fully saturated filament (1:1 ratio of long tailed subunits, NF-M and/or NF-H, to NF-L) the dimers are arranged into a mature filament as shown in the schematic (c; x, y axis is not to scale), which represents one side of a filament composed of 32 total subunits or 16 per side. Each “X” is one dimer and the solid ovals symbolize the sidearm protrusions of either NF-M or NF-H; the open ovals signify NF-L tails.

FIGURE 2

Estimate of the charge distribution of the NF subunits with a rolling sum over 10 amino acids (18). The head, body, and tail domains are represented by the blocked regions along the plot from N-terminus to C-terminus, respectively. The charge distribution was calculated by assuming the charge of a single amino acid in solution at pH 6.8. Plots for NF-L (bovine), NF-M (bovine), and NF-H (mouse) without phosphorylation show the slightly negative tail of NF-M and overall charge neutral NF-H. NF-M and NF-H are highly phosphorylated, which changes each modified amino acid from neutral to approximately a −1.8 charge (sequences from expasy.org protein database). The bar between the NF-M and NF-H curves indicates the hydrophobicity of the subunits. The rolling sum over 10 amino acids of the average phosphorylation state is also plotted for NF-M (11) and NF-H, showing how dramatically the charge distribution changes with posttranslational modification. The curves show that NF subunits are polyampholytes, having both positively and negatively charged regions along the sidearm projection.

FIGURE 3

Curves showing the number of long-tailed subunits NF-M (a) and NF-H (b) that assemble (86 mM monovalent salt) with NF-L into a filament for a given initial composition before dialysis. The saturation point of NF-LM and NF-LH networks is the maximum number of NF-M or NF-H subunits that can assemble into a filament and is given by the point where the curves deviate from the initial linear slope. The maximum grafting density for NF-LM networks is 12 NF-M subunits per 32 total subunits and for NF-LH it is 8 NF-H subunits per 32 total subunits. The assembly efficiency is given by the slopes of the initial linear regions. The insets show a schematic representation of the distribution of the subunit tails for the saturation point as described in Fig. 1.

FIGURE 4

(a) Phase diagram showing the phase behavior, as determined via polarized microscopy, of NF-LM networks composed of increasing weight % NF-M as a function of total protein concentration (NF-L + NF-M) for a monovalent salt concentration of 86 mM. At high weight % total protein, the NFs form a nematic gel (N_G). The nematic gel is stabilized even at low weight % total protein where a two-phase nematic gel and isotropic sol (N_G + I_S) is reached. The transition to the two-phase region occurs at a higher weight % total protein as the NF-M grafting density is increased (solid line is a guide for the eye). Exemplary polarized micrographs (b) show the behavior of NF-LM networks at ∼1 wt % total protein (samples from the dashed line on the phase diagram from a) immediately after dilution and after equilibration of 30 days. Gel swelling can be seen in both the 0 wt % NF-M (100:0, NF-L/NF-M) and 20 wt % NF-M (80:20, NF-L/NF-M) whereas no gel swelling is seen upon dilution of the 35 wt % NF-M (65:35, NF-L/NF-M) networks. Bright field image (inset) shows a pellet with an asymmetric interface and a clear, not milky, appearance, indicating that the network is a gel.

FIGURE 5

(a) Phase diagram showing the phase behavior, as determined via polarized microscopy, of NF-LH networks composed of increasing weight % NF-H as a function of total protein concentration (NF-L + NF-H) for a monovalent salt concentration of 86 mM. At high weight % total protein, the NFs form a nematic gel (N_G) for all compositions. For low grafting densities of NF-H (<15 wt % NF-H), the nematic phase is stabilized at low weight % protein where the system becomes two-phase (N_G + I_S). Increasing weight % NF-H has the opposite effect of adding NF-M sidearms, resulting in a disordered isotropic gel + isotropic sol (I_G + I_S) at low weight % total protein (solid lines are guides for the eyes). Exemplary polarized micrographs show the behavior of NF-LH (b) networks at ∼1 wt % total protein (samples from the dashed line on the phase diagram from a) immediately after dilution and after equilibration of 30 days. The bright field image (inset) shows an asymmetric interface, indicating that the network is a gel.

FIGURE 6

SAXS scans of dilution series of NF-LM networks (a) at 86 mM monovalent salt (shown on a log-log scale with dilution series at additional compositions in the Supplementary Material figures, Data S1). The sharp correlation peak is related to the interfilament spacing, and an inward shift of the peak position corresponds to an increase in interfilament spacing. The curves are labeled with the weight % total protein. A compilation of the center-to-center interfilament distance (d spacing) versus weight % total protein shows a decrease in spacing with increasing NF-M grafting density (b). The d spacing was found by subtracting a linear background from SAXS intensity plots and fitting the resulting first order peak to a Lorentzian curve. Plotting the d spacing versus volume fraction^−1/2 (Φ^−1/2) for various weight ratios of NF-L:NF-M (c) shows that the filaments dilute like a two-dimensional solid, linear region, at the low weight % NF-M, and the slope is related to the NF diameter. Deviation from linearity corresponds to reaching the two-phase region in the dilution phase diagram (Fig. 4 a).

FIGURE 7

Exemplary SAXS scans of dilution series of NF-LH networks (a) for increasing grafting densities of NF-H (86 mM monovalent salt) labeled with weight % total protein (shown on a log-log scale in the Supplementary Material figures, Data S1). The primary correlation peak becomes much broader at high weight % total protein with increasing NF-H grafting density and disappears when the samples enter the isotropic gel phase (I_G) at low weight % total protein. A compilation of the center-to-center interfilament distance (d spacing) for NF-LH networks as determined with SAXS versus weight % total protein (b) shows that the interfilament spacing increases upon dilution until a correlation peak is no longer detectable when the isotropic phase is reached (as seen in the dilution phase diagram). The linear dependence of the d spacing versus volume fraction^−1/2 (Φ^−1/2) plot for various weight ratios of NF-L/NF-H indicates that the filaments dilute like a two-dimensional solid and the slope is related to the NF diameter.

FIGURE 8

Ternary phase diagram showing the phase behavior of filaments composed of all three NF subunits at 86 mM monovalent salt. The aligned nematic phase is stabilized with increasing weight % NF-M. Small angle synchrotron x-ray diffraction scans show the onset of a correlation peak with increasing weight % NF-M coinciding with nematic ordering.

FIGURE 9

NF-LM and NF-LH phase behavior as a function of monovalent salt in excess solvent. The y axis refers to total ionic strength comprised of 43 mM monovalent ions from the MES buffer plus added KCl. The isotropic phase is induced in all compositions at low monovalent salt concentration, and nematic ordering occurs with increasing ionic strength. A higher electrolyte concentration is needed to stabilize the nematic phase with high grafting densities of NF-H, whereas nematic anisotropy appears at low salt concentrations, well below physiological monovalent salt conditions, for NF-LM networks. Dashed lines are at 86 mM monovalent salt and solid lines are guides for the eyes.