Liquid crystal assemblies in biologically inspired systems.

In this paper, which is part of a collection in honor of Noel Clark's remarkable career on liquid crystal and soft matter research, we present examples of biologically inspired systems, which form liquid crystal (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). Here, we describe studies on neurofilaments (NFs), the intermediate filaments of neurons, which form open network nematic liquid crystal hydrogels in axons. Synchrotron small-angle-x-ray scattering studies of NF-protein dilution experiments and NF hydrogels subjected to osmotic stress show that neurofilament networks are stabilized by competing long-range repulsion and attractions mediated by the neurofilament's polyampholytic sidearms. The attractions are present both at very large interfilament spacings, in the weak sidearm-interpenetrating regime, and at smaller interfilament 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 nucleic acid (DNA, RNA) in therapeutic applications.

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.

Synchrotron X-ray diffraction study of microtubules buckling and bundling under osmotic stress: a probe of interprotofilament interactions.

Microtubules are hollow cylinders composed of tubulin heterodimers that stack into linear protofilaments that interact laterally to form the microtubule wall. Synchrotron x-ray diffraction of microtubules under increasing osmotic stress shows they transition to rectangular bundles with noncircular buckled cross sections, followed by hexagonally packed bundles. This new technique probes the strength of interprotofilamen bonds, yielding insight into the mechanism by which associated proteins and the chemotherapy drug taxol stabilize microtubules.