Cell Mechanical Behavior Research Articles

Head/tail interaction of vinculin influences cell mechanical behavior

This study evaluates the influence of vinculin in closed conformation on the mechanical properties of cells. We demonstrate that MEFvin −/− cells transfected with the eGFP-vinculin mutant A50I (talin-binding-deficient-vinculin in a constitutively closed conformation) show 2-fold lower stiffness and focal adhesion density compared to MEFvin +/+ and MEF Rescue cells. MEF A50I cells are as stiff as MEFvin −/− cells with similar focal adhesion density. Further, 2D traction microscopy indicates that MEF A50I and MEFvin −/− cells generate 3- to 4-fold less strain energy than MEFvin +/+ and MEF Rescue cells. These results demonstrate that vinculin’s mechano-coupling function is dependent on its conformational state.

The Origin and Limits of Natural Variation in Cell Mechanical Behavior

Investigations of natural variation in cell mechanics within a population are essential for understanding the stochastic nature of cell deformation under applied load. Historically, a wide range of methods to measure cell stiffness have revealed a weak power law that governs the relationship between complex elastic modulus and excitation frequency; similarly, measurements of creep compliance find a corresponding power-law dependence on load application time. In both experimental regimes, researchers have reported a Gaussian distribution of power-law exponent along with a log-normal distribution of cell stiffness/compliance. However, the underlying analytical relationship between these two distributions has not yet been fully explored. Do these mechanical distributions stem from inherent variations within a cell population, from stochastic mechanisms of single-cell deformation, or both? Here, we develop this relationship to generate new predictions regarding the evolution of mechanical parameters during testing in the frequency and time domains. These predictions are in agreement with literature reports that were originally presented as empirical findings lacking theoretical explanation, as well as with our own studies of creep compliance in stem cell populations. Our work thus serves to link two fundamental findings in the cell rheology literature, produce nontrivial predictions supported by experiment, and motivate further study into constitutive laws describing the mechanical variation of living matter.

Tensile Mechanical Characterization of Cell Stiffness Improves Correlation to Metastatic Potential in Models of Osteosarcoma

A correlative link between decreased cancer cell stiffness and increased aggressiveness (metatstatic potential) has been established in a wide range of cancers, such as leukemia, breast cancer and chondrosarcoma. Nearly all studies establishing this link in adherent cells have utilized cell compression (indentation) or shearing (e.g. magnetic cyto-twisting). In this study we applied a novel tensile assay of cell stiffness to investigate the mechanical behavior of two osteosarcoma models (SAOS-2/LM5 and HUO9/M132), with each system comprising a parental cell line and its highly metastatic derivative form. The goal was to find a common trend linking cell stiffness and metastatic potential, further hypothesizing that a tensile assay could provide additional information compared to traditional compressive measures. Cells were therefore tested in two different mechanical configurations: in compression using atomic force micro-indentation; and in tension using a custom functional imaging platform that applies a controlled bi-axial deformation to the cell substrate while imaging and tracking substrate deformation at high spatial resolution. These measurements were coupled with an inverse finite element model to more precisely estimate cell tensile constitutive behavior.Both parental lines proved to be significantly (p<0.01) stiffer than their metastatic variants in tension: 2.5±1.6 kPa (SAOS-2) vs. 1.4±1.0 kPa (LM5) and 5.3±2.5 kPa (HUO9) vs. 2.5±0.7 kPa (M132). Although a compressive characterization of the SAOS-2/LM5 model confirmed this trend (3.5±1.7 kPa vs. 1.8±0.8 kPa, p<0.001), the compressive (cytosolic) stiffness of the HUO9/M132 was unexpectedly higher in the highly metastatic cells (2.0±0.7 kPa vs. 4.9±2.1 kPa, p<0.001). These results indicate that the relationship between “cell stiffness” and phenotypic aggressiveness is not straight forward, and that tensional mechanical characterization provides additional, and perhaps more pertinent, information linking cell mechanical behavior to metastatic potential.

Cell Motility and Mechanics in Three-Dimensional Collagen Matrices

Fibrous connective tissues provide mechanical support and frameworks for other tissues of the body and play an integral role in normal tissue physiology and pathology. Three-dimensional collagen matrices exhibit mechanical and structural features that resemble fibrous connective tissue and have become an important model system to study cell behavior in a tissue-like environment. This review focuses on motile and mechanical interactions between cells&#x2014;especially fibroblasts&#x2014;and collagen matrices. We describe several matrix contraction models, the interactions between fibroblasts and collagen fibrils at global and subcellular levels, unique features of mechanical feedback between cells and the matrix, and the impact of the cell-matrix tension state on cell morphology and mechanical behavior. We develop a conceptual framework to explain the balance between cell migration and collagen translocation including the concept of promigratory and procontractile growth factor environments. Finally, we review the significance of these concepts for the physiology of wound repair.

Modeling and Characterization of the Relationship between Cell Size and Mechanical Behavior of Microcellular PP/Mica Composites

Abstract The microcellular foamed polypropylene (PP)/mica composites were prepared through chemical microcellular injection to investigate the relationship between the mechanical behavior and the cell size. The mechanical behavior model of the microcellular foams is built based on the elastic stress/strain field. The mechanical behavior of the microcellular composites coincides well with the theoretical model. Under loading the large and non uniform cells are in a state of plane strain, which leads to low tensile and impact strengths, whereas small and uniform cells are in a state of plane stress, which contributes to the transition from brittle to tough fracture behavior in the microcellular PP/mica composite.

Tyrosine phosphorylation of vinculin at position 1065 modifies focal adhesion dynamics and cell tractions

Focal adhesions (FAs) connect the cellular actin cytoskeleton via integrin with the extracellular matrix. They comprise of many structural and signaling proteins which are highly dynamic, well regulated, and responsible for the sensing of physical properties from the environment. Vinculin is a protein that incorporates all these functions. Here, we investigated the phosphorylation of Y1065 in the activation/regulation of vinculin. We used different vinculin mutants mimicking either a permanently activated or inhibited phosphorylation site at position 1065. Using these mutants, we determined their influence on the exchange dynamics and cell forces using fluorescence recovery after photobleaching and traction microscopy. The results indicate that phosphorylation at Y1065 significantly increases the amount of freely exchanging vinculin within FAs whereas inhibition of this phosphorylation site leads to an uncontrolled exchange of vinculin and reduced adhesive cell forces. In conclusion, we show that phosphorylation on position Y1065 is essential for accurate incorporation of vinculin into FAs and mechanical behavior of cells.

Correlation between the interaction of the vinculin tail domain with lipid membranes, its phosphorylation and cell mechanical behaviour

Cell adhesion and cell-cell contacts are a pre-requisite for proper metabolism, protein synthesis and cell survival. Integrins are the transmembrane receptors that link the extracellular matrix via the FAC (focal adhesion complex) with the cytoskeleton. Vinculin is a pivotal FAC protein that has not only been implicated in regulating FAC formation and transmitting mechanical forces, but also in associating with membranous lipids in biological systems.

From Cellular Mechanotransduction to Biologically Inspired Engineering

This article is based on a lecture I presented as the recipient of the 2009 Pritzker Distinguished Lecturer Award at the Biomedical Engineering Society annual meeting in October 2009. Here, I review more than thirty years of research from my laboratory, beginning with studies designed to test the theory that cells use tensegrity (tensional integrity) architecture to stabilize their shape and sense mechanical signals, which I believed to be critical for control of cell function and tissue development. Although I was trained as a cell biologist, I found that the tools I had at my disposal were insufficient to experimentally test these theories, and thus I ventured into engineering to find critical solutions. This path has been extremely fruitful as it has led to confirmation of the critical role that physical forces play in developmental control, as well as how cells sense and respond to mechanical signals at the molecular level through a process known as cellular mechanotransduction. Many of the predictions of the cellular tensegrity model relating to cell mechanical behaviors have been shown to be valid, and this vision of cell structure led to discovery of the central role that transmembrane adhesion receptors, such as integrins, and the cytoskeleton play in mechanosensing and mechanochemical conversion. In addition, these fundamental studies have led to significant unexpected technology fallout, including development of micromagnetic actuators for non-invasive control of cellular signaling, microfluidic systems as therapeutic extracorporeal devices for sepsis therapy, and new DNA-based nanobiotechnology approaches that permit construction of artificial tensegrities that mimic properties of living materials for applications in tissue engineering and regenerative medicine.

Intermediate filament-deficient cells are mechanically softer at large deformation: A multi-scale simulation study

The cell’s cytoskeleton, providing cells with structure and shape, consists of different structural proteins, including microtubules, actin microfilaments and intermediate filaments. It has been suggested that intermediate filaments play a crucial role in providing mechanical stability to cells. By utilizing a simple coarse-grained computational model of the intermediate filament network in eukaryotic cells, we show here that intermediate filaments play a significant role in the cell mechanical behavior at large deformation, and reveal mechanistic insight into cell deformation under varying intermediate filament densities. We find that intermediate filament-deficient cells display an altered mechanical behavior, featuring a softer mechanical response at large deformation while the mechanical properties remain largely unchanged under small deformation. We compare the results with experimental studies in vimentin-deficient cells, showing good qualitative agreement. Our results suggest that intermediate filaments contribute to cell stiffness and deformation at large deformation, and thus play a significant role in maintaining cell structural integrity in response to applied stress and strain, in agreement with earlier hypotheses. The simulation results also suggest that changes in the filament density result in profound alterations of the deformation state of the cell nucleus, leading to greater stretch in the direction of loading and greater contraction in the orthogonal direction as the intermediate filament density is increased. Our model opens the door to future studies to investigate disease states, the effects of amino acid mutations and how structural changes at different levels in the cell’s structural makeup influence biomechanical properties.

Anchorage of Vinculin to Lipid Membranes Influences Cell Mechanical Properties

The focal adhesion protein vinculin (1066 residues) can be separated into a 95-kDa head and a 30-kDa tail domain. Vinculin's lipid binding sites localized on the tail, helix 3 (residues 944–978) and the unstructured C-terminal arm (residues 1052–1066, the so-called lipid anchor), influence focal adhesion turnover and are important for cell migration and adhesion. Using magnetic tweezers, we characterized the cell mechanical behavior in mouse embryonic fibroblast (MEF)-vin(−/−) cells transfected with EGFP-linked-vinculin deficient of the lipid anchor (vinΔC, residues 1–1051). MEF-vinΔC cells incubated with fibronectin-coated paramagnetic beads were less stiff, and more beads detached during these experiments compared to MEF-rescue cells. Cells expressing vinΔC formed fewer focal contacts as determined by confocal microscopy. Two-dimensional traction measurements showed that MEF-vinΔC cells generate less force compared to rescue cells. Attenuated traction forces were also found in cells that expressed vinculin with point mutations (R1060 and K1061 to Q) of the lipid anchor that impaired lipid binding. However, traction generation was not diminished in cells that expressed vinculin with impaired lipid binding caused by point mutations on helix 3. Mutating the src-phosphorylation site (Y1065 to F) resulted in reduced traction generation. These observations show that both the lipid binding and the src-phosphorylation of vinculin's C-terminus are important for cell mechanical behavior.

Mechanical testing of single yeast cells in liquid environment: Effect of the extracellular osmotic conditions on the failure behavior

Abstract In industrial biotechnology microbial cells are utilized to produce diverse products (e. g., enzymes, food and animal feed additives, pharmaceutical agents). Whilst relationships on genetic and molecular levels are reasonably understood there are still numerous unsolved questions in matters of the mechanical properties of those cells. Recently, a compression test in liquid environment using a nanoindentation device was established to address the cell mechanical behavior of Saccharomyces cerevisiae (baker's yeast) cells. At higher deformations compressive stressing yielded failure of the cell wall. In the present work, the influence of the extracellular osmotic conditions on the failure characteristics (bursting force, bursting energy, and relative deformation at bursting) was studied. Moreover, a simple mechanical model was applied to characterize the mechanical properties of the cell wall.

Compressive testing of single yeast cells in liquid environment using a nanoindentation system

Due to their versatility and accuracy, nanoindentation systems are increasingly used for the characterization of micron-sized particles. Single microbial cells (e.g., yeast cells) can be regarded as micron-sized, liquid-filled biological particles. Applying a nanoindentation system for the compressive testing of those cells offers many options, such as testing in liquid environment. However, diverse experimental problems have to be resolved, especially the visualization of the cells in liquid and the alignment of the surfaces between which the cell is compressed. Single yeast cells were tested using a nanoindenter equipped with a flat punch tip. The deformation behavior of the cells during loading as well as the shape recovery behavior during unloading was investigated. A bursting force was determined as the cell wall was failing at higher deformations. Moreover, the influence of the compression speed on the cell mechanical behavior was characterized.

Disparate effects of different mutations in plakoglobin on cell mechanical behavior

Mutations in genes encoding desmosomal proteins have been implicated in the pathogenesis of heart and skin diseases. This has led to the hypothesis that defective cell-cell adhesion is the underlying cause of injury in tissues that repeatedly bear high mechanical loads. In this study, we examined the effects of two different mutations in plakoglobin on cell migration, stiffness, and adhesion. One is a C-terminal mutation causing Naxos disease, a recessive syndrome of arrhythmogenic right ventricular cardiomyopathy (ARVC) and abnormal skin and hair. The other is an N-terminal mutation causing dominant inheritance of ARVC without cutaneous abnormalities. To assess the effects of plakoglobin mutations on a broad range of cell mechanical behavior, we characterized a model system consisting of stably transfected HEK cells which are particularly well suited for analyses of cell migration and adhesion. Both mutations increased the speed of wound healing which appeared to be related to increased cell motility rather than increased cell proliferation. However, the C-terminal mutation led to dramatically decreased cell-cell adhesion, whereas the N-terminal mutation caused a decrease in cell stiffness. These results indicate that different mutations in plakoglobin have markedly disparate effects on cell mechanical behavior, suggesting complex biomechanical roles for this protein.

The nonlinear mechanical response of the red blood cell

We measure the dynamical mechanical properties of human red blood cells. A single cell response is measured with optical tweezers. We investigate both the stress relaxation following a fast deformation and the effect of varying the strain rate. We find a power-law decay of the stress as a function of time, down to a plateau stress, and a power-law increase of the cell's elasticity as a function of the strain rate. Interestingly, the exponents of these quantities violate the linear superposition principle, indicating a nonlinear response. We propose that this is due to the breaking of a fraction of the crosslinks during the deformation process. The soft glassy rheology model accounts for the relation between the exponents we observe experimentally. This picture is consistent with recent models of bond remodeling in the red blood cell's molecular structure. Our results imply that the blood cell's mechanical behavior depends critically on the deformation process.

PH and expansin action on single suspension-cultured tomato (Lycopersicon esculentum) cells

The aim of this study was to measure key material properties of the cell walls of single suspension-cultured plant cells and relate these to cell-wall biochemistry. To this end, micromanipulation was used to compress single tomato cells between two flat surfaces until they ruptured, and force-deformation data were obtained. In addition to measuring the bursting force, we also determined the elastic (Young's) modulus of the cell walls by matching low strain (< or = 20% deformation) experimental data with a cell compression model, assuming linear elastic cell walls. The walls were most elastic at pH 4.5, the pH optimum for expansin activity, with an elastic modulus of 2.0 +/- 0.1 GPa. Following the addition of exogenous expansins, cell walls became more elastic at all pH values. Western blot analysis of proteins from walls of cultured cells revealed the presence of expansin epitopes, suggesting that the inherent pH dependence of elasticity and other compression phenomena is related to the presence of endogenous expansin proteins and their wall-loosening ability. Although strict application of the linear-elastic model could not be applied to large deformations-for example, up to cell bursting-because of irreversible behaviour, the deviation of the data from the model was generally small enough to allow estimation of the strain in the cell wall at failure. This strain was greater at pH 4.5 and when expansins were added to the suspension. The changes in elasticity are consistent with suggestions about the mode of expansin action. The estimated strains at failure are compatible with data on the failure of Acetobacter-derived cellulose-xyloglucan composites and proposed mechanisms of such failure. Through the measurement of cell-wall material properties using micromanipulation, it may be possible to understand more fully how cell-wall composition, structure and biochemistry lead to cell mechanical behaviour.

Atomistic-based continuum constitutive relation for microtubules: elastic modulus prediction

Mechanical behavior of cells is primarily governed by the cytoskeleton (CSK), a remarkable system of filaments consisting of microtubules, actin filaments and intermediate filaments. This system defines the shape and bulk mechanical properties of the cell. In order to understand how the CSK network influences the mechanical behavior of living cells from a theoretical perspective, the mechanical properties of an individual CSK filament must first be properly described. Existing atomistic simulation methods have computational size limitations; conversely, conventional continuum mechanics lack fundamental nanoscale information. Here a new simulation method is developed that bridges the gap between these two simulation regimes using an atomistic-based continuum constitutive relation for microtubules based on the interatomic potential for proteins and specific atomic structures. This theory is used to predict the elastic modulus of microtubules, which agrees with the range of experimentally measured values without any parameter fitting. The proposed method is applicable to other biopolymers if the subunits are bonded through noncovalent bonds.

Microfabricated Force Sensors and Their Applications in the Study of Cell Mechanical Response

Living cells are sensitive to their mechanical environments and they transduce mechanical stimuli into biological responses. Developing suitable experimental techniques is essential to explore the question on how cells respond to mechanical stimuli. The current major techniques normally induce small cell deformations and measure their corresponding cell force response (small) in the range of 1 pN to 10nN. However, in many physiological conditions, cell deformations can be large (comparable to the cell sizes) inducing large force response. In order to explore cell mechanical behavior under large deformations, we introduce a class of microfabricated force sensors. The sensors, consisting of a probe and flexible beams, normally measure cell force response in the range of 1nN to 1μN. Both the one- and two-component force sensors have been developed, and have been used in cell experiments. These experiments showed the versatility of the force sensors. Representative experimental results on cell stretch force response, cell indentation force response, and in situ observation of the actin cytoskeleton during indentation, will be given. These results provide significant insight on cell mechanical response under large deformations.

Rheological Behavior of Living Cells Is Timescale-Dependent

The dynamic mechanical behavior of living cells has been proposed to result from timescale-invariant processes governed by the soft glass rheology theory derived from soft matter physics. But this theory is based on experimental measurements over timescales that are shorter than those most relevant for cell growth and function. Here we report results measured over a wider range of timescales which demonstrate that rheological behaviors of living cells are not timescale-invariant. These findings demonstrate that although soft glass rheology appears to accurately predict certain cell mechanical behaviors, it is not a unified model of cell rheology under biologically relevant conditions and thus, alternative mechanisms need to be considered.

Characterizing mechanical behavior of cells and soft tissues by indentation: Potential and limitations

Characterizing mechanical behavior of cells and soft tissues by indentation: Potential and limitations

An Integrative Model of the Self-Sustained Oscillating Contractions of Cardiac Myocytes

Computational cell models appear as necessary tools for handling the complexity of intracellular cell dynamics, especially calcium dynamics. However, while oscillating intracellular calcium oscillations are well documented and modelled, a simple enough virtual cell taking into account the mechano-chemical coupling between calcium oscillations and cell mechanical properties is still lacking. Considering the spontaneous periodic contraction of isolated cardiac myocytes, we propose here a virtual cardiac cell model in which the cellular contraction is modelled using an hyperelastic description of the cell mechanical behaviour. According to the experimental data, the oscillating cytosolic calcium concentrations trigger the spatio-temporal variation of the anisotropic intracellular stresses. The finite element simulations of the virtual cell deformations are compared to the self-sustained contractions of isolated rat cardiomyocytes recorded by time-lapse video-microscopy.