Alteration of viscoelastic properties is associated with a change in cytoskeleton components of ageing chondrocytes from rabbit knee articular cartilage

The cytoskeleton network is believed to play an important role in the biomechanical properties of the chondrocyte. Ours and other laboratories have demonstrated that chondrocytes exhibit a viscoelastic solid creep behavior in vitro and that viscoelastic properties decrease in osteoarthritic chondrocytes. In this study, we aimed to understand whether the alteration of viscoelastic properties is associated with changes in cytoskeleton components of ageing chondrocytes from rabbit knee articular cartilage. Three age groups were used for this study: young (2-months-old, N=23), adult (8-months-old, N=23), and old (31-months-old, N=23) rabbit groups. Cartilage structure and proteoglycan and type II collagen content were determined by H&E and Toluidine Blue staining, and type II collagen antibody. The detailed structure of the chondrocytes in all groups was visualized using transmission electron microscopy (TEM). Chondrocytes were isolated from full-thickness knee cartilage of rabbits from all groups and their viscoelastic properties were quantified within 2 hours of isolation using a micropipette aspiration technique combined with a standard linear viscoelastic solid model. The components and network of the cytoskeleton within the cells were analyzed by laser scanning confocal microscopy (LSCM) with immunofluorescence staining as well as real time PCR and western blotting. With ageing, articular cartilage contained less chondrocytes and less proteoglycans and type II collagen. TEM observations showed that the cell membranes were not clearly defined, organelles were fewer and the nuclei were deformed or shrunk in the old cells compared with the young and adult cells. In suspension, chondrocytes from all three age groups showed significant viscoelastic creep behavior, but the deformation rate and amplitude of old chondrocytes were increased under the same negative pressure when compared to young and adult chondrocytes. Viscoelastic properties of the old cells, including equilibrium modulus (E infinity), instantaneous modulus (E0) and apparent viscosity (mu) were significantly lower than that those of the young and adult ones (P < 0.001). No significant differences were detected between young and adult chondrocytes (P > 0.05). Moreover, we found that the cytoskeletal networks of old cells were sparser, and that the contents of the various components of the intracellular networks were reduced in old cells, compared with adult and young cells. Aged chondrocytes had a different response to mechanical stimulation when compared to young and adult chondrocytes due to alteration of their viscoelastic properties, which was in turn associated with changes in cell structure and cytoskeleton composition.     

Mitochondrial dynamics in chondrocytes and their connection to the mechanical properties of the cytoplasm

BACKGROUND: The motion and redistribution of intracellular organelles is a fundamental process in cells. Organelle motion is a complex phenomenon that depends on a large number of variables including the shape of the organelle, the type of motors with which the organelles are associated, and the mechanical properties of the cytoplasm. This paper presents a study that characterizes the diffusive motion of mitochondria in chondrocytes seeded in agarose constructs and what this implies about the mechanical properties of the cytoplasm. METHOD OF APPROACH: Images showing mitochondrial motion in individual cells at 30 s intervals for 15 min were captured with a confocal microscope. Digital image correlation was used to quantify the motion of the mitochondria, and the mean square displacement (MSD) was calculated. Statistical tools for testing whether the characteristic motion of mitochondria varied throughout the cell were developed. Calculations based on statistical mechanics were used to establish connections between the measured MSDs and the mechanical nature of the cytoplasm. RESULTS: The average MSD of the mitochondria varied with time according to a power law with the power term greater than 1, indicating that mitochondrial motion can be viewed as a combination of diffusion and directional motion. Statistical analysis revealed that the motion of the mitochondria was not uniform throughout the cell, and that the diffusion coefficient may vary by over 50%, indicating intracellular heterogeneity. High correlations were found between movements of mitochondria when they were less than 2 microm apart. The correlation is probably due to viscoelastic properties of the cytoplasm. Theoretical analysis based on statistical mechanics suggests that directed diffusion can only occur in a material that behaves like a fluid on large time scales. CONCLUSIONS: The study shows that mitochondria in different regions of the cell experience different characteristic motions. This suggests that the cytoplasm is a heterogeneous viscoelastic material. The study provides new insight into the motion of mitochondria in chondrocytes and its connection with the mechanical properties of the cytoplasm.     

Micromechanical mapping of live cells by multiple-particle-tracking microrheology

This paper introduces the method of live-cell multiple-particle-tracking microrheology (MPTM), which quantifies the local mechanical properties of living cells by monitoring the Brownian motion of individual microinjected fluorescent particles. Particle tracking of carboxylated microspheres imbedded in the cytoplasm produce spatial distributions of cytoplasmic compliances and frequency-dependent viscoelastic moduli. Swiss 3T3 fibroblasts are found to behave like a stiff elastic material when subjected to high rates of deformations and like a soft liquid at low rates of deformations. By analyzing the relative contributions of the subcellular compliances to the mean compliance, we find that the cytoplasm is much more mechanically heterogeneous than reconstituted actin filament networks. Carboxylated microspheres embedded in cytoplasm through endocytosis and amine-modified polystyrene microspheres, which are microinjected or endocytosed, often show directed motion and strong nonspecific interactions with cytoplasmic proteins, which prevents computation of local moduli from the microsphere displacements. Using MPTM, we investigate the mechanical function of alpha-actinin in non-muscle cells: alpha-actinin-microinjected cells are stiffer and yet mechanically more heterogeneous than control cells, in agreement with models of reconstituted cross-linked actin filament networks. MPTM is a new type of functional microscopy that can test the local, rate-dependent mechanical and ultrastructural properties of living cells.     

Extracellular Matrix Stiffness and Architecture Govern Intracellular Rheology in Cancer

Little is known about the complex interplay between the extracellular mechanical environment and the mechanical properties that characterize the dynamic intracellular environment. To elucidate this relationship in cancer, we probe the intracellular environment using particle-tracking microrheology. In three-dimensional (3D) matrices, intracellular effective creep compliance of prostate cancer cells is shown to increase with increasing extracellular matrix (ECM) stiffness, whereas modulating ECM stiffness does not significantly affect the intracellular mechanical state when cells are attached to two-dimensional (2D) matrices. Switching from 2D to 3D matrices induces an order-of-magnitude shift in intracellular effective creep compliance and apparent elastic modulus. However, for a given matrix stiffness, partial blocking of β1 integrins mitigates the shift in intracellular mechanical state that is invoked by switching from a 2D to 3D matrix architecture. This finding suggests that the increased cell-matrix engagement inherent to a 3D matrix architecture may contribute to differences observed in viscoelastic properties between cells attached to 2D matrices and cells embedded within 3D matrices. In total, our observations show that ECM stiffness and architecture can strongly influence the intracellular mechanical state of cancer cells.     

Diffusing wave spectroscopy microrheology of actin filament networks.

Filamentous actin (F-actin), one of the constituents of the cytoskeleton, is believed to be the most important participant in the motion and mechanical integrity of eukaryotic cells. Traditionally, the viscoelastic moduli of F-actin networks have been measured by imposing a small mechanical strain and quantifying the resulting stress. The magnitude of the viscoelastic moduli, their concentration dependence and strain dependence, as well as the viscoelastic nature (solid-like or liquid-like) of networks of uncross-linked F-actin, have been the subjects of debate. Although this paper helps to resolve the debate and establishes the extent of the linear regime of F-actin networks' rheology, we report novel measurements of the high-frequency behavior of networks of F-actin, using a noninvasive light-scattering based technique, diffusing wave spectroscopy (DWS). Because no external strain is applied, our optical assay generates measurements of the mechanical properties of F-actin networks that avoid many ambiguities inherent in mechanical measurements. We observe that the elastic modulus has a small magnitude, no strain dependence, and a weak concentration dependence. Therefore, F-actin alone is not sufficient to generate the elastic modulus necessary to sustain the structural rigidity of most cells or support new cellular protrusions. Unlike previous studies, our measurements show that the mechanical properties of F-actin are highly dependent on the frequency content of the deformation. We show that the loss modulus unexpectedly dominates the elastic modulus at high frequencies, which are key for fast transitions. Finally, the measured mean square displacement of the optical probes, which is also generated by DWS measurements, offers new insight into the local bending fluctuations of the individual actin filaments and shows how they generate enhanced dissipation at short time scales.     

Spatial distribution of filament elasticity determines the migratory behaviors of a cell

Any cellular response leading to morphological changes is highly tuned to balance the force generated from structural reorganization, provided by actin cytoskeleton. Actin filaments serve as the backbone of intracellular force, and transduce external mechanical signal via focal adhesion complex into the cell. During migration, cells not only undergo molecular changes but also rapid mechanical modulation. Here we focus on determining, the role of spatial distribution of mechanical changes of actin filaments in epithelial, mesenchymal, fibrotic and cancer cells with non-migration, directional migration, and non-directional migration behaviors using the atomic force microscopy. We found 1) non-migratory cells only generated one type of filament elasticity, 2) cells generating spatially distributed two types of filament elasticity showed directional migration, and 3) pathologic cells that autonomously generated two types of filament elasticity without spatial distribution were actively migrating non-directionally. The demonstration of spatial regulation of filament elasticity of different cell types at the nano-scale highlights the coupling of cytoskeletal function with physical characters at the sub-cellular level, and provides new research directions for migration related disease.     

Dynamics of mechanical signal transmission through prestressed stress fibers

Transmission of mechanical stimuli through the actin cytoskeleton has been proposed as a mechanism for rapid long-distance mechanotransduction in cells; however, a quantitative understanding of the dynamics of this transmission and the physical factors governing it remains lacking. Two key features of the actin cytoskeleton are its viscoelastic nature and the presence of prestress due to actomyosin motor activity. We develop a model of mechanical signal transmission through prestressed viscoelastic actin stress fibers that directly connect the cell surface to the nucleus. The analysis considers both temporally stationary and oscillatory mechanical signals and accounts for cytosolic drag on the stress fibers. To elucidate the physical parameters that govern mechanical signal transmission, we initially focus on the highly simplified case of a single stress fiber. The results demonstrate that the dynamics of mechanical signal transmission depend on whether the applied force leads to transverse or axial motion of the stress fiber. For transverse motion, mechanical signal transmission is dominated by prestress while fiber elasticity has a negligible effect. Conversely, signal transmission for axial motion is mediated uniquely by elasticity due to the absence of a prestress restoring force. Mechanical signal transmission is significantly delayed by stress fiber material viscosity, while cytosolic damping becomes important only for longer stress fibers. Only transverse motion yields the rapid and long-distance mechanical signal transmission dynamics observed experimentally. For simple networks of stress fibers, mechanical signals are transmitted rapidly to the nucleus when the fibers are oriented largely orthogonal to the applied force, whereas the presence of fibers parallel to the applied force slows down mechanical signal transmission significantly. The present results suggest that cytoskeletal prestress mediates rapid mechanical signal transmission and allows temporally oscillatory signals in the physiological frequency range to travel a long distance without significant decay due to material viscosity and/or cytosolic drag.     

A master relation defines the nonlinear viscoelasticity of single fibroblasts

Cell mechanical functions such as locomotion, contraction, and division are controlled by the cytoskeleton, a dynamic biopolymer network whose mechanical properties remain poorly understood. We perform single-cell uniaxial stretching experiments on 3T3 fibroblasts. By superimposing small amplitude oscillations on a mechanically prestressed cell, we find a transition from linear viscoelastic behavior to power law stress stiffening. Data from different cells over several stress decades can be uniquely scaled to obtain a master relation between the viscoelastic moduli and the average force. Remarkably, this relation holds independently of deformation history, adhesion biochemistry, and intensity of active contraction. In particular, it is irrelevant whether force is actively generated by the cell or externally imposed by stretching. We propose that the master relation reflects the mechanical behavior of the force-bearing actin cytoskeleton, in agreement with stress stiffening known from semiflexible filament networks.     

Effect of Age and Cytoskeletal Elements on the Indentation-Dependent Mechanical Properties of Chondrocytes

Articular cartilage chondrocytes are responsible for the synthesis, maintenance, and turnover of the extracellular matrix, metabolic processes that contribute to the mechanical properties of these cells. Here, we systematically evaluated the effect of age and cytoskeletal disruptors on the mechanical properties of chondrocytes as a function of deformation. We quantified the indentation-dependent mechanical properties of chondrocytes isolated from neonatal (1-day), adult (5-year) and geriatric (12-year) bovine knees using atomic force microscopy (AFM). We also measured the contribution of the actin and intermediate filaments to the indentation-dependent mechanical properties of chondrocytes. By integrating AFM with confocal fluorescent microscopy, we monitored cytoskeletal and biomechanical deformation in transgenic cells (GFP-vimentin and mCherry-actin) under compression. We found that the elastic modulus of chondrocytes in all age groups decreased with increased indentation (15–2000 nm). The elastic modulus of adult chondrocytes was significantly greater than neonatal cells at indentations greater than 500 nm. Viscoelastic moduli (instantaneous and equilibrium) were comparable in all age groups examined; however, the intrinsic viscosity was lower in geriatric chondrocytes than neonatal. Disrupting the actin or the intermediate filament structures altered the mechanical properties of chondrocytes by decreasing the elastic modulus and viscoelastic properties, resulting in a dramatic loss of indentation-dependent response with treatment. Actin and vimentin cytoskeletal structures were monitored using confocal fluorescent microscopy in transgenic cells treated with disruptors, and both treatments had a profound disruptive effect on the actin filaments. Here we show that disrupting the structure of intermediate filaments indirectly altered the configuration of the actin cytoskeleton. These findings underscore the importance of the cytoskeletal elements in the overall mechanical response of chondrocytes, indicating that intermediate filament integrity is key to the non-linear elastic properties of chondrocytes. This study improves our understanding of the mechanical properties of articular cartilage at the single cell level.     

A model for cytoplasmic rheology consistent with magnetic twisting cytometry.

Magnetic twisting cytometry is gaining wide applicability as a tool for the investigation of the rheological properties of cells and the mechanical properties of receptor-cytoskeletal interactions. Current technology involves the application and release of magnetically induced torques on small magnetic particles bound to or inside cells, with measurements of the resulting angular rotation of the particles. The properties of purely elastic or purely viscous materials can be determined by the angular strain and strain rate, respectively. However, the cytoskeleton and its linkage to cell surface receptors display elastic, viscous, and even plastic deformation, and the simultaneous characterization of these properties using only elastic or viscous models is internally inconsistent. Data interpretation is complicated by the fact that in current technology, the applied torques are not constant in time, but decrease as the particles rotate. This paper describes an internally consistent model consisting of a parallel viscoelastic element in series with a parallel viscoelastic element, and one approach to quantitative parameter evaluation. The unified model reproduces all essential features seen in data obtained from a wide variety of cell populations, and contains the pure elastic, viscoelastic, and viscous cases as subsets.     

Real-time monitoring of cell elasticity reveals oscillating myosin activity

The cytoskeleton is the physical and biochemical interface for a large variety of cellular processes. Its complex regulation machinery is involved upstream and downstream in various signaling pathways. The cytoskeleton determines the mechanical properties of a cell. Thus, cell elasticity could serve as a parameter reflecting the behavior of the system rather than reflecting the specific properties of isolated components. In this study, we used atomic force microscopy to perform real-time monitoring of cell elasticity unveiling cytoskeletal dynamics of living bronchial epithelial cells. In resting cells, we found a periodic activity of the cytoskeleton. Amplitude and frequency of this spontaneous oscillation were strongly affected by intracellular calcium. Experiments reveal that basal cell elasticity and superimposed elasticity oscillations are caused by the collective action of myosin motor proteins. We characterized the cell as a mechanically multilayered structure, and followed cytoskeletal dynamics in the different layers with high time resolution. In conclusion, the collective activities of the myosin motor proteins define overall mechanical cell dynamics, reflecting specific changes of the chemical and mechanical environment.     

Rheology of the vitreous gel: effects of macromolecule organization on the viscoelastic properties

The macromolecular organization of vitreous gel is responsible for its viscoelastic properties. Knowledge of this correlation enables us to relate the physical properties of vitreous to its pathology, as well as optimize surgical procedures such as vitrectomy. Herein, we studied the rheological properties (e.g. dynamic deformation, shear stress-strain flow, and creep compliance) of porcine vitreous humor using a stressed-control shear rheometer. All experiments were performed in a closed environment with the temperature set to that of the human body (i.e. 37°C) to mimic in-vivo conditions. We modeled the creep deformation using the two-element retardation spectrum model. By associating each element of the model to an individual biopolymeric system in the vitreous gel, a distinct response to the applied stress was observed from each component. We hypothesized that the first viscoelastic response with the short time scale (~1 s) is associated with the collagen structure, while the second viscoelastic response with longer time scale (~100 s) is related to the microfibrilis and hyaluronan network. Consequently, we were able to differentiate the role of each main component from the overall viscoelastic properties.     

Influence of divalent cations on the cytoskeletal dynamics of K562 cells determined by nano-scale bead tracking

Cytoskeletal reorganization processes can be analyzed by studying the nanometer-scale spontaneous motion of beads bound to the cytoskeleton. The bead motion is determined by force fluctuations within the cytoskeletal network that originates from myosin motor activity and dynamic restructuring of cytoskeletal filaments. We investigated to what extend the spontaneous bead motion is influenced by the dynamics of the link between the bead and the cytoskeleton in the presence of divalent cations. Our data show that, when K562 cells expressing constitutively (alpha 5 beta 1) integrin and when stably transfected with (alpha v beta 3) integrin, spontaneous bead motion is dramatically affected by the presence of 1mM Mn(2+) (integrin, activate state) compared to 1mM Ca(2+)/Mg(2+) ions (integrin, inactive state). The directionality of the bead motion, which is influenced by the overall stability of the cytoskeletal network and by actomyosin-generated forces, is markedly different, whilst the persistence remained similar due to the specific binding of either Mn(2+) or Mg(2+)/Ca(2+) ions.     

Surprisingly simple mechanical behavior of a complex embryonic tissue

Background

Previous studies suggest that mechanical feedback could coordinate morphogenetic events in embryos. Furthermore, embryonic tissues have complex structure and composition and undergo large deformations during morphogenesis. Hence we expect highly non-linear and loading-rate dependent tissue mechanical properties in embryos.

Methodology/Principal Findings

We used micro-aspiration to test whether a simple linear viscoelastic model was sufficient to describe the mechanical behavior of gastrula stage Xenopus laevis embryonic tissue in vivo. We tested whether these embryonic tissues change their mechanical properties in response to mechanical stimuli but found no evidence of changes in the viscoelastic properties of the tissue in response to stress or stress application rate. We used this model to test hypotheses about the pattern of force generation during electrically induced tissue contractions. The dependence of contractions on suction pressure was most consistent with apical tension, and was inconsistent with isotropic contraction. Finally, stiffer clutches generated stronger contractions, suggesting that force generation and stiffness may be coupled in the embryo.

Conclusions/Significance

The mechanical behavior of a complex, active embryonic tissue can be surprisingly well described by a simple linear viscoelastic model with power law creep compliance, even at high deformations. We found no evidence of mechanical feedback in this system. Together these results show that very simple mechanical models can be useful in describing embryo mechanics.     

应用原子力显微镜分析正常淋巴细胞和Jurkat细胞的形态和机械性质

淋巴细胞形态和机械性质的变化与人的健康、疾病的治疗和诊断有着密切关系。本研究利用原子力显微镜研究淋巴细胞和Jurkat细胞形态和机械性质。结果显示,这2种细胞的形态较为相似,但通过对力曲线的分析得出这2种细胞的机械性质明显不同。正常淋巴细胞粘弹力范围大致为(796.7±248.5)pN,而Jurkat细胞分布于(158.5±37.5)pN;正常淋巴细胞的杨氏模量(0.471kPa±0.081kPa)近4倍于Jurkat细胞(0.0964kPa±0.0229kPa);而Jurkat细胞(4.322mN/m±0.382mN/m)的硬度近2倍于正常淋巴细胞(2.278mN/m±0.488mN/m)。结果表明原子力显微镜能可在临床诊断上区分正常细胞与肿瘤细胞,即使两者形态区别不明显。     

Viscoelastic properties of skin in Mov-13 and Tsk mice

Viscoelastic properties of skin samples were measured in three types of mice (tight skin, Tsk, control and Mov-13), that are known to differ with regard to content of type I collagen. The experimental design used uniaxial stretching and measured the creep response and the complex compliance. The creep response was measured directly. The complex compliance was determined using a Wiener-Volterra constitutive model for each sample. The models were calculated from data obtained by applying a stress input having a pseudo-Gaussian waveform and measuring the strain response. The storage compliance of Mov-13 and control skin were similar and were greater than Tsk (p<0.001). The loss compliance of each group was significantly different (p<0.001) from each other group; Tsk had the lowest and control had the highest loss compliance. The phase angle of the Mov-13 and Tsk were similar and were less than the controls (p<0.001). The creep response was fit with a linear viscoelastic model. None of the parameters in the creep model differed between groups. The results indicate that gene-targeted and mutant animals have soft tissue mechanical phenotypes that differ in complex ways. Caution should be exercised when using such animals as models to explore the role of specific constituents on tissue properties.     

Viscoelastic retraction of single living stress fibers and its impact on cell shape, cytoskeletal organization, and extracellular matrix mechanics

Cells change their form and function by assembling actin stress fibers at their base and exerting traction forces on their extracellular matrix (ECM) adhesions. Individual stress fibers are thought to be actively tensed by the action of actomyosin motors and to function as elastic cables that structurally reinforce the basal portion of the cytoskeleton; however, these principles have not been directly tested in living cells, and their significance for overall cell shape control is poorly understood. Here we combine a laser nanoscissor, traction force microscopy, and fluorescence photobleaching methods to confirm that stress fibers in living cells behave as viscoelastic cables that are tensed through the action of actomyosin motors, to quantify their retraction kinetics in situ, and to explore their contribution to overall mechanical stability of the cell and interconnected ECM. These studies reveal that viscoelastic recoil of individual stress fibers after laser severing is partially slowed by inhibition of Rho-associated kinase and virtually abolished by direct inhibition of myosin light chain kinase. Importantly, cells cultured on stiff ECM substrates can tolerate disruption of multiple stress fibers with negligible overall change in cell shape, whereas disruption of a single stress fiber in cells anchored to compliant ECM substrates compromises the entire cellular force balance, induces cytoskeletal rearrangements, and produces ECM retraction many microns away from the site of incision; this results in large-scale changes of cell shape (> 5% elongation). In addition to revealing fundamental insight into the mechanical properties and cell shape contributions of individual stress fibers and confirming that the ECM is effectively a physical extension of the cell and cytoskeleton, the technologies described here offer a novel approach to spatially map the cytoskeletal mechanics of living cells on the nanoscale.     

Mechanosensitivity of Cancer Cells in Contact with Soft Substrates Using AFM

Cancer cells are usually found to be softer than normal cells, but their stiffness changes when they are in contact with different environments because of mechanosensitivity. For example, they adhere to a given substrate by tuning their cytoskeleton, thus affecting their rheological properties. This mechanism could become efficient when cancer cells invade the surrounding tissues, and they have to remodel their cytoskeleton in order to achieve particular deformations. Here we use an atomic force microscope in force modulation mode to study how local rheological properties of cancer cells are affected by a change of the environment. Cancer cells were plated on functionalized polyacrylamide substrates of different stiffnesses as well as on an endothelium substrate. A new correction of the Hertz model was developed because measurements require one to account for the precise properties of the thin, layered viscoelastic substrates. The main results show the influence of local cell rheology (the nucleus, perinuclear region, and edge locations) and the role of invasiveness. A general mechanosensitive trend is found by which the cell elastic modulus and transition frequency increase with substrate elasticity, but this tendency breaks down with a real endothelium substrate. These effects are investigated further during cell transmigration, when the actin cytoskeleton undergoes a rapid reorganization process necessary to push through the endothelial gap, in agreement with the local viscoelastic changes measured by atomic force microscopy. Taken together, these results introduce a paradigm for a new—to our knowledge—possible extravasation mechanism.     

Measuring cell viscoelastic properties using a force-spectrometer: influence of protein-cytoplasm interactions

Cell adhesive and rheological properties play a very important role in cell transmigration through the endothelial barrier, in particular in the case of inflammation (leukocytes) or cancer metastasis (cancer cells). In order to characterize cell viscoelastic properties, we have designed a force spectrometer (AFM) which can stretch cells thereby allowing measurement of their rheological properties. This custom-made force spectrometer allows two different visualizations, one lateral and one from below. It allows investigation of the effects of rheology involved during cell stretching. To test the ability of our system to characterize such viscoelastic properties, ICAM-1 transfected CHO cells were analyzed. Two forms of ICAM-1 were tested; wild type ICAM-1, which can interact with the cytoskeleton, and a mutant form which lacks the cytoplasmic domain, and is unable to associate with the cytoskeleton. Stretching experiments carried out on these cells show the formation of long filaments. Using a previous model of filament elongation, we could determine the viscoelastic properties of a single cell. As expected, different viscoelastic components were found between the wild type and the mutant, which reveal that the presence of interactions between ICAM-1 and the cytoskeleton increases the stiffness of the cell.