Composite bottlebrush mechanics: α-internexin fine-tunes neurofilament network properties (original) (raw)

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

Nature Materials, 2010

NF-H (high), assembled to form mature filaments with protruding unstructured C-terminus side arms 1-5 . Liquidcrystal 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 sidearm interactions, are a hallmark of motor-neuron diseases including amyotrophic lateral sclerosis 3-9 . 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 ≈ 10 kPa, an abrupt non-reversible gel-expanded to gel-condensed transition. The transition indicates side-armmediated 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.

Relating Interactions between Neurofilaments to the Structure of Axonal Neurofilament Distributions through Polymer Brush Models

Biophysical Journal, 2002

Neurofilaments (NFs) have been proposed to interact with one another through mutual steric exclusion of their unstructured C-terminal "sidearm" domains, producing order in axonal NF distributions and conferring mechanical strength to the axon. Here we apply theory developed for polymer brushes to examine the relationship between the brush properties of the sidearms and NF organization in axons. We first measure NF-NF radial distribution functions and occupancy probability distributions for adult mice. Interpreting the probability distributions using information theory, we show that the NF distributions may be represented by a single pair potential of mean force. Then, to explore the relationship between model parameters and NF architecture, we conduct two-dimensional Monte Carlo simulations of NF cross-sectional distributions. We impose purely repulsive interaction potentials in which the sidearms are represented as neutral and polyelectrolyte chains. By treating the NFs as telechelic polymer brushes, we also incorporate cross-bridging interactions. Both repulsive potentials are capable of reproducing NF cross-sectional densities and their pair correlations. We find that NF structure is sensitive to changes in brush thickness mediated by chain charge, consistent with the experimental observation that sidearm phosphorylation regulates interfilament spacing. The presence of attractive cross-bridging interactions contributes only modestly to structure for moderate degrees of cross-bridging and leads to NF aggregation for extensive cross-bridging.

The regulative role of neurite mechanical tension in network development

Biophysical journal, 2009

A bewildering series of dynamical processes take part in the development of the nervous system. Neuron branching dynamics, the continuous formation and elimination of neural interconnections, are instrumental in constructing distinct neuronal networks, which are the functional building blocks of the nervous system. In this study, we investigate and validate the important regulative role of mechanical tension in determining the final morphology of neuronal networks. To single out the mechanical effect, we cultured relatively large ...

Structures and Interactions in Neurofilament: Gel Expanded To Gel Condensed Transition

2010

NF-H (high), assembled to form mature filaments with protruding unstructured C-terminus side arms 1-5 . Liquidcrystal 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 sidearm interactions, are a hallmark of motor-neuron diseases including amyotrophic lateral sclerosis 3-9 . 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 ≈ 10 kPa, an abrupt non-reversible gel-expanded to gel-condensed transition. The transition indicates side-armmediated 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.

Probing Mechanical Adaptation of Neurite Outgrowth on a Hydrogel Material Using Atomic Force Microscopy

Annals of Biomedical Engineering, 2011

In this study, we describe the design and initial results of probing mechanical adaptation of neurite growth of lightly fixed neurons on a hydrogel substrate by using atomic force microscopy (AFM). It has been shown previously that cells are responsive to the physical conditions of their micro-environment, and that certain cells can adjust their own stiffness as part of the adaptation to the substrate. AFM, a powerful tool to probe micro- and nano-scale structures, has been utilized in assessing topography, morphology, and structural change of neuronal cells. We used AFM with a robust force analysis approach in this study to probe the mechanical properties of both neurites and the substrate at close proximity. We first confirmed the robustness and consistency of the approach specific to soft materials by comparing measurements made on the same reference material using different methods. Subsequently, it was found that the primary spinal cord neurons that were lightly fixed exhibited different stiffnesses between the cell body and neurites. Furthermore, in comparison to the rigidity of the substrate, the stiffness of the neurites was lower, whereas that of the neuronal cell body was higher.

Hydrogels for 3D Neural Tissue Models: Understanding Cell-Material Interactions at a Molecular Level

Frontiers in Bioengineering and Biotechnology, 2020

The development of 3D neural tissue analogs is of great interest to a range of biomedical engineering applications including tissue engineering of neural interfaces, treatment of neurodegenerative diseases and in vitro assessment of cell-material interactions. Despite continued efforts to develop synthetic or biosynthetic hydrogels which promote the development of complex neural networks in 3D, successful long-term 3D approaches have been restricted to the use of biologically derived constructs. In this study a poly (vinyl alcohol) biosynthetic hydrogel functionalized with gelatin and sericin (PVA-SG), was used to understand the interplay between cell-cell communication and cell-material interaction. This was used to probe critical shortterm interactions that determine the success or failure of neural network growth and ultimately the development of a useful model. Complex primary ventral mesencephalic (VM) neural cells were encapsulated in PVA-SG hydrogels and critical molecular cues that demonstrate mechanosensory interaction were examined. Neuronal presence was constant over the 10 day culture, but the astrocyte population decreased in number. The lack of astrocytic support led to a reduction in neural process outgrowth from 24.0 ± 1.3 µm on Day 7 to 7.0 ± 0.1 µm on Day 10. Subsequently, purified astrocytes were studied in isolation to understand the reasons behind PVA-SG hydrogel inability to support neural network development. It was proposed that the spatially restrictive nature (or tight mesh size) of PVA-SG hydrogels limited the astrocytic actin polymerization together with a cytoplasmic-nuclear translocation of YAP over time, causing an alteration in their cell cycle. This was confirmed by the evaluation of p27 /Kip1 gene that was found to be upregulated by a twofold increase in expression at both Days 7 and 10 compared to Day 3, indicating the quiescent stage of the astrocytes in PVA-SG hydrogel. Cell migration was further studied by the quantification of MMP-2 production that was negligible compared to 2D controls, ranging from 2.7 ± 2.3% on Day 3 to 5.3 ± 2.9% on Day 10. This study demonstrates the importance of understanding astrocyte-material interactions at the molecular level, with the need to address spatial constraints in the 3D hydrogel environment. These findings will inform the design of future hydrogel constructs with greater capacity for remodeling by the cell population to create space for cell migration and neural process extension.

Molecular mechanisms for organizing the neuronal cytoskeleton

BioEssays, 2004

Neurofilaments and microtubules are important components of the neuronal cytoskeleton. In axons or dendrites, these filaments are aligned in parallel arrays, and separated from one another by nonrandom distances. This distinctive organization has been attributed to cross bridges formed by NF side arms or microtubuleassociated proteins. We recently proposed a polymerbrush-based mechanism for regulating interactions between neurofilaments and between microtubules. In this model, the side arms of neurofilaments and the projection domains of microtubule-associated proteins are highly unstructured and exert long-range repulsive forces that are largely entropic in origin; these forces then act to organize the cytoskeleton in axons and dendrites. Here, we review the biochemical, biophysical, genetic and cell biological data for the polymer-brush and crossbridging models. We explore how the data traditionally used to support cross bridging may be reconciled with a polymer-brush mechanism and compare the implications of recent experimental insights into axonal transport and physiology for each model.

Brushes, cables, and anchors: Recent insights into multiscale assembly and mechanics of cellular structural networks

Cell Biochemistry and Biophysics, 2007

The remarkable ability of living cells to mechanical stimuli in their environment depends on the rapid and efficient interconversion of mechanical and chemical energy at specific times and places within the cell. For example, application of force to cells leads to conformational changes in specific mechanosensitive molecules which then trigger cellular signaling cascades that may alter cellular structure, mechanics, and migration and profoundly influence gene expression. Similarly, the sensitivity of cells to mechanical stresses is governed by the composition, architecture, and mechanics of the cellular cytoskeleton and extracellular matrix (ECM), which are in turn driven by molecular-scale forces between the constituent biopolymers. Understanding how these mechanochemical systems coordinate over multiple length and time scales to produce orchestrated cell behaviors represents a fundamental challenge in cell biology. Here, we review recent advances in our understanding of these complex processes in three experimental systems: the assembly of axonal neurofilaments, generation of tensile forces by actomyosin stress fiber bundles, and mechanical control of adhesion assembly.

Elasticity and Rupture of a Multi-Domain Neural Cell Adhesion Molecule Complex

Biophysical Journal, 2009

The neural cell adhesion molecule (NCAM) plays an important role in nervous system development. NCAM forms a complex between its terminal domains Ig1 and Ig2. When NCAM of cell A and of cell B connect to each other through complexes Ig12(A)/Ig12(B), the relative mobility of cells A and B and membrane tension exerts a force on the Ig12(A)/Ig12(B) complex. In this study, we investigated the response of the complex to force, using steered molecular dynamics. Starting from the structure of the complex from the Ig1-Ig2-Ig3 fragment, we first demonstrated that the complex, which differs in dimensions from a previous structure from the Ig1-Ig2 fragment in the crystal environment, assumes the same extension when equilibrated in solvent. We then showed that, when the Ig12(A)/Ig12(B) complex is pulled apart with forces 30-70 pN, it exhibits elastic behavior (with a spring constant of~0.03 N/m) because of the relative reorientation of domains Ig1 and Ig2. At higher forces, the complex ruptures; i.e., Ig12(A) and Ig12(B) separate. The interfacial interactions between Ig12(A) and Ig12(B), monitored throughout elastic extension and rupture, identify E16, F19, K98, and L175 as key side chains stabilizing the complex.