How Cells Feel: Stochastic Model for a Molecular Mechanosensor

Understanding mechanosensitivity (i.e., how cells sense the stiffness of their environment) is very important, yet there is a fundamental difficulty in understanding its mechanism: to measure an elastic modulus one requires two points of application of force—a measuring and a reference point. The cell in contact with substrate has only one (adhesion) point to work with, and thus a new method of measurement needs to be invented. The aim of this theoretical work is to develop a self-consistent physical model for mechanosensitivity, a process by which a cell detects the mechanical stiffness of its environment (e.g., a substrate it is attached to via adhesion points) and generates an appropriate chemical signaling to remodel itself in response to this environment. The model uses the molecular mechanosensing complex of latent TGF-β attached to the adhesion point as the biomarker. We show that the underlying Brownian motion in the substrate is the reference element in the measuring process. The model produces a closed expression for the rate of release of active TGF-β, which depends on the substrate stiffness and the pulling force coming from the cell in a subtle and nontrivial way. It is consistent with basic experimental data showing an increase in signal for stiffer substrates and higher pulling forces. In addition, we find that for each cell there is a range of stiffness where a homeostatic configuration of the cell can be achieved, outside of which the cell either relaxes its cytoskeletal forces and detaches from the very weak substrate, or generates an increasingly strong pulling force through stress fibers with a positive feedback loop on very stiff substrates. In this way, the theory offers the underlying mechanism for the myofibroblast conversion in wound healing and smooth muscle cell dysfunction in cardiac disease.     

Mechanosensitivity of the 2nd Kind: TGF-β Mechanism of Cell Sensing the Substrate Stiffness

Cells can sense forces applied to them, but also the stiffness of their environment. These are two different phenomena, and here we investigate the mechanosensitivity of the 2^nd kind: how the cell can measure an elastic modulus at a single point of adhesion—and how the cell can receive and interpret the chemical signal released from the sensor. Our model uses the example of large latent complex of TGF-β as a sensor. Stochastic theory gives the rate of breaking of latent complex, which initiates the signaling feedback loop after the active TGF-β release and leads to a change of cell phenotype driven by the α-smooth muscle actin. We investigate the dynamic and steady-state behaviors of the model, comparing them with experiments. In particular, we analyse the timescale of approach to the steady state, the stability of the non-linear dynamical system, and how the steady-state concentrations of the key markers vary depending on the elasticity of the substrate. We discover a crossover region for values of substrate elasticity closely corresponding to that of the fibroblast to myofibroblast transition. We suggest that the cell could actively vary the parameters of its dynamic feedback loop to ‘choose’ the position of the transition region and the range of substrate elasticity that it can detect. In this way, the theory offers the unifying mechanism for a variety of phenomena, such as the myofibroblast conversion in fibrosis of wounds and lungs and smooth muscle cell dysfunction in cardiac disease.     

Tunable cell-surface mimetics as engineered cell substrates

Most recent breakthroughs in understanding cell adhesion, cell migration, and cellular mechanosensitivity have been made possible by the development of engineered cell substrates of well-defined surface properties. Traditionally, these substrates mimic the extracellular matrix (ECM) environment by the use of ligand-functionalized polymeric gels of adjustable stiffness. However, such ECM mimetics are limited in their ability to replicate the rich dynamics found at cell-cell contacts. This review focuses on the application of cell surface mimetics, which are better suited for the analysis of cell adhesion, cell migration, and cellular mechanosensitivity across cell-cell interfaces. Functionalized supported lipid bilayer systems were first introduced as biomembrane-mimicking substrates to study processes of adhesion maturation during adhesion of functionalized vesicles (cell-free assay) and plated cells. However, while able to capture adhesion processes, the fluid lipid bilayer of such a relatively simple planar model membrane prevents adhering cells from transducing contractile forces to the underlying solid, making studies of cell migration and cellular mechanosensitivity largely impractical. Therefore, the main focus of this review is on polymer-tethered lipid bilayer architectures as biomembrane-mimicking cell substrate. Unlike supported lipid bilayers, these polymer-lipid composite materials enable the free assembly of linkers into linker clusters at cellular contacts without hindering cell spreading and migration and allow the controlled regulation of mechanical properties, enabling studies of cellular mechanosensitivity. The various polymer-tethered lipid bilayer architectures and their complementary properties as cell substrates are discussed.     

Attenuation of Cell Mechanosensitivity in Colon Cancer Cells during In Vitro Metastasis

Human colon carcinoma (HCT-8) cells show a stable transition from low to high metastatic state when cultured on appropriately soft substrates (21 kPa). Initially epithelial (E) in nature, the HCT-8 cells become rounded (R) after seven days of culture on soft substrate. R cells show a number of metastatic hallmarks [1]. Here, we use gradient stiffness substrates, a bio-MEMS force sensor, and Coulter counter assays to study mechanosensitivity and adhesion of E and R cells. We find that HCT-8 cells lose mechanosensitivity as they undergo E-to-R transition. HCT-8 R cells'' stiffness, spread area, proliferation and migration become insensitive to substrate stiffness in contrast to their epithelial counterpart. They are softer, proliferative and migratory on all substrates. R cells show negligible cell-cell homotypic adhesion, as well as non-specific cell-substrate adhesion. Consequently they show the same spread area on all substrates in contrast to E cells. Taken together, these results indicate that R cells acquire autonomy and anchorage independence, and are thus potentially more invasive than E cells. To the best of our knowledge, this is the first report of quantitative data relating changes in cancer cell adhesion and stiffness during the expression of an in vitro metastasis-like phenotype.     

Substrate Deformation Predicts Neuronal Growth Cone Advance

Although pulling forces have been observed in axonal growth for several decades, their underlying mechanisms, absolute magnitudes, and exact roles are not well understood. In this study, using two different experimental approaches, we quantified retrograde traction force in Aplysia californica neuronal growth cones as they develop over time in response to a new adhesion substrate. In the first approach, we developed a novel method, to our knowledge, for measuring traction forces using an atomic force microscope (AFM) with a cantilever that was modified with an Aplysia cell adhesion molecule (apCAM)-coated microbead. In the second approach, we used force-calibrated glass microneedles coated with apCAM ligands to guide growth cone advance. The traction force exerted by the growth cone was measured by monitoring the microneedle deflection using an optical microscope. Both approaches showed that Aplysia growth cones can develop traction forces in the 10⁰–10² nN range during adhesion-mediated advance. Moreover, our results suggest that the level of traction force is directly correlated to the stiffness of the microneedle, which is consistent with a reinforcement mechanism previously observed in other cell types. Interestingly, the absolute level of traction force did not correlate with growth cone advance toward the adhesion site, but the amount of microneedle deflection did. In cases of adhesion-mediated growth cone advance, the mean needle deflection was 1.05 ± 0.07 μm. By contrast, the mean deflection was significantly lower (0.48 ± 0.06 μm) when the growth cones did not advance. Our data support a hypothesis that adhesion complexes, which can undergo micron-scale elastic deformation, regulate the coupling between the retrogradely flowing actin cytoskeleton and apCAM substrates, stimulating growth cone advance if sufficiently abundant.     

Mechanosensitivity of a Rapid Bioluminescence Reporter System Assessed by Atomic Force Microscopy

Cells are sophisticated integrators of mechanical stimuli that lead to physiological, biochemical, and genetic responses. The bioluminescence of dinoflagellates, alveolate protists that use light emission for predator defense, serves as a rapid noninvasive whole-cell reporter of mechanosensitivity. In this study, we used atomic force microscopy (AFM) to explore the relationship between cell mechanical properties and mechanosensitivity in live cells of the dinoflagellate Pyrocystis lunula. Cell stiffness was 0.56 MPa, consistent with cells possessing a cell wall. Cell response depended on both the magnitude and velocity of the applied force. At the maximum stimulation velocity of 390 μm s⁻¹, the threshold response occurred at a force of 7.2 μN, resulting in a contact time of 6.1 ms and indentation of 2.1 μm. Cells did not respond to a low stimulation velocity of 20 μm s⁻¹, indicating a velocity dependent response that, based on stress relaxation experiments, was explained by the cell viscoelastic properties. This study demonstrates the use of AFM to study mechanosensitivity in a cell system that responds at fast timescales, and provides insights into how viscoelastic properties affect mechanosensitivity. It also provides a comparison with previous studies using hydrodynamic stimulation, showing the discrepancy in cell response between direct compressive forces using AFM and those within flow fields based on average flow properties.     

Integrin cross-talk modulates stiffness-independent motility of CD4+ T lymphocytes

To carry out their physiological responsibilities, CD4+ T lymphocytes interact with various tissues of different mechanical properties. Recent studies suggest that T cells migrate upstream on surfaces expressing intracellular adhesion molecule-1 (ICAM-1) through interaction with leukocyte function-associated antigen-1 (α_Lβ₂) (LFA-1) integrins. LFA-1 likely behaves as a mechanosensor, and thus we hypothesized that substrate mechanics might affect the ability of LFA-1 to support upstream migration of T cells under flow. Here we measured motility of CD4+ T lymphocytes on polyacrylamide gels with predetermined stiffnesses containing ICAM-1, vascular cell adhesion molecule-1 (VCAM-1), or a 1:1 mixture of VCAM-1/ICAM-1. Under static conditions, we found that CD4+ T cells exhibit an increase in motility on ICAM-1, but not on VCAM-1 or VCAM-1/ICAM-1 mixed, surfaces as a function of matrix stiffness. The mechanosensitivity of T-cell motility on ICAM-1 is overcome when VLA-4 (very late antigen-4 [α₄β₁]) is ligated with soluble VCAM-1. Last, we observed that CD4+ T cells migrate upstream under flow on ICAM–1-functionalized hydrogels, independent of substrate stiffness. In summary, we show that CD4+ T cells under no flow respond to matrix stiffness through LFA-1, and that the cross-talk of VLA-4 and LFA-1 can compensate for deformable substrates. Interestingly, CD4+ T lymphocytes migrated upstream on ICAM-1 regardless of the substrate stiffness, suggesting that flow can compensate for substrate stiffness.     

Migration in Confined 3D Environments Is Determined by a Combination of Adhesiveness,Nuclear Volume,Contractility, and Cell Stiffness

In cancer metastasis and other physiological processes, cells migrate through the three-dimensional (3D) extracellular matrix of connective tissue and must overcome the steric hindrance posed by pores that are smaller than the cells. It is currently assumed that low cell stiffness promotes cell migration through confined spaces, but other factors such as adhesion and traction forces may be equally important. To study 3D migration under confinement in a stiff (1.77 MPa) environment, we use soft lithography to fabricate polydimethylsiloxane (PDMS) devices consisting of linear channel segments with 20 μm length, 3.7 μm height, and a decreasing width from 11.2 to 1.7 μm. To study 3D migration in a soft (550 Pa) environment, we use self-assembled collagen networks with an average pore size of 3 μm. We then measure the ability of four different cancer cell lines to migrate through these 3D matrices, and correlate the results with cell physical properties including contractility, adhesiveness, cell stiffness, and nuclear volume. Furthermore, we alter cell adhesion by coating the channel walls with different amounts of adhesion proteins, and we increase cell stiffness by overexpression of the nuclear envelope protein lamin A. Although all cell lines are able to migrate through the smallest 1.7 μm channels, we find significant differences in the migration velocity. Cell migration is impeded in cell lines with larger nuclei, lower adhesiveness, and to a lesser degree also in cells with lower contractility and higher stiffness. Our data show that the ability to overcome the steric hindrance of the matrix cannot be attributed to a single cell property but instead arises from a combination of adhesiveness, nuclear volume, contractility, and cell stiffness.     

Role of Suspended Fiber Structural Stiffness and Curvature on Single-Cell Migration,Nucleus Shape,and Focal-Adhesion-Cluster Length

It has been shown that cellular migration, persistence, and associated cytoskeletal arrangement are highly dependent on substrate stiffness (modulus: N/m² and independent of geometry), but little is known on how cells respond to subtle changes in local geometry and structural stiffness (N/m). Here, using fibers of varying diameter (400, 700, and 1200 nm) and length (1 and 2 mm) deposited over hollow substrates, we demonstrate that single mouse C2C12 cells attached to single suspended fibers form spindle morphologies that are sensitive to fiber mechanical properties. Over a wide range of increasing structural stiffness (2 to 100+ mN/m), cells exhibited decreases in migration speed and average nucleus shape index of ∼57% (from 58 to 25 μm/h) and ∼26% (from 0.78 to 0.58), respectively, whereas the average paxillin focal-adhesion-cluster (FAC, formed at poles) length increased by ∼38% (from 8 to 11 μm). Furthermore, the increase in structural stiffness directly correlates with cellular persistence, with 60% of cells moving in the direction of increasing structural stiffness. At similar average structural stiffness (25 ± 5 mN/m), cells put out longer FAC lengths on smaller diameters, suggesting a conservation of FAC area, and also exhibited higher nucleus shape index and migration speeds on larger-diameter fibers. Interestingly, cells were observed to deform fibers locally or globally through forces applied through the FAC sites and cells undergoing mitosis were found to be attached to the FAC sites by single filamentous tethers. These varied reactions have implications in developmental and disease biology models as they describe a strong dependence of cellular behavior on the cell’s immediate mechanistic environment arising from alignment and geometry of fibers.     

Nucleation and decay initiation are the stiffness-sensitive phases of focal adhesion maturation

A cell plated on a two-dimensional substrate forms adhesions with that surface. These adhesions, which consist of aggregates of various proteins, are thought to be important in mechanosensation, the process by which the cell senses and responds to the mechanical properties of the substrate (e.g., stiffness). On the basis of experimental measurements, we model these proteins as idealized molecules that can bind to the substrate in a strain-dependent manner and can undergo a force-dependent state transition. The model forms molecular aggregates that are similar to adhesions. Substrate stiffness affects whether a simulated adhesion is initially formed and how long it grows, but not how that adhesion grows or shrinks. Our own experimental tests support these predictions, suggesting that the mechanosensitivity of adhesions is an emergent property of a simple molecular-mechanical system.     

The consequence of substrates of large-scale rigidity on actin network tension in adherent cells

Abstract

There is compelling evidence that substrate stiffness affects cell adhesion as well as cytoskeleton organization and contractile activity. This work was designed to study the cytoskeletal contractile activity of single cells plated on micropost substrates of different stiffness using a numerical model simulating the intracellular tension of individual cells. We allowed cells to adhere onto micropost substrates of various rigidities and used experimental traction force data to infer cell contractility using a numerical model. The model shows that higher substrate stiffness leads to an increase in intracellular tension. The strength of this model is its ability to calculate the mechanical state of each cell in accordance to its individual cytoskeletal structure. This is achieved by regenerating a numerical cytoskeleton based on microscope images of the actin network of each cell. The resulting numerical structure consequently represents pulling characteristics on its environment similar to those generated by the cell in-vivo. From actin imaging we can calculate and better understand how forces are transmitted throughout the cell.     

The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 ºC - a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 ºC. Next, the temperature is decreased again to 4 ºC to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 °C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 ºC as in the endocytic assay. Next, cells are incubated again at 37 °C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 ºC, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.     

Cell-cell interactions mediate the response of vascular smooth muscle cells to substrate stiffness

The vessel wall experiences progressive stiffening with age and the development of cardiovascular disease, which alters the micromechanical environment experienced by resident vascular smooth muscle cells (VSMCs). In vitro studies have shown that VSMCs are sensitive to substrate stiffness, but the exact molecular mechanisms of their response to stiffness remains unknown. Studies have also shown that cell-cell interactions can affect mechanotransduction at the cell-substrate interface. Using flexible substrates, we show that the expression of proteins associated with cell-matrix adhesion and cytoskeletal tension is regulated by substrate stiffness, and that an increase in cell density selectively attenuates some of these effects. We also show that cell-cell interactions exert a strong effect on cell morphology in a substrate-stiffness dependent manner. Collectively, the data suggest that as VSMCs form cell-cell contacts, substrate stiffness becomes a less potent regulator of focal adhesion signaling. This study provides insight into the mechanisms by which VSMCs respond to the mechanical environment of the blood vessel wall, and point to cell-cell interactions as critical mediators of VSMC response to vascular injury.     

TGF-β1 regulates cell fate during epithelial–mesenchymal transition by upregulating survivin

Members of the transforming growth factor beta (TGF-β) superfamily are multifunctional cytokines that regulate several cellular processes, including cell cycle arrest, differentiation, morphogenesis, and apoptosis. TGF-β promotes extracellular matrix production and morphological change. Morphogenetic responses to TGF-β include cell migration and epithelial–mesenchymal transition (EMT), which are critical during embryogenesis, development of fibrotic diseases, and the spreading of advanced carcinomas. The purpose of this study was to clarify how TGF-β regulates the fate of retinal pigment epithelial (RPE) cells. TGF-β1 promoted cell cycle progression and phosphorylation of retinoblastoma protein (Rb) in ARPE-19 cells. TGF-β1 induced survivin expression, which in turn stabilized tubulin and Aurora B. RT-PCR and western blot analysis revealed that survivin expression increased in ARPE-19 cells following TGF-β1 treatment. When survivin was depleted, TGF-β1 induced cell cycle arrest and apoptosis and also reduced Rb phosphorylation. In conclusion, the present study shows that induction of EMT in human RPE cells upregulates survivin, leading to survivin-dependent inhibition of cell cycle arrest and apoptosis. Whether cells undergo EMT or apoptosis in response to TGF-β1 is dependent on their cell cycle state, and TGF-β1 regulates the cell cycle via survivin.     

Integrin inhibition promotes atypical anoikis in glioma cells

Integrins regulate cellular adhesion and transmit signals important for cell survival, proliferation and motility. They are expressed by glioma cells and may contribute to their malignant phenotype. Integrin inhibition may therefore represent a promising therapeutic strategy. GL-261 and SMA-560 glioma cells grown under standard conditions uniformly detached and formed large cell clusters after integrin gene silencing or pharmacological inhibition using EMD-121974, a synthetic Arg-Gly-Asp-motif peptide, or GLPG0187, a nonpeptidic integrin inhibitor. After 120 h, the clusters induced by integrin inhibition decayed and cells died. In contrast, when cells were cultured under stem cell (sphere) conditions, no disaggregation became apparent upon integrin inhibition, and cell death was not observed. As poly-HEMA-mediated detachment had similar effects on cell viability as integrin inhibition, we postulated that cell death may result from detachment alone, which was confirmed using various permissive and nonpermissive substrates. No surrogate markers of apoptosis were detected and electron microscopy confirmed that necrosis represents the dominant morphology of detachment-induced cell death. In addition, integrin inhibition resulted in the induction of autophagy that represents a survival signal. When integrins were inhibited in nonsphere glioma cells, the TGF-β pathway was strongly impaired, whereas no such effect was observed in glioma cells cultured under sphere conditions. Cell death induced by integrin inhibition was rescued by the addition of recombinant transforming growth factor-β (TGF-β) and accelerated by exposure to the TGF-β receptor inhibitor, SD-208. In summary, cell death following integrin inhibition is detachment mediated, represents an atypical form of anoikis involving necrosis as well as autophagy, and is modulated by TGF-β pathway activity.     

The Physical Interaction of Myoblasts with the Microenvironment during Remodeling of the Cytoarchitecture

Integrins, focal adhesions, the cytoskeleton and the extracellular matrix, form a structural continuum between the external and internal environment of the cell and mediate the pathways associated with cellular mechanosensitivity and mechanotransduction. This continuum is important for the onset of muscle tissue generation, as muscle precursor cells (myoblasts) require a mechanical stimulus to initiate myogenesis. The ability to sense a mechanical cue requires an intact cytoskeleton and strong physical contact and adhesion to the microenvironment. Importantly, myoblasts also undergo reorientation, alignment and large scale remodeling of the cytoskeleton when they experience mechanical stretch and compression in muscle tissue. It remains unclear if such dramatic changes in cell architecture also inhibit physical contact and adhesion with the tissue microenvironment that are clearly important to myoblast physiology. In this study, we employed interference reflection microscopy to examine changes in the close physical contact of myoblasts with a substrate during induced remodeling of the cytoarchitecture (de-stabilization of the actin and microtubule cytoskeleton and inhibition of acto-myosin contractility). Our results demonstrate that while each remodeling pathway caused distinct effects on myoblast morphology and sub-cellular structure, we only observed a ∼13% decrease in close physical contact with the substrate, regardless of the pathway inhibited. However, this decrease did not correlate well with changes in cell adhesion strength. On the other hand, there was a close correlation between cell adhesion and β1-integrin expression and the presence of cell-secreted fibronectin, but not with the presence of intact focal adhesions. In this study, we have shown that myoblasts are able to maintain a large degree of physical contact and adhesion to the microenvironment, even during shot periods (<60 min) of large scale remodeling and physiological stress, which is essential to their in-vivo functionality.     

Prostate apoptosis response-4 mediates TGF-β-induced epithelial-to-mesenchymal transition

A growing body of evidence supports that the epithelial-to-mesenchymal transition (EMT), which occurs during cancer development and progression, has a crucial role in metastasis by enhancing the motility of tumor cells. Transforming growth factor-β (TGF-β) is known to induce EMT in a number of cancer cell types; however, the mechanism underlying this transition process is not fully understood. In this study we have demonstrated that TGF-β upregulates the expression of tumor suppressor protein Par-4 (prostate apoptosis response-4) concomitant with the induction of EMT. Mechanistic investigations revealed that exogenous treatment with each TGF-β isoform upregulates Par-4 mRNA and protein levels in parallel levels of phosphorylated Smad2 and IκB-α increase. Disruption of TGF-β signaling by using ALK5 inhibitor, neutralizing TGF-β antibody or phosphoinositide 3-kinase inhibitor reduces endogenous Par-4 levels, suggesting that both Smad and NF-κB pathways are involved in TGF-β-mediated Par-4 upregulation. NF-κB-binding sites in Par-4 promoter have previously been reported; however, using chromatin immunoprecipitation assay we showed that Par-4 promoter region also contains Smad4-binding site. Furthermore, TGF-β promotes nuclear localization of Par-4. Prolonged TGF-β3 treatment disrupts epithelial cell morphology, promotes cell motility and induces upregulation of Snail, vimentin, zinc-finger E-box binding homeobox 1 and N-Cadherin and downregulation of Claudin-1 and E-Cadherin. Forced expression of Par-4, results in the upregulation of vimentin and Snail expression together with increase in cell migration. In contrast, small interfering RNA-mediated silencing of Par-4 expression results in decrease of vimentin and Snail expression and prevents TGF-β-induced EMT. We have also uncovered a role of X-linked inhibitor of apoptosis protein in the regulation of endogenous Par-4 levels through inhibition of caspase-mediated cleavage. In conclusion, our findings suggest that Par-4 is a novel and essential downstream target of TGF-β signaling and acts as an important factor during TGF-β-induced EMT.     

Confinement-Dependent Friction in Peptide Bundles

Friction within globular proteins or between adhering macromolecules crucially determines the kinetics of protein folding, the formation, and the relaxation of self-assembled molecular systems. One fundamental question is how these friction effects depend on the local environment and in particular on the presence of water. In this model study, we use fully atomistic MD simulations with explicit water to obtain friction forces as a single polyglycine peptide chain is pulled out of a bundle of k adhering parallel polyglycine peptide chains. The whole system is periodically replicated along the peptide axes, so a stationary state at prescribed mean sliding velocity V is achieved. The aggregation number is varied between k = 2 (two peptide chains adhering to each other with plenty of water present at the adhesion sites) and k = 7 (one peptide chain pulled out from a close-packed cylindrical array of six neighboring peptide chains with no water inside the bundle). The friction coefficient per hydrogen bond, extrapolated to the viscous limit of vanishing pulling velocity V → 0, exhibits an increase by five orders of magnitude when going from k = 2 to k = 7. This dramatic confinement-induced friction enhancement we argue to be due to a combination of water depletion and increased hydrogen-bond cooperativity.     

Cardiac myocyte remodeling mediated by N-cadherin-dependent mechanosensing

Cell-to-cell adhesions are crucial in maintaining the structural and functional integrity of cardiac cells. Little is known about the mechanosensitivity and mechanotransduction of cell-to-cell interactions. Most studies of cardiac mechanotransduction and myofibrillogenesis have focused on cell-extracellular matrix (ECM)-specific interactions. This study assesses the direct role of intercellular adhesion, specifically that of N-cadherin-mediated mechanotransduction, on the morphology and internal organization of neonatal ventricular cardiac myocytes. The results show that cadherin-mediated cell attachments are capable of eliciting a cytoskeletal network response similar to that of integrin-mediated force response and transmission, affecting myofibrillar organization, myocyte shape, and cortical stiffness. Traction forces mediated by N-cadherin were shown to be comparable to those sustained by ECM. The directional changes in predicted traction forces as a function of imposed loads (gel stiffness) provide the added evidence that N-cadherin is a mechanoresponsive adhesion receptor. Strikingly, the mechanical sensitivity response (gain) in terms of the measured cell-spread area as a function of imposed load (adhesive substrate rigidity) was consistently higher for N-cadherin-coated surfaces compared with ECM protein-coated surfaces. In addition, the cytoskeletal architecture of myocytes on an N-cadherin adhesive microenvironment was characteristically different from that on an ECM environment, suggesting that the two mechanotransductive cell adhesion systems may play both independent and complementary roles in myocyte cytoskeletal spatial organization. These results indicate that cell-to-cell-mediated force perception and transmission are involved in the organization and development of cardiac structure and function.     

Universal Kinetics of the Onset of Cell Spreading on Substrates of Different Stiffness

When plated onto substrates, cell morphology and even stem-cell differentiation are influenced by the stiffness of their environment. Stiffer substrates give strongly spread (eventually polarized) cells with strong focal adhesions and stress fibers; very soft substrates give a less developed cytoskeleton and much lower cell spreading. The kinetics of this process of cell spreading is studied extensively, and important universal relationships are established on how the cell area grows with time. Here, we study the population dynamics of spreading cells, investigating the characteristic processes involved in the cell response to the substrate. We show that unlike the individual cell morphology, this population dynamics does not depend on the substrate stiffness. Instead, a strong activation temperature dependence is observed. Different cell lines on different substrates all have long-time statistics controlled by the thermal activation over a single energy barrier ΔG ≈ 18 kcal/mol, whereas the early-time kinetics follows a power law ～t⁵. This implies that the rate of spreading depends on an internal process of adhesion complex assembly and activation; the operational complex must have five component proteins, and the last process in the sequence (which we believe is the activation of focal adhesion kinase) is controlled by the binding energy ΔG.