The role of mechanics in axonal stability and development.

Much of the focus of neuronal cell biology has been devoted to growth cone guidance, synaptogenesis, synaptic activity, plasticity, etc. The axonal shaft too has received much attention, mainly for its astounding ability to transmit action potentials and the transport of material over long distances. For these functions, the axonal cytoskeleton and membrane have been often assumed to play static structural roles. Recent experiments have changed this view by revealing an ultrastructure much richer in features than previously perceived and one that seems to be maintained at a dynamic steady state. The role of mechanics in this is only beginning to be broadly appreciated and appears to involve passive and active modes of coupling different biopolymer filaments, filament turnover dynamics and membrane biophysics. Axons, being unique cellular processes in terms of high aspect ratios and often extreme lengths, also exhibit unique passive mechanical properties that might have evolved to stabilize them under mechanical stress. In this review, we summarize the experiments that have exposed some of these features. It is our view that axonal mechanics deserves much more attention not only due to its significance in the development and maintenance of the nervous system but also due to the susceptibility of axons to injury and neurodegeneration.

Mechanical role of the submembrane spectrin scaffold in red blood cells and neurons.

Spectrins are large, evolutionarily well-conserved proteins that form highly organized scaffolds on the inner surface of eukaryotic cells. Their organization in different cell types or cellular compartments helps cells withstand mechanical challenges with unique strategies depending on the cell type. This Review discusses our understanding of the mechanical properties of spectrins, their very distinct organization in red blood cells and neurons as two examples, and the contribution of the scaffolds they form to the mechanical properties of these cells.

Cell adhesion strength and tractions are mechano-diagnostic features of cellular invasiveness.

The adhesion of cells to substrates occurs via integrin clustering and binding to the actin cytoskeleton. Oncogenes modify anchorage-dependent mechanisms in cells during cancer progression. Fluid shear devices provide a label-free way to characterize cell-substrate interactions and heterogeneities in cell populations. We quantified the critical adhesion strengths of MCF-7, MDAMB-231, A549, HPL1D, HeLa, and NIH3T3 cells using a custom fluid shear device. The detachment response was sigmoidal for each cell type. A549 and MDAMB-231 cells had significantly lower critical adhesion strengths (τ50) than their non-invasive counterparts, HPL1D and MCF-7. Detachment dynamics inversely correlated with cell invasion potentials. A theoretical model, based on τ50 values and the distribution of cell areas on substrates, provided good fits to results from de-adhesion experiments. Quantification of cell tractions, using the Reg-FTTC method on 10 kPa polyacrylamide gels, showed highest values for invasive, MDAMB-231 and A549, cells compared to non-invasive cells. Immunofluorescence studies show differences in vinculin distributions; non-invasive cells have distinct vinculin puncta, whereas invasive cells have more dispersed distributions. The cytoskeleton in non-invasive cells was devoid of well-developed stress fibers, and had thicker cortical actin bundles in the boundary. Fluorescence intensity of actin was significantly lower in invasive cells as compared to non invasive cells. These correlations in adhesion strengths and traction stresses with cell invasiveness may be useful in cancer diagnostics and other pathologies featuring mis-regulation in adhesion.

Investigation of soft and living matter using a micro-extensional rheometer.

Rheological properties of a material often require to be probed under extensional deformation. Examples include fibrous materials such as spider-silk, high-molecular weight polymer melts, and the contractile response of living cells. Such materials have strong molecular-level anisotropies which are either inherent or are induced by an imposed extension. However, unlike shear rheology, which is well-established, techniques to perform extensional rheology are currently under development and setups are often custom-designed for the problem under study. In this article, we present a versatile device that can be used to conduct extensional deformation studies of samples at microscopic scales with simultaneous imaging. We discuss the operational features of this device and present a number of applications.

The axonal actin-spectrin lattice acts as a tension buffering shock absorber.

Axons span extreme distances and are subject to significant stretch deformations during limb movements or sudden head movements, especially during impacts. Yet, axon biomechanics, and its relation to the ultrastructure that allows axons to withstand mechanical stress, is poorly understood. Using a custom developed force apparatus, we demonstrate that chick dorsal root ganglion axons exhibit a tension buffering or strain-softening response, where its steady state elastic modulus decreases with increasing strain. We then explore the contributions from the various cytoskeletal components of the axon to show that the recently discovered membrane-associated actin-spectrin scaffold plays a prominent mechanical role. Finally, using a theoretical model, we argue that the actin-spectrin skeleton acts as an axonal tension buffer by reversibly unfolding repeat domains of the spectrin tetramers to release excess mechanical stress. Our results revise the current viewpoint that microtubules and their associated proteins are the only significant load-bearing elements in axons.

Strain softening and stiffening responses of spider silk fibers probed using a Micro-Extension Rheometer.

Spider silk possesses unique mechanical properties like large extensibility, high tensile strength, super-contractility, etc. Understanding these mechanical responses requires characterization of the rheological properties of silk beyond the simple force-extension relations which are widely reported. Here we study the linear and non-linear viscoelastic properties of dragline silk obtained from social spider Stegodyphus sarasinorum using a Micro-Extension Rheometer that we have developed. Unlike continuous extension data, our technique allows for the probing of the viscoelastic response by applying small perturbations about sequentially increasing steady-state strain values. In addition, we extend our analysis to obtain the characteristic stress relaxation times and the frequency responses of the viscous and elastic moduli. Using these methods, we show that in a small strain regime (0-4%) dragline silk of social spiders shows a strain softening response followed by a strain stiffening response at higher strains (>4%). The stress relaxation time, on the other hand, increases monotonically with increasing strain for the entire range. We also show that the silk stiffens while ageing within the typical lifetime of a web. Our results demand the inclusion of the kinetics of domain unfolding and refolding in the existing models to account for the relaxation time behavior.

Biophysics of Cell-Substrate Interactions Under Shear.

Cells adhere to substrates through mechanosensitive focal adhesion complexes. Measurements that probe how cells detach from substrates when they experience an applied force connect molecular-scale aspects of cell adhesion with the biophysical properties of adherent cells. Such forces can be applied through shear devices that flow fluid in a controlled manner across cells. The signaling pathways associated with focal adhesions, in particular those that involve integrins and receptor tyrosine kinases, are complex, receiving mechano-chemical feedback from the sensing of substrate stiffness as well as of external forces. This article reviews the signaling processes involved in mechanosensing and mechanotransduction during cell-substrate interactions, describing the role such signaling plays in cancer metastasis. We examine some recent progress in quantifying the strength of these interactions, describing a novel fluid shear device that allows for the visualization of the cell and its sub-cellular structures under a shear flow. We also summarize related results from a biophysical model for cellular de-adhesion induced by applied forces. Quantifying cell-substrate adhesions under shear should aid in the development of mechano-diagnostic techniques for diseases in which cell-adhesion is mis-regulated, such as cancers.

The Roles of Microtubules and Membrane Tension in Axonal Beading, Retraction, and Atrophy.

Axonal beading, or the formation of a series of swellings along the axon, and retraction are commonly observed shape transformations that precede axonal atrophy in Alzheimer's disease, Parkinson's disease, and other neurodegenerative conditions. The mechanisms driving these morphological transformations are poorly understood. Here, we report controlled experiments that can induce either beading or retraction and follow the time evolution of these responses. By making quantitative analysis of the shape modes under different conditions, measurement of membrane tension, and using theoretical considerations, we argue that membrane tension is the main driving force that pushes cytosol out of the axon when microtubules are degraded, causing axonal thinning. Under pharmacological perturbation, atrophy is always retrograde, and this is set by a gradient in the microtubule stability. The nature of microtubule depolymerization dictates the type of shape transformation, vis-à-vis beading or retraction. Elucidating the mechanisms of these shape transformations may facilitate development of strategies to prevent or arrest axonal atrophy due to neurodegenerative conditions.

Mechanochemical feedback control of dynamin independent endocytosis modulates membrane tension in adherent cells.

Plasma membrane tension regulates many key cellular processes. It is modulated by, and can modulate, membrane trafficking. However, the cellular pathway(s) involved in this interplay is poorly understood. Here we find that, among a number of endocytic processes operating simultaneously at the cell surface, a dynamin independent pathway, the CLIC/GEEC (CG) pathway, is rapidly and specifically upregulated upon a sudden reduction of tension. Moreover, inhibition (activation) of the CG pathway results in lower (higher) membrane tension. However, alteration in membrane tension does not directly modulate CG endocytosis. This requires vinculin, a mechano-transducer recruited to focal adhesion in adherent cells. Vinculin acts by controlling the levels of a key regulator of the CG pathway, GBF1, at the plasma membrane. Thus, the CG pathway directly regulates membrane tension and is in turn controlled via a mechano-chemical feedback inhibition, potentially leading to homeostatic regulation of membrane tension in adherent cells.

Cytoskeletal Mechanisms of Axonal Contractility.

Mechanotransduction is likely to be an important mechanism of signaling in thin, elongated cells such as neurons. Maintenance of prestress or rest tension may facilitate mechanotransduction in these cells. In recent years, functional roles for mechanical tension in neuronal development and physiology are beginning to emerge, but the cellular mechanisms regulating neurite tension remain poorly understood. Active contraction of neurites is a potential mechanism of tension regulation. In this study, we have explored cytoskeletal mechanisms mediating active contractility of neuronal axons. We have developed a simple assay in which we evaluate contraction of curved axons upon trypsin-mediated detachment. We show that curved axons undergo contraction and straighten upon deadhesion. Axonal straightening was found to be actively driven by actomyosin contractility, whereas microtubules may subserve a secondary role. We find that although axons show a monotonous decrease in length upon contraction, subcellularly, the cytoskeleton shows a heterogeneous contractile response. Further, using an assay for spontaneous development of tension without trypsin-induced deadhesion, we show that axons are intrinsically contractile. These experiments, using novel experimental approaches, implicate the axonal cytoskeleton in tension homeostasis. Our data suggest that although globally, the axon behaves as a mechanical continuum, locally, the cytoskeleton is remodeled heterogeneously.

Modeling cell-substrate de-adhesion dynamics under fluid shear.

Changes in cell-substrate adhesion are believed to signal the onset of cancer metastasis, but such changes must be quantified against background levels of intrinsic heterogeneity between cells. Variations in cell-substrate adhesion strengths can be probed through biophysical measurements of cell detachment from substrates upon the application of an external force. Here, we investigate, theoretically and experimentally, the detachment of cells adhered to substrates when these cells are subjected to fluid shear. We present a theoretical framework within which we calculate the fraction of detached cells as a function of shear stress for fast ramps as well as the decay in this fraction at fixed shear stress as a function of time. Using HEK and 3T3 fibroblast cells as experimental model systems, we extract characteristic force scales for cell adhesion as well as characteristic detachment times. We estimate force-scales of ∼500 pN associated to a single focal contact, and characteristic time-scales of [Formula: see text] s representing cell-spread-area dependent mean first passage times to the detached state at intermediate values of the shear stress. Variations in adhesion across cell types are especially prominent when cell detachment is probed by applying a time-varying shear stress. These methods can be applied to characterizing changes in cell adhesion in a variety of contexts, including metastasis.

Dynamics of membrane tethers reveal novel aspects of cytoskeleton-membrane interactions in axons.

Mechanical properties of cell membranes are known to be significantly influenced by the underlying cortical cytoskeleton. The technique of pulling membrane tethers from cells is one of the most effective ways of studying the membrane mechanics and the membrane-cortex interaction. In this article, we show that axon membranes make an interesting system to explore as they exhibit both free membrane-like behavior where the tether-membrane junction is movable on the surface of the axons (unlike many other cell membranes) as well as cell-like behavior where there are transient and spontaneous eruptions in the tether force that vanish when F-actin is depolymerized. We analyze the passive and spontaneous responses of axonal membrane tethers and propose theoretical models to explain the observed behavior.

Role of actin filaments in correlating nuclear shape and cell spreading.

It is well known that substrate properties like stiffness and adhesivity influence stem cell morphology and differentiation. Recent experiments show that cell morphology influences nuclear geometry and hence gene expression profile. The mechanism by which surface properties regulate cell and nuclear properties is only beginning to be understood. Direct transmission of forces as well as chemical signalling are involved in this process. Here, we investigate the formal aspect by studying the correlation between cell spreading and nuclear deformation using Mesenchymal stem cells under a wide variety of conditions. It is observed that a robust quantitative relation holds between the cell and nuclear projected areas, irrespective of how the cell area is modified or when various cytoskeletal or nuclear components are perturbed. By studying the role of actin stress fibers in compressing the nucleus we propose that nuclear compression by stress fibers can lead to enhanced cell spreading due to an interplay between elastic and adhesion factors. The significance of myosin-II in regulating this process is also explored. We demonstrate this effect using a simple technique to apply external compressive loads on the nucleus.

Optical fiber-based force transducer for microscale samples.

We discuss the design, instrumentation, and calibration of a versatile force transducer with feedback control, called the Micro-Extensional Rheometer (MER). A force range of eight decades (1-10(8) pN) and a displacement range of four decades (10-10(5) nm) with a spatial resolution of the order of nanometers are accessible with the instrument. A feedback-loop algorithm is used to control the commanded force or the extensional strain on the sample and implement different rheometric protocols such as step-strain, step-force, exponential strain, among others. The device may also be used to measure the forces exerted by active suspensions, pulling neurons, etc.

The role of the cytoskeleton in volume regulation and beading transitions in PC12 neurites.

We present investigations on volume regulation and beading shape transitions in PC12 neurites, conducted using a flow-chamber technique. By disrupting the cell cytoskeleton with specific drugs, we investigate the role of its individual components in the volume regulation response. We find that microtubule disruption increases both swelling rate and maximum volume attained, but does not affect the ability of the neurite to recover its initial volume. In addition, investigation of axonal beading-also known as pearling instability-provides additional clues on the mechanical state of the neurite. We conclude that volume recovery is driven by passive diffusion of osmolites, and propose that the initial swelling phase is mechanically slowed down by microtubules. Our experiments provide a framework to investigate the role of cytoskeletal mechanics in volume homeostasis.

Drag force as a tool to test the active mechanical response of PC12 neurites.

We investigate the mechanical response of PC12 neurites subjected to a drag force imposed by a laminar flow perpendicular to the neurite axis. The curvature of the catenary shape acquired by an initially straight neurite under the action of the drag force provides information on both elongation and tension of the neurite. This method allows us to measure the rest tension and viscoelastic parameters of PC12 neurites and active behavior of neurites. Measurement of oscillations in the strain rate of neurites at constant flow rate provides insight on the response of molecular motors and additional support for the presence of a negative strain-rate sensitivity region in the global mechanical response of PC12 neurites.

Coarsening through directed droplet coalescence in fluid-fluid phase separation.

Phase-separation dynamics of an asymmetric mixture of an isotropic dopant in a nematogenic fluid is presented. We show that, on steady cooling, the nucleating nematic drops move down the dopant concentration gradient, with a velocity that is dependent on the cooling rate and concentration gradient. This propulsion of the drops leads to a mechanism of droplet coarsening, where radius of a drop scales with time as R(t) approximately t. Various mechanisms for droplet propulsion are discussed.

Mechanogenetic coupling of Hydra symmetry breaking and driven Turing instability model.

The freshwater polyp Hydra can regenerate from tissue fragments or random cell aggregates. We show that the axis-defining step ("symmetry breaking") of regeneration requires mechanical inflation-collapse oscillations of the initial cell ball. We present experimental evidence that axis definition is retarded if these oscillations are slowed down mechanically. When biochemical signaling related to axis formation is perturbed, the oscillation phase is extended and axis formation is retarded as well. We suggest that mechanical oscillations play a triggering role in axis definition. We extend earlier reaction-diffusion models for Hydra regrowth by coupling morphogen transport to mechanical stress caused by the oscillations. The modified reaction-diffusion model reproduces well two important experimental observations: 1), the existence of an optimum size for regeneration, and 2), the dependence of the symmetry breaking time on the properties of the mechanical oscillations.

An osmoregulatory basis for shape oscillations in regenerating hydra.

The freshwater polyp Hydra has considerable regeneration capabilities. A small fragment of tissue excised from an adult animal is sufficient to regenerate an entire Hydra in the course of a few days. During the initial stages of the regeneration process, the tissue forms a hollow sphere. Then the sphere exhibits shape oscillations in the form of repeated cycles of swelling and collapse. We propose a biophysical model for the swelling mechanism. Our model takes the osmotic pressure difference between Hydra's inner and outer media and the elastic forces of the Hydra shell into account. We validate the model by a comprehensive experimental study including variations in initial medium concentrations, Hydra sphere sizes and temperatures. Numerical simulations of the model provide values for the swelling rates that are in agreement with the ones measured experimentally. Based on our results we argue that the shape oscillations are a consequence of Hydra's osmoregulation.

Mechanical properties of axons.

The mechanical response of PC12 neurites under tension is investigated using a microneedle technique. Elastic response, viscoelastic relaxation, and active contraction are observed. The mechanical model proposed by Dennerll et al. [J. Cell Biol. 109, 3073 (1989).10.1083/jcb.109.6.3073], which involves three mechanical devices--a stiff spring kappa coupled with a Voigt element that includes a less stiff spring k and a dashpot gamma--has been improved by adding a new element to describe the main features of the contraction of axons. This element, which represents the action of molecular motors, acts in parallel with viscous forces defining a global tension response of axons T against elongation rates delta(k). Under certain conditions, axons show a transition from a viscoelastic elongation to active contraction, suggesting the presence of a negative elongation rate sensitivity in the curve T vs delta(k).