Joanna Deek

Tunable, High Modulus Hydrogels Driven by Ionic Coacervation

The need for robust and responsive hydrogels in numerous pharmaceutical, biomedical, and industrial applications has motivated intense research efforts in these important polymeric materials. [ 1–6 ] The defi ning feature of hydrogels is that the vast majority of their mass consists of water, yet they still exhibit solid-like mechanical properties due to the presence of a three-dimensional network structure that, classically, is created through in situ covalent bond formation between multifunctional, reactive precursors. [ 6 , 7 ] A wide variety of chemistries have been utilized for covalent crosslinking of hydrogel-forming materials, e.g. free radical polymerization, Michael addition, and thiol-ene coupling, with the resulting hydrogels having good mechanical properties arising from the strong covalently bonded framework. [ 1 , 6–10 ] Limitations of the covalent approach are that the hydrogels are not re-moldable once formed, have limited responsiveness to external stimuli, and may require organic co-solvents/reagents during their formation. To overcome these limitations, hydrogels formed through non-covalent, physical associations arising from intermolecular interactions, in lieu of covalent crosslinks, have attracted signifi cant interest recently, particularly as responsive materials and injectable gels. [ 5 , 6 ] Typically, a drawback of such physically-associated hydrogels is their poor mechanical properties due to generally weak intermolecular interactions. [ 11 , 12 ] However, recent work by Gong et al. [ 13 ] and Yasuda et al . [ 14 ] on double network gels and Wang et al . [ 15 ] on the development of “aquamaterials” has demonstrated that signifi cant improvement in hydrogel mechanical properties is possible through careful design of the intermolecular interactions and length-scales between crosslinks or physical associations. In addressing new strategies to yield high performance, physically associated hydrogels, the role of dynamic materials formed via electrostatic interactions serves as a powerful model. While block copolyelectrolytes are widely used in the construction of hydrogel materials, the majority of these systems are based on block copolymers where the ionic blocks serve as the water soluble component and neutral, hydrophobic blocks

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.

Unconventional salt trend from soft to stiff in single neurofilament biopolymers.

We present persistence length measurements on neurofilaments (NFs), an intermediate filament with protruding side arms, of the neuronal cytoskeleton. Tapping mode atomic force microscopy enabled us to visualize and trace at subpixel resolution photoimmobilized NFs, assembled at various subunit protein ratios, thereby modifying the side-arm length and chain density charge distribution. We show that specific polyampholyte sequences of the side arms can form salt-switchable intrafilament attractions that compete with the net electrostatic and steric repulsion and can reduce the total persistence length by half. The results are in agreement with present X-ray and microscopy data yet present a theoretical challenge for polyampholyte interchain interactions.

Structures and interactions in ‘bottlebrush’ neurofilaments: the role of charged disordered proteins in forming hydrogel networks

NFs (neurofilaments), the major cytoskeletal constituent of myelinated axons in vertebrates, consist of three different molecular-mass subunit proteins, NF-L (low), NF-M (medium) and NF-H (high), assembled to form mature filaments with protruding intrinsically disordered C-terminal side-arms. Liquid crystal gel networks of side-arm-mediated NF assemblies play a key role in the mechanical stability of neuronal processes. Disruptions of the NF network, due to NF overaccumulation or incorrect side-arm interactions, are a hallmark of motor neuron diseases including amyotrophic lateral sclerosis. Using synchrotron small-angle X-ray scattering and various microscopy techniques, we have investigated the role of the peptide charges in the subunit side-arms on the structure and interaction of NFs. Our findings, which delineate the distinct roles of NF-M and NF-H in regulating NF interactions, shed light on possible mechanisms of disruption of optimal mechanical network properties.

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.

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

BACKGROUND Neurofilaments (NFs) - the neuron-specific intermediate filament proteins - are assembled into 10nm wide filaments in a tightly controlled ratio of three different monomer types: NF-Low (NF-L), NF-Medium (NF-M), and NF-High (NF-H). Previous work on reconstituted bovine NF hydrogels has shown the dependence of network properties, including filament alignment and spacing, on the subunit composition. METHODS We use polarized optical microscopy and SAXS to explore the full salt-dependent phase behavior of reconstituted bovine NF networks as a function of various binary and ternary subunit ratios. RESULTS We observe three salt-induced liquid crystalline phases: the liquid-ordered B(G) and N(G) phases, and the disordered I(G) phase. We note the emergent sidearm roles, particularly that of NF-H in driving the parallel to cross-filament transition, and the counter-role of NF-M in suppressing the I(G) phase. CONCLUSIONS In copolymers of NF-LH, NF-H shifts the I(G) to N(G) transition to nearer physiological salt concentrations, as compared to NF-M in copolymers of NF-LM. For ternary mixtures, the role of NF-H is modulated by the ratio of NF-M, where beneath 10wt.% NF-M, NF-H drives the transition to the disordered phase, and above which NF-H increases interfilament spacing. GENERAL SIGNIFICANCE Understanding the role of individual subunits in regulating the network structure will enable us to understand the mechanisms that drive the dysfunction of these networks, as observed in diseased conditions.

Mechanics of soft epithelial keratin networks depend on modular filament assembly kinetics

UNLABELLED Structural adaptability is a pivotal requirement of cytoskeletal structures, enabling their reorganization to meet the cellular needs. Shear stress, for instance, results in large morphological network changes of the human soft epithelial keratin pair K8:K18, and is accompanied by an increase in keratin phosphorylation levels. Yet the mechanisms responsible for the disruption of the network structure in vivo remain poorly understood. To understand the effect of the stress-related site-specific phosphorylation of the K8:K18 pair, we created phosphomimicry mutants - K8(S431E), K8(S73E), K18(S52E) - in vitro, and investigated the various steps of keratin assembly from monomer to network structure using fluorescence and electron microscopy, and using rheology characterized their network mechanical properties. We find that the addition of a charged group produces networks with depleted intra-connectivity, which translates to a mechanically weaker and more deformable network. This large variation in network structure is achieved by the formation of shorter mutant filaments, which exhibit differing assembly kinetics and a manifestly reduced capacity to form the extended structures characteristic of the wild-type system. The similarity in outcome for all the phosphomimicry mutants explored points to a more general mechanism of structural modulation of intermediate filaments via phosphorylation. Understanding the role of kinetic effects in the construction of these cytoskeletal biopolymer networks is critical to elucidating their structure-function properties, providing new insight for the design of keratin-inspired biomaterials. STATEMENT OF SIGNIFICANCE Structural remodeling of cytoskeletal networks accompanies many cellular processes. Interestingly, levels of phosphorylation of the human soft epithelial keratin pair K8:K18 increase during their stress-related structural remodeling. Our multi-scale study sheds light on the poorly understood mechanism with which site-specific phosphorylation induces disruption of the keratin network structure in vivo. We show how phosphorylation reduces keratin filament length, an effect that propagates through to the mesoscopic structure, resulting in the formation of connectivity-depleted and mechanically weaker networks. We determine that the intrinsically-set filament-to-filament attractions that drive bundle assembly give rise to the structural variability by enabling the formation of kinetically-arrested structures. Overall, our results shed light on how self-assembled intermediate filament structures can be tailored to exhibit different structural functionalities.

Responsive and Tunable Neurofilament Protein Hydrogel Assemblies - A Synchrotron X-Ray Scattering Study of Composition and Salt Dependent Response

Neurofilaments (NFs) are the intermediate filaments in neuronal cells that along with other cytoskeletal network structures play a major role in the mechanical integrity of neuronal processes. NFs are assembled from three different subunits (NF-Low (NF-L), NF-Medium (NF-M), NF-High (NF-H)) that differ mainly in the sequence length of their unstructured C-terminal sidearms. The sidearms direct the lateral associations between filaments thus forming the NF hydrogel networks (the physiologically relevant NF protein assembly state in the axoplasm). The interfilament lateral associations are predominantly electrostatic, enabled by the polyampholytic nature of the sidearms. We examine their strength and range by varying the salinity of the in vitro buffer. Furthermore, motivated by variable in vivo subunit expression in axons versus dendrites that results in variable network packing, reassembled (in vitro) binary system NF-hydrogels have revealed the different contributions of individual subunits to interfilament interactions and to network interfilament spacings [1]. Synchrotron x-ray scattering experiments have allowed us to study the changes in the microscopic structure of NF hydrogels as a function of salt and sidearm density. At high weight ratios of NF-M and NF-H, and as a function of increasing salt concentrations, NF gels exhibit an unexpectedly abrupt transition from a weakly oriented (nearly isotropic) low filament density gel with interfilament spacing d=1000A to a highly oriented liquid crystalline gel with high filament density and d=500A (NF-M) and 700A (NF-H). 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 one with orientational plasticity.Funded by DOE-BES-DE-FG-02-06ER46314 and NSF-DMR-1101900[1] R. Beck, J. Deek, J.B. Jones, C.R. Safinya. Nature Materials. 9, 40 (2010).

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)