Jayna B. Jones

Gel-expanded to gel-condensed transition in neurofilament networks revealed by direct force measurements

Neurofilaments (NF)--the principal cytoskeletal constituent of myelinated axons in vertebrates--consist of three molecular-weight subunit proteins NF-L (low), NF-M (medium) and NF-H (high), assembled to form mature filaments with protruding unstructured C-terminus side arms. Liquid-crystal gel networks of side-arm-mediated neurofilament assemblies have a key role in the mechanical stability of neuronal processes. Disruptions of the neurofilament network, owing to neurofilament over-accumulation or incorrect side-arm interactions, are a hallmark of motor-neuron diseases including amyotrophic lateral sclerosis. Using synchrotron X-ray scattering, we report on a direct measurement of forces in reconstituted neurofilament gels under osmotic pressure (P). With increasing pressure near physiological salt and average phosphorylation conditions, NF-LMH, comprising the three subunits near in vivo composition, or NF-LH gels, undergo for P > P(c) approximately 10 kPa, an abrupt non-reversible gel-expanded to gel-condensed transition. The transition indicates side-arm-mediated attractions between neurofilaments consistent with an electrostatic model of interpenetrating chains. In contrast, NF-LM gels remain in a collapsed state for P < P(c) and transition to the gel-condensed state at P > P(c). These findings, which delineate the distinct roles of NF-M and NF-H in regulating neurofilament interactions, shed light on possible mechanisms for disruptions of optimal mechanical network properties.

Interplay Between Liquid Crystalline And Isotropic Gels in Self-Assembled Neurofilament Networks

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.

Direct Imaging of Aligned Neurofilament Networks Assembled Using In Situ Dialysis in Microchannels

We report a technique to produce aligned neurofilament networks for direct imaging and diffraction studies using in situ dialysis in a microfluidic device. The alignment is achieved by assembling neurofilaments from protein subunits confined within microchannels. Resulting network structure was probed by polarized optical microscopy and atomic force microscopy, which confirmed a high degree of protein alignment inside the microchannels. This technique can be expanded to facilitate structural studies of a wide range of filamentous proteins and their hierarchical assemblies under varying assembly conditions.

Liquid crystal assemblies in biologically inspired systems

In this paper, which is part of a collection in honour of Noel Clark’s remarkable career on liquid crystal (LC) and soft matter research, we present examples of biologically inspired systems, which form LC phases with their LC nature impacting biological function in cells or being important in biomedical applications. One area focuses on understanding network and bundle formation of cytoskeletal polyampholytes (filamentous actin, microtubules and neurofilaments (NFs)). Here, we describe studies on NFs, the intermediate filaments of neurons, which form open network nematic LC hydrogels in axons. Synchrotron small-angle-X-ray scattering studies of NF protein dilution experiments and NF hydrogels subjected to osmotic stress show that NF networks are stabilised by competing long-range repulsion and attractions mediated by the NF’s polyampholytic sidearms. The attractions are present both at very large inter-filament spacings, in the weak sidearm-interpenetrating regime, and at smaller inter-filament spacings, in the strong sidearm-interpenetrating regime. A second series of experiments will describe the structure and properties of cationic liposomes (CLs) complexed with nucleic acids (NAs). CL-NA complexes form liquid crystalline phases, which interact in a structure-dependent manner with cellular membranes enabling the design of complexes for efficient delivery of NA (DNA and RNA) in therapeutic applications.

Supramolecular assembly of biological molecules purified from bovine nerve cells: from microtubule bundles and necklaces to neurofilament networks

With the completion of the human genome project, the biosciences community is beginning the daunting task of understanding the structures and functions of a large number of interacting biological macromolecules. Examples include the interacting molecules involved in the process of DNA condensation during the cell cycle, and in the formation of bundles and networks of filamentous actin proteins in cell attachment, motility and cytokinesis. In this proceedings paper we present examples of supramolecular assembly based on proteins derived from the vertebrate nerve cell cytoskeleton. The axonal cytoskeleton in vertebrate neurons provides a rich example of bundles and networks of neurofilaments, microtubules (MTs) and filamentous actin, where the nature of the interactions, structures, and structure–function correlations remains poorly understood. We describe synchrotron x-ray diffraction, electron microscopy, and optical imaging data, in reconstituted protein systems purified from bovine central nervous system, which reveal unexpected structures not predicted by current electrostatic theories of polyelectrolyte bundling, including three-dimensional MT bundles and two-dimensional MT necklaces.

Tuning of Neurofilament Hydrogel Network Features - a Synchrotron X-Ray Scattering Study of Salt Dependent Network Response

Neurofilaments (NFs) are cytoskeletal proteins expressed in neuronal cells, with a role in the maintenance and mechanical integrity of neuronal processes. NFs assemble as flexible cylinders from 3 protein subunits: NF-Low (NF-L), NF-Medium (NF-M), and NF-High (NF-H). The variable length and charge of the subunits sets the strength and range of the interactions, which are predominantly electrostatic. Reassembled (in vitro) binary system hydrogels have shown us the different contributions of individual subunits to interfilament interactions and thus to network characteristics [1,2]. We emulate cellular conditions by varying the salinity of the in vitro buffer: low salt conditions parallel higher inherent charge of the subunits, and high salt conditions parallel the lower inherent charge states of the subunits. The tunability of the network in vitro mirrors in vivo cellular control of the NF network via subunit phosphorylation, which may transition the network from a highly oriented rigid state to an isotropic gel with orientational plasticity. We describe synchrotron x-ray scattering experiments that have allowed us to quantitatively study the changes in the microscopic structure of the NF gels as a function of salt and sidearm density. At low NF-M and NF-H sidearm weight ratios, NF gels exhibit weak salt dependence. In contrast, at high weight ratios, and as a function of decreasing salt concentrations, NF gels exhibit an unexpectedly abrupt transition from highly oriented liquid crystalline gels with high filament density (α 1/d , d = interfilament spacing) to a weakly oriented (nearly isotropic) low filament density gel.Funded by DOE DE-FG-02-06ER46314, NSF DMR-0503347.[1] R. Beck, J. Deek, J.B. Jones, C.R. Safinya. Nature Materials, In Press[2] J.B. Jones, C.R. Safinya, Biophys. J. 95, 823 (2008)