Neurofilament Biophysics: From Structure to Biomechanics.

Neurofilaments (NFs) are multisubunit, neuron-specific intermediate filaments consisting of a 10-nm diameter filament "core" surrounded by a layer of long intrinsically disordered protein (IDP) "tails." NFs are thought to regulate axonal caliber during development and then stabilize the mature axon, with NF subunit misregulation, mutation, and aggregation featuring prominently in multiple neurological diseases. The field's understanding of NF structure, mechanics, and function has been deeply informed by a rich variety of biochemical, cell biological, and mouse genetic studies spanning more than four decades. These studies have contributed much to our collective understanding of NF function in axonal physiology and disease. In recent years, however, there has been a resurgence of interest in NF subunit proteins in two new contexts: as potential blood- and cerebrospinal fluid-based biomarkers of neuronal damage, and as model IDPs with intriguing properties. Here, we review established principles and more recent discoveries in NF structure and function. Where possible, we place these findings in the context of biophysics of NF assembly, interaction, and contributions to axonal mechanics.

Viscoelastic high-molecular-weight hyaluronic acid hydrogels support rapid glioblastoma cell invasion with leader-follower dynamics.

Hyaluronic acid (HA), the primary component of brain extracellular matrix, is increasingly used to model neuropathological processes, including glioblastoma (GBM) tumor invasion. While elastic hydrogels based on crosslinked low-molecular-weight (LMW) HA are widely exploited for this purpose and have proven valuable for discovery and screening, brain tissue is both viscoelastic and rich in high-MW (HMW) HA, and it remains unclear how these differences influence invasion. To address this question, hydrogels comprised of either HMW (1.5 MDa) or LMW (60 kDa) HA are introduced, characterized, and applied in GBM invasion studies. Unlike LMW HA hydrogels, HMW HA hydrogels relax stresses quickly, to a similar extent as brain tissue, and to a greater extent than many conventional HA-based scaffolds. GBM cells implanted within HMW HA hydrogels invade much more rapidly than in their LMW HA counterparts and exhibit distinct leader-follower dynamics. Leader cells adopt dendritic morphologies, similar to invasive GBM cells observed in vivo. Transcriptomic, pharmacologic, and imaging studies suggest that leader cells exploit hyaluronidase, an enzyme strongly enriched in human GBMs, to prime a path for followers. This study offers new insight into how HA viscoelastic properties drive invasion and argues for the use of highly stress-relaxing materials to model GBM.

Effects of Ionic Strength on the Morphology, Scattering, and Mechanical Response of Neurofilament-Derived Protein Brushes.

Protein brushes not only play a key role in the functionality of neurofilaments but also have wide applications in biomedical materials. Here, we investigate the effect of ionic strength on the morphology of protein brushes using continuous-space self-consistent field theory. A coarse-grained multiblock charged macromolecular model is developed to capture the chemical identity of amino acid sequences. For neurofilament heavy (NFH) brushes at pH 2.4, we predict three morphological regimes: swollen brushes, condensed brushes, and coexisting brushes, which consist of both a dense inner layer and a diffuse outer layer. The brush height predicted by our theory is in good agreement with the experimental data for a wide range of ionic strengths. The dramatic height decrease is a result of the electrostatic screening-induced transition from the overlapping state to the isolated state of the coexisting brushes. We also studied the evolution of the scattering and mechanical responses accompanying the morphological change. The oscillation in the reflectivity spectra characterizes the existence and microstructure of the inner condensed layer, whereas the shoulder in the force spectra signifies a swollen morphology.

A Balance between Inter- and Intra-Microgel Mechanics Governs Stem Cell Viability in Injectable Dynamic Granular Hydrogels.

Injectable hydrogels are increasingly explored for the delivery of cells to tissue. These materials exhibit both liquid-like properties, protecting cells from mechanical stress during injection, and solid-like properties, providing a stable 3D engraftment niche. Many strategies for modulating injectable hydrogels tune liquid- and solid-like material properties simultaneously, such that formulation changes designed to improve injectability can reduce stability at the delivery site. The ability to independently tune liquid- and solid-like properties would greatly facilitate formulation development. Here, such a strategy is presented in which cells are ensconced in the pores between microscopic granular hyaluronic acid (HA) hydrogels (microgels), where elasticity is tuned with static covalent intra-microgel crosslinks and flowability with mechanosensitive adamantane-cyclodextrin (AC) inter-microgel crosslinks. Using the same AC-free microgels as a 3D printing support bath, the location of each cell is preserved as it exits the needle, allowing identification of the mechanism driving mechanical trauma-induced cell death. The microgel AC concentration is varied to find the threshold from microgel yielding- to AC interaction-dominated injectability, and this threshold is exploited to fabricate a microgel with better injection-protecting performance. This delivery strategy, and the balance between intra- and inter-microgel properties it reveals, may facilitate the development of new cell injection formulations.

Glioma Cells Secrete Collagen VI to Facilitate Invasion.

While glioblastoma (GBM) progression is associated with extensive extracellular matrix (ECM) secretion, the causal contributions of ECM secretion to invasion remain unclear. Here we investigate these contributions by combining engineered materials, proteomics, analysis of patient data, and a model of bevacizumab-resistant GBM. We find that GBM cells cultured in engineered 3D hyaluronic acid hydrogels secrete ECM prior to invasion, particularly in the absence of exogenous ECM ligands. Proteomic measurements reveal extensive secretion of collagen VI, and collagen VI-associated transcripts are correspondingly enriched in microvascular proliferation regions of human GBMs. We further show that bevacizumab-resistant GBM cells deposit more collagen VI than their responsive counterparts, which is associated with marked cell-ECM stiffening. COL6A3 deletion in GBM cells reduces invasion, ß-catenin signaling, and expression of mesenchymal markers, and these effects are amplified in hypoxia. Our studies strongly implicate GBM cell-derived collagen VI in microenvironmental remodeling to facilitate invasion.