Rolling adhesion of leukocytes on soft substrates: Does substrate stiffness matter?

Cell rolling on vascular endothelium under hydrodynamic blood flow is critical for realization of many physiological and pathological processes, such as inflammatory response and tumor metastasis. The blood-borne cells are in direct contact with the inner layer of endothelium, formed by a highly compliant layer of endothelial cells. The effect of endothelial stiffness on the adhesion and motion of rolling cells is poorly understood. Inspired by recent in vitro studies, here we implemented a computational method to model the specific adhesion of a rolling cell onto a soft substrate, subjected to a creeping shear flow. The substrate is modeled as an elastic half-space, coated with P- and E-selectin receptors with specific affinity for the complementary ligands located on the moving cell. Of particular importance is to predict the effect of substrate stiffness on cell adhesion and its kinematics and kinetics of motion. Simulation results show that the effect of substrate compliance is minimal when coated with P-selectin. Conversely, the trajectory of rolling cells on E-selectin coated substrates is sensitive to the substrate compliance. This is attributed to the moderation of binding forces applied by the soft substrate which leads to a higher average translational velocity of cells.     

Integrin-dependent actomyosin contraction regulates epithelial cell scattering

The scattering of Madin-Darby canine kidney cells in vitro mimics key aspects of epithelial-mesenchymal transitions during development, carcinoma cell invasion, and metastasis. Scattering is induced by hepatocyte growth factor (HGF) and is thought to involve disruption of cadherin-dependent cell-cell junctions. Scattering is enhanced on collagen and fibronectin, as compared with laminin1, suggesting possible cross talk between integrins and cell-cell junctions. We show that HGF does not trigger any detectable decrease in E-cadherin function, but increases integrin-mediated adhesion. Time-lapse imaging suggests that tension on cell-cell junctions may disrupt cell-cell adhesion. Varying the density and type of extracellular matrix proteins shows that scattering correlates with stronger integrin adhesion and increased phosphorylation of the myosin regulatory light chain. To directly test the role of integrin-dependent traction forces, substrate compliance was varied. Rigid substrates that produce high traction forces promoted scattering, in comparison to more compliant substrates. We conclude that integrin-dependent actomyosin traction force mediates the disruption of cell-cell adhesion during epithelial cell scattering.     

AFM-based dual nano-mechanical phenotypes for cancer metastasis

An enhanced mechanical compliance is considered to be a mechanical indicator for metastatic cancer cells. Our study using atomic force microscopy (AFM) revealed that breast cancer cells agreed well with this hypothesis. However, prostate cancer cells displayed a reverse correlation; less metastatic prostate cancer cells were more mechanically compliant. Two-dimensional AFM force spectroscopy was performed to characterize dual mechanical properties—the cell–substrate adhesion as well as the mechanical compliance. Interestingly, prostate cancer cells displayed a strong positive correlation between the cell–substrate adhesion and metastatic potential. However, there was no clearly observable correlation between the cell–substrate adhesion and the metastatic potential despite variations in mechanical compliance of breast cancer cells. These results suggest that the correlation between the dual mechanical signatures and metastatic potential be uniquely identified for cancer cells originating from different organs. We postulate that this correlation could reveal which step of cancer progression is favorable in terms of physical interaction between cancer cells and micro-environments. We expect that based on the “seed and soil hypothesis”, the identification of the dual mechanical phenotypes, could provide a new insight for understanding how a dominant metastatic site is determined for cancer cells originating from specific organs.     

Simulation of detachment of specifically bound particles from surfaces by shear flow.

The receptor-mediated adhesion of cells to ligand-coated surfaces is important in many physiological and biotechnological processes. Previously, we measured the detachment of antibody-coated spheres from counter-antibody- and protein A-coated substrates using a radial-flow detachment assay and were able to relate mechanical adhesion strength to chemical binding affinity (Kuo and Lauffenburger, Biophys. J. 65:2191-2200 (1993)). In this paper, we use "adhesive dynamics" to simulate the detachment of antibody-coated hard spheres from a ligand-coated substrate. We modeled the antibody-ligand (either counter-antibody or protein A) bonds as adhesive springs. In the simulation as in the experiments, beads attach to the substrate under static conditions. Flow is then initiated, and detachment is measured by the significant displacement of previously bound particles. The model can simulate the effects of many parameters on cell detachment, including hydrodynamic stresses, receptor number, ligand density, reaction rates between receptor and ligand, and stiffness and reactive compliance of the adhesive springs. The simulations are compared with experimental detachment data, thus relating measured bead adhesion strength to molecular properties of the adhesion molecules. The simulations accurately recreated the logarithmic dependence of adhesion strength on affinity of receptor-ligand recognition, which was seen in experiments and predicted by analytic theory. In addition, we find the value of the reactive compliance, the parameter which relates the strain of a bond to its rate of breakage, that gives the best match between theory and experiment to be 0.01. Finally, we analyzed the effect of varying either the forward or reverse rate constants as different ways to achieve the same affinity, and showed that adhesion strength depends uniquely on the equilibrium affinity, not on the kinetics of binding. Given that attachment is independent of affinity, detachment and attachment are distinct adhesive phenomena.     

Sticking around: Cell adhesion patterning for energy minimization and substrate mechanosensing

Tissue stiffness (Young’s modulus) is a key control parameter in cell behavior and bioengineered gels where defined mechanical properties have become an essential part of the toolkit for interrogating mechanotransduction. Here, we show using a mechanical cell model that the effective substrate stiffness experienced by a cell depends, not just on the engineered mechanical properties of the substrate but critically also on the particular arrangement of adhesions between cell and substrate. In particular, we find that cells with different adhesion patterns can experience two different gel stiffnesses as equivalent and will generate the same mean cell deformations. In considering small patches of adhesion, which mimic focal adhesion complexes, we show how the experimentally observed focal adhesion growth and elongation on stiff substrates can be explained by energy considerations. Relatedly, energy arguments also provide a reason why nascent adhesions do not establish into focal adhesions on soft substrates, as has been commonly observed. Fewer and larger adhesions are predicted to be preferred over more and smaller, an effect enhanced by random spot placing with the simulations predicting qualitatively realistic cell shapes in this case.     

Three-dimensional analysis of the effect of epidermal growth factor on cell-cell adhesion in epithelial cell clusters

The effect that growth factors such as epidermal growth factor (EGF) have on cell-cell adhesion is of interest in the study of cellular processes such as epithelial-mesenchymal transition. Because cell-cell adhesions cannot be measured directly, we use three-dimensional traction force microscopy to measure the tractions applied by clusters of MCF-10A cells to a compliant substrate beneath them before and after stimulating the cells with EGF. To better interpret the results, a finite element model, which simulates a cluster of individual cells adhered to one another and to the substrate with linear springs, is developed to better understand the mechanical interaction between the cells in the experiments. The experiments and simulations show that the cluster of cells acts collectively as a single unit, indicating that cell-cell adhesion remains strong before and after stimulation with EGF. In addition, the experiments and model emphasize the importance of three-dimensional measurements and analysis in these experiments.     

Mechanochemical manipulation of hepatocyte aggregation can selectively induce or repress liver-specific function

Controlled activation of hepatocyte aggregation is critical to three-dimensional (3D) multicellular morphogenesis during native regeneration of liver as well as tissue reconstruction therapies. In this work, we quantify the stimulatory effects of two model hepatotrophic activators, epidermal growth factor (EGF) and hepatocyte growth factor (HGF), on the aggregation kinetics and liver-specific function of hepatocytes cultured on organotypic substrates with differing mechanical resistivity. Substrate-specific morphogenesis of cultured hepatocytes is induced on a tissue basement membrane extract, Matrigel, formulated at two distinct levels of mechanical compliance (storage modulus G', at oscillatory shear rate 1 rad/s, was 34 Pa for basal Matrigel and 118 Pa for crosslinked Matrigel). Overall, we report that growth factor stimulation selectively promotes the kinetics of aggregation in the form of two-dimensional corded aggregates on basal Matrigel and three-dimensional spheroidal aggregates on crosslinked Matrigel. Our analysis also indicates that costimulation with EGF and HGF (20 ng/mL each) cooperatively maximizes the kinetics of aggregation in a substrate-specific manner. In addition, we show that the role of growth factor stimulation on hepatocyte function is sensitively governed by the mechanical compliance of the substrate. In particular, on matrices with high compliance, costimulatory aggregation is shown to elicit a marked increase in albumin secretion rate, whereas on matrices with low compliance aggregation results in effective functional repression to basal, unstimulated levels. Thus, our studies highlight a novel interplay of physicochemical parameters of the culture microenvironment, leading to selective enhancement or repression of differentiated functions of hepatocytes, in concert with the activation of cellular morphogenesis.     

Serine and Threonine Phosphorylation of the Paxillin LIM Domains Regulates Paxillin Focal Adhesion Localization and Cell Adhesion to Fibronectin

We have previously shown that the LIM domains of paxillin operate as the focal adhesion (FA)-targeting motif of this protein. In the current study, we have identified the capacity of paxillin LIM2 and LIM3 to serve as binding sites for, and substrates of serine/threonine kinases. The activities of the LIM2- and LIM3-associated kinases were stimulated after adhesion of CHO.K1 cells to fibronectin; consequently, a role for LIM domain phosphorylation in regulating the subcellular localization of paxillin after adhesion to fibronectin was investigated. An avian paxillin-CHO.K1 model system was used to explore the role of paxillin phosphorylation in paxillin localization to FAs. We found that mutations of paxillin that mimicked LIM domain phosphorylation accelerated fibronectin-induced localization of paxillin to focal contacts. Further, blocking phosphorylation of the LIM domains reduced cell adhesion to fibronectin, whereas constitutive LIM domain phosphorylation significantly increased the capacity of cells to adhere to fibronectin. The potentiation of FA targeting and cell adhesion to fibronectin was specific to LIM domain phosphorylation as mutation of the amino-terminal tyrosine and serine residues of paxillin that are phosphorylated in response to fibronectin adhesion had no effect on the rate of FA localization or cell adhesion. This represents the first demonstration of the regulation of protein localization through LIM domain phosphorylation and suggests a novel mechanism of regulating LIM domain function. Additionally, these results provide the first evidence that paxillin contributes to “inside-out” integrin-mediated signal transduction.     

Src SH2 arginine 175 is required for cell motility: specific focal adhesion kinase targeting and focal adhesion assembly function

Src kinase is a crucial mediator of adhesion-related signaling and motility. Src binds to focal adhesion kinase (FAK) through its SH2 domain and subsequently activates it for phosphorylation of downstream substrates. In addition to this binding function, data suggested that the SH2 domain might also perform an important role in targeting Src to focal adhesions (FAs) to enable further substrate phosphorylations. To examine this, we engineered an R175L mutation in cSrc to prevent the interaction with FAK pY397. This constitutively open Src kinase mediated up-regulated substrate phosphorylation in SYF cells but was unable to promote malignant transformation. Significantly, SrcR175L cells also had a profound motility defect and an impaired FA generation capacity. Importantly, we were able to recapitulate wild-type motile behavior and FA formation by directing the kinase to FAs, clearly implicating the SH2 domain in recruitment to FAK and indicating that this targeting capacity, and not simply Src-FAK scaffolding, was critical for normal Src function.     

Dynamics of cellular focal adhesions on deformable substrates: consequences for cell force microscopy

Cell focal adhesions are micrometer-sized aggregates of proteins that anchor the cell to the extracellular matrix. Within the cell, these adhesions are connected to the contractile, actin cytoskeleton; this allows the adhesions to transmit forces to the surrounding matrix and makes the adhesion assembly sensitive to the rigidity of their environment. In this article, we predict the dynamics of focal adhesions as a function of the rigidity of the substrate. We generalize previous theories and include the fact that the dynamics of proteins that adsorb to adhesions are also driven by their coupling to cell contractility and the deformation of the matrix. We predict that adhesions reach a finite size that is proportional to the elastic compliance of the substrate, on a timescale that also scales with the compliance: focal adhesions quickly reach a relatively small, steady-state size on soft materials. However, their apparent sliding is not sensitive to the rigidity of the substrate. We also suggest some experimental probes of these ideas and discuss the nature of information that can be extracted from cell force microscopy on deformable substrates.     

Fibroblast polarization is a matrix-rigidity-dependent process controlled by focal adhesion mechanosensing

Cell elongation and polarization are basic morphogenetic responses to extracellular matrix adhesion. We demonstrate here that human cultured fibroblasts readily polarize when plated on rigid, but not on compliant, substrates. On rigid surfaces, large and uniformly oriented focal adhesions are formed, whereas cells plated on compliant substrates form numerous small and radially oriented adhesions. Live-cell monitoring showed that focal adhesion alignment precedes the overall elongation of the cell, indicating that focal adhesion orientation may direct cell polarization. siRNA-mediated knockdown of 85 human protein tyrosine kinases (PTKs) induced distinct alterations in the cell polarization response, as well as diverse changes in cell traction force generation and focal adhesion formation. Remarkably, changes in rigidity-dependent traction force development, or focal adhesion mechanosensing, were consistently accompanied by abnormalities in the cell polarization response. We propose that the different stages of cell polarization are regulated by multiple, PTK-dependent molecular checkpoints that jointly control cell contractility and focal-adhesion-mediated mechanosensing.     

The regulation of osteogenesis by ECM rigidity in MC3T3-E1 cells requires MAPK activation

Once thought to provide only structural support to tissues by acting as a scaffold to which cells bind, it is now widely recognized that the extracellular matrix (ECM) provides instructive signals that dictate cell behavior. Recently we demonstrated that mechanical cues intrinsic to the ECM directly regulate the behavior of pre-osteoblastic MC3T3-E1 cells. We hypothesized that one possible mechanism by which ECM compliance exerts its influence on osteogenesis is by modulating the mitogen-activated protein kinase (MAPK) pathway. To address this hypothesis, the differentiation of MC3T3-E1 cells cultured on poly(ethylene glycol) (PEG)-based model substrates with tunable mechanical properties was assessed. Alkaline phosphatase (ALP) levels at days 7 and 14 were found to be significantly higher in cells grown on stiffer substrates (423.9 kPa hydrogels and rigid tissue culture polystyrene (TCPS) control) than on a soft hydrogel (13.7 kPa). Osteocalcin (OCN) and bone sialoprotein (BSP) gene expression levels followed a similar trend. In parallel, MAPK activity was significantly higher in cells cultured on stiffer substrates at both time points. Inhibiting this activation pharmacologically, using PD98059, resulted in significantly lower ALP levels, OCN, and BSP gene expression levels on the hydrogels. Interestingly, the effectiveness of PD98059 was itself dependent on substrate stiffness, with marked inhibition of MAPK phosphorylation in cells grown on compliant hydrogels but insignificant reduction in cells grown on TCPS. Together, these data confirm a role for MAPK in the regulation of osteogenic differentiation by ECM compliance.     

Mechanosensitive axon outgrowth mediated by L1-laminin clutch interface

Mechanical properties of the extracellular environment modulate axon outgrowth. Growth cones at the tip of extending axons generate traction force for axon outgrowth by transmitting the force of actin filament retrograde flow, produced by actomyosin contraction and F-actin polymerization, to adhesive substrates through clutch and cell adhesion molecules. A molecular clutch between the actin filament flow and substrate is proposed to contribute to cellular mechanosensing. However, the molecular identity of the clutch interface responsible for mechanosensitive growth cone advance is unknown. We previously reported that mechanical coupling between actin filament retrograde flow and adhesive substrates through the clutch molecule shootin1a and the cell adhesion molecule L1 generates traction force for axon outgrowth and guidance. Here, we show that cultured mouse hippocampal neurons extend longer axons on stiffer substrates under elastic conditions that correspond to the soft brain environments. We demonstrate that this stiffness-dependent axon outgrowth requires actin-adhesion coupling mediated by shootin1a, L1, and laminin on the substrate. Speckle imaging analyses showed that L1 at the growth cone membrane switches between two adhesive states: L1 that is immobilized and that undergoes retrograde movement on the substrate. The duration of the immobilized phase was longer on stiffer substrates; this was accompanied by increases in actin-adhesion coupling and in the traction force exerted on the substrate. These data suggest that the interaction between L1 and laminin is enhanced on stiffer substrates, thereby promoting force generation for axon outgrowth.     

Engineering hepatocyte functional fate through growth factor dynamics: the role of cell morphologic priming.

We have reported previously that cellular stimulation induced by variable mechanochemical properties of the extracellular microenvironment can significantly alter liver-specific function in cultured hepatocytes (Semler et al., Biotech Bioeng 69:359-369, 2000). Cell activation via time-invariant presentation of biochemical growth factors was found to either enhance or repress cellular differentiation of cultured hepatocytes depending on the mechanical properties of the underlying substrate. In this work, we investigated the effects of dynamic growth factor stimulation on the cell growth and differentiation behavior of hepatocytes cultured on either compliant or rigid substrates. Specifically, hepatotrophic growth factors (epidermal and hepatocyte) were either temporally added or withdrawn from hepatocyte cultures on Matrigel that was crosslinked to yield differential degrees of mechanical compliance. We determined that the functional responsiveness of hepatocytes to fluctuations in GF stimulation is substrate specific but only in conditions in which the initial mechanochemical environment induced significant cell morphogenesis. Our studies indicate that in conditions under which hepatocytes adopted a "rounded" phenotype, they exhibited increased levels of differentiated function upon soluble stimulation and markedly decreased function upon the depletion of GF stimulation. In contrast, hepatocytes that assumed a "spread" phenotype exhibited slightly increased function upon the depletion of GF stimulation. By examining the functional responsiveness of hepatocytes of differential morphology to varied fluctuations in GF activation, insights into the ability of cell shape to "prime" hepatocyte behavior in dynamic microenvironments were elucidated. We report on the possibility of uncoupling and, thus, selectively manipulating, the concerted contributions of GF-induced cellular activation and substrate- and GF-induced cell morphogenesis toward induction of cell function.     

Matrix elasticity regulates the secretory profile of human bone marrow-derived multipotent mesenchymal stromal cells (MSCs)

The therapeutic efficacy of multipotent mesenchymal stromal cells (MSCs) is attributed to particular MSC-derived cytokines and growth factors. As MSCs are applied locally to target organs or home there after systemic administration, they experience diverse microenvironments that are biochemically and biophysically distinct. Here we use well-defined in vitro conditions to study the impact of substrate elasticity on MSC-derived trophic factors. By varying hydrogel compliance, the elasticity of brain and muscle tissue was mimicked. We screened >90 secreted factors at the protein level, finding a diverse elasticity-dependent expression pattern. In particular, IL-8 was up-regulated as much as 90-fold in MSCs cultured for 2 days on hard substrates, whereas levels were consistently low on soft substrates. In summary, we show substrate elasticity directly affects MSC paracrine expression, a relevant finding for therapies administering MSCs in vivo.     

Effects of contact-induced membrane stiffening on platelet adhesion

The adhesion of platelets plays an essential role in thrombogenesis. Adhesion occurs at sites called focal adhesions (FA), where cell-membrane receptors bind specifically to substrate proteins and couple to each other and to the cytoskeleton via various cellular proteins. The resulting molecular structure suggests that the cortex stiffens at the FA, which likely affects platelet adhesion. This hypothesis is explored by structural analysis and parametric investigation. The cortex is modeled as a shell anchored to the substrate by adhesion forces and subjected to a detachment force. Equilibrium considerations result in a non-linear, two-point boundary value problem that is solved numerically. The results show that cortex stiffening significantly influences the force required for detachment as well as the cell-membrane internal stresses. The magnitude of these effects depends on the ratio of adhesion-to-bending energies and on the inclination of the detachment force. Because the cortex stiffening depends on cellular events, these results suggest a possible mechanism by which platelets can control their adhesion and protect themselves from damage.     

Identification and characterization of an ATP.Mg-dependent protein phosphatase from pig brain

Substantial amounts of ATP.Mg-dependent phosphorylase phosphatase (Fc. M) and its activator (kinase FA) were identified and extensively purified from pig brain, in spite of the fact that glycogen metabolism in the brain is of little importance. The brain Fc.M was completely inactive and could only be activated by ATP.Mg and FA, isolated either from rabbit muscle or pig brain. Kinetical analysis of the dephosphorylation of endogenous brain protein indicates that Fc.M could dephosphorylate 32P-labeled myelin basic protein (MBP) and [32P]phosphorylase alpha at a comparable rate and moreover, this associated MBP phosphatase activity was also strictly kinase FA/ATP.Mg-dependent, demonstrating that MBP is a potential substrate for Fc.M in the brain. By manipulating MBP and inhibitor-2 as specific potent phosphorylase phosphatase inhibitors, we further demonstrate that 1) Fc.M contains two distinct catalytic sites to dephosphorylate different substrates, and 2) brain MBP may be a physiological trigger involved in the regulation of protein phosphatase substrate specificity in mammalian nervous tissues.     

The ideal free distribution and bacterial growth on two substrates

A population dynamical model describing growth of bacteria on two substrates is analyzed. The model assumes that bacteria choose substrates in order to maximize their per capita population growth rate. For batch bacterial growth, the model predicts that as the concentration of the preferred substrate decreases there will be a time at which both substrates provide bacteria with the same fitness and both substrates will be used simultaneously thereafter. Preferences for either substrate are computed as a function of substrate concentrations. The predicted time of switching is calculated for some experimental data given in the literature and it is shown that the fit between predicted and observed values is good. For bacterial growth in the chemostat, the model predicts that at low dilution rates bacteria should feed on both substrates while at higher dilution rates bacteria should feed on the preferred substrate only. Adaptive use of substrates permits bacteria to survive in the chemostat at higher dilution rates when compared with non-adaptive bacteria.