Strength Dependence of Cadherin-Mediated Adhesions

Traction forces between adhesive cells play an important role in a number of collective cell processes. Intercellular contacts, in particular cadherin-based intercellular junctions, are the major means of transmitting force within tissues. We investigated the effect of cellular tension on the formation of cadherin-cadherin contacts by spreading cells on substrates with tunable stiffness coated with N-cadherin homophilic ligands. On the most rigid substrates, cells appear well-spread and present cadherin adhesions and cytoskeletal organization similar to those classically observed on cadherin-coated glass substrates. However, when cells are cultured on softer substrates, a change in morphology is observed: the cells are less spread, with a more disorganized actin network. A quantitative analysis of the cells adhering on the cadherin-coated surfaces shows that forces are correlated with the formation of cadherin adhesions. The stiffer the substrates, the larger are the average traction forces and the more developed are the cadherin adhesions. When cells are treated with blebbistatin to inhibit myosin II, the forces decrease and the cadherin adhesions disappear. Together, these findings are consistent with a mechanosensitive regulation of cadherin-mediated intercellular junctions through the cellular contractile machinery.     

A model for cell motility on soft bio-adhesive substrates

Mechanical stiffness of bio-adhesive substrates has been recognized as a major regulator of cell motility. We present a simple physical model to study the crawling locomotion of a contractile cell on a soft elastic substrate. The mechanism of rigidity sensing is accounted for using Schwarz's two-spring model Schwarz et al. (2006). The predicted dependency between the speed of motility and substrate stiffness is qualitatively consistent with experimental observations. The model demonstrates that the rigidity dependent motility of cells is rooted in the regulation of actomyosin contractile forces by substrate deformation at each anchorage point. On stiffer substrates, the traction forces required for cell translocation acquire larger magnitude but show weaker asymmetry which leads to slower cell motility. On very soft substrates, the model predicts a biphasic relationship between the substrate rigidity and the speed of locomotion, over a narrow stiffness range, which has been observed experimentally for some cell types.     

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.     

Decoupling Substrate Stiffness, Spread Area, and Micropost Density: A Close Spatial Relationship between Traction Forces and Focal Adhesions

Mechanical cues can influence the manner in which cells generate traction forces and form focal adhesions. The stiffness of a cell's substrate and the available area on which it can spread can influence its generation of traction forces, but to what extent these factors are intertwined is unclear. In this study, we used microcontact printing and micropost arrays to control cell spreading, substrate stiffness, and post density to assess their effect on traction forces and focal adhesions. We find that both the spread area and the substrate stiffness influence traction forces in an independent manner, but these factors have opposite effects: cells on stiffer substrates produce higher average forces, whereas cells with larger spread areas generate lower average forces. We show that post density influences the generation of traction forces in a manner that is more dominant than the effect of spread area. Additionally, we observe that focal adhesions respond to spread area, substrate stiffness, and post density in a manner that closely matches the trends seen for traction forces. This work supports the notion that traction forces and focal adhesions have a close relationship in their response to mechanical cues.     

Calpain 2 controls turnover of LFA-1 adhesions on migrating T lymphocytes

The immune cells named T lymphocytes circulate around the body fulfilling their role in immunosurveillance by monitoring the tissues for injury or infection. To migrate from the blood into the tissues, they make use of the integrin LFA-1 which is exclusively expressed by immune cells. These highly motile cells attach and migrate on substrates expressing the LFA-1 ligand ICAM-1. The molecular events signaling LFA-1 activation and adhesion are now reasonably well identified, but the process of detaching LFA-1 adhesions is less understood. The cysteine protease calpain is involved in turnover of integrin-mediated adhesions in less motile cell types. In this study we have explored the involvement of calpain in turnover of LFA-1-mediated adhesions of T lymphocytes. Using live cell imaging and immunohistochemistry, we demonstrate that turnover of adhesions depends on the Ca2+-dependent enzyme, calpain 2. Inhibition of calpain activity by means of siRNA silencing or pharmacological inhibition results in inefficient disassembly of LFA-1 adhesions causing T lymphocyte elongation and shedding of LFA-1 clusters behind the migrating T lymphocytes. We show that calpain 2 is distributed throughout the T lymphocyte, but is most active at the trailing edge as detected by expression of its fluorescent substrate CMAC,t-BOC-Leu-Met. Extracellular Ca2+ entry is essential for the activity of calpain 2 that is constantly maintained as the T lymphocytes migrate. Use of T cells from a patient with mutation in ORAI1 revealed that the major calcium-release-activated-calcium channel is not the ion channel delivering the Ca2+. We propose a model whereby Ca2+ influx, potentially through stretch activated channels, is sufficient to activate calpain 2 at the trailing edge of a migrating T cell and this activity is essential for the turnover of LFA-1 adhesions.     

NBT-II cell locomotion is modulated by restricting the size of focal contacts and is improved through EGF and ROCK signaling

Focal contacts, large macromolecular complexes that link the extracellular matrix and the internal cell cytoskeleton, are thought to govern cell locomotion. However, the maturation process through which focal contacts control the cellular migratory machinery by changes in size and molecular composition remain unclear. Here, we fabricated cell growth substrates that contained linear ECM strips of micron- or submicron-width in order to limit the enlargement of focal contacts. We found that NBT-II cells plated on the submicron substrate possessed smaller focal complexes that exhibited a highly dynamic turnover. These cells possessed various leading edges at multiple sites of the cell periphery, which prevented the cell from advancing. In contrast, cells grown on the micron-width substrate possessed large and stable focal adhesions. Most of these cells were elongated bipolar cells that were tethered at both ends and were immobile. Further, EGF and ROCK signaling pathways can modulate the cellular migratory responses according to the substrate guidance. On the submicron-width substrate, EGF treatment increased the focal contact size and the contractile force, causing these cells to develop one leading edge and migrate along the submicron-sized ECM paths. In contrast, inhibition of ROCK signaling decreased the focal contact size for cells plated on the micron substrate. These cells became less tethered and were able to migrate along or even across the micron-sized ECM paths. Our results indicate that formation and maturation of focal contacts is controlled by both ECM cues and intracellular signaling and they play a central role in directed cell motion.     

Collagen type V modulates fibroblast behavior dependent on substrate stiffness

Collagen type V is highly expressed during tissue development and wound repair, but its exact function remains unclear. Cell binding to collagen V affects various basic cell functions and increased collagen V levels alter the structural organization and the stiffness of the ECM. We studied the combined effects of collagen V and substrate stiffness on the morphology, focal adhesion formation, and actin organization of fibroblasts. We found that a hybrid collagen I/V coating impairs fibroblast spreading on soft substrates (<10 kPa), but not on stiffer substrates (68 kPa or glass). In sharp contrast, a pure collagen I coating does not impair cell spreading on soft substrates. The impairment of cell spreading by collagen V is accompanied by diffuse actin staining patterns and small focal adhesions. These observations suggest that collagen V plays an essential role in modifying cell behavior during development and remodeling, when very soft tissues are present.     

Substrate Topography Induces a Crossover from 2D to 3D Behavior in Fibroblast Migration

In a three-dimensional environment, cells migrate through complex topographical features. Using microstructured substrates, we investigate the role of substrate topography in cell adhesion and migration. To do so, fibroblasts are plated on chemically identical substrates composed of microfabricated pillars. When the dimensions of the pillars (i.e., the diameter, length, and spacing) are varied, migrating cells encounter alternating flat and rough surfaces that depend on the spacing between the pillars. Consequently, we show that substrate topography affects cell shape and migration by modifying cell-to-substrate interactions. Cells on micropillar substrates exhibit more elongated and branched shapes with fewer actin stress fibers compared with cells on flat surfaces. By analyzing the migration paths in various environments, we observe different mechanisms of cell migration, including a persistent type of migration, that depend on the organization of the topographical features. These responses can be attributed to a spatial reorganization of the actin cytoskeleton due to physical constraints and a preferential formation of focal adhesions on the micropillars, with an increased lifetime compared to that observed on flat surfaces. By changing myosin II activity, we show that actomyosin contractility is essential in the cellular response to micron-scale topographic signals. Finally, the analysis of cell movements at the frontier between flat and micropillar substrates shows that cell transmigration through the micropillar substrates depends on the spacing between the pillars.     

Both contractile axial and lateral traction force dynamics drive amoeboid cell motility

Chemotaxing Dictyostelium discoideum cells adapt their morphology and migration speed in response to intrinsic and extrinsic cues. Using Fourier traction force microscopy, we measured the spatiotemporal evolution of shape and traction stresses and constructed traction tension kymographs to analyze cell motility as a function of the dynamics of the cell’s mechanically active traction adhesions. We show that wild-type cells migrate in a step-wise fashion, mainly forming stationary traction adhesions along their anterior–posterior axes and exerting strong contractile axial forces. We demonstrate that lateral forces are also important for motility, especially for migration on highly adhesive substrates. Analysis of two mutant strains lacking distinct actin cross-linkers (mhcA⁻ and abp120⁻ cells) on normal and highly adhesive substrates supports a key role for lateral contractions in amoeboid cell motility, whereas the differences in their traction adhesion dynamics suggest that these two strains use distinct mechanisms to achieve migration. Finally, we provide evidence that the above patterns of migration may be conserved in mammalian amoeboid cells.     

Guidance of Cell Migration by Substrate Dimension

There is increasing evidence to suggest that physical parameters, including substrate rigidity, topography, and cell geometry, play an important role in cell migration. As there are significant differences in cell behavior when cultured in 1D, 2D, or 3D environments, we hypothesize that migrating cells are also able to sense the dimension of the environment as a guidance cue. NIH 3T3 fibroblasts were cultured on micropatterned substrates where the path of migration alternates between 1D lines and 2D rectangles. We found that 3T3 cells had a clear preference to stay on 2D rather than 1D substrates. Cells on 2D surfaces generated stronger traction stress than did those on 1D surfaces, but inhibition of myosin II caused cells to lose their sensitivity to substrate dimension, suggesting that myosin-II-dependent traction forces are the determining factor for dimension sensing. Furthermore, oncogene-transformed fibroblasts are defective in mechanosensing while generating similar traction forces on 1D and 2D surfaces. Dimension sensing may be involved in guiding cell migration for both physiological functions and tissue engineering, and for maintaining normal cells in their home tissue.     

Embryonal carcinoma cell adhesion: the role of surface galactosyltransferase and its 90K lactosaminoglycan substrate

Embryonal carcinoma (EC) cells possess a complex cell surface glycoconjugate called lactosaminoglycan, whose core structure is composed of repeating N-acetyllactosamine (Gal leads to GlcNAc) disaccharides. Recent studies suggest that the cell surface receptor for lactosaminoglycan is galactosyltransferase, which binds terminal GlcNAc residues on various side chains, thus anchoring the glycoconjugate to the cell surface (Shur, B. D. (1982). J. Biol. Chem. 257, 6871-6878.). The results described in this paper suggest that multivalent lactosaminoglycans mediate EC cell adhesions by binding to their surface galactosyltransferase receptors. In the presence of UDPgalactose, but not other sugar nucleotides, EC cell adhesion is reduced and preformed cell adhesions are dissociated. UDPgalactose interferes with EC cell adhesion by forcing the galactosyltransferase reaction to completion, thus dissociating the enzyme from its galactosylated substrate (i.e., lactosaminoglycan), and thereby dissociating EC cells from one another. Lactosaminoglycans purified from EC cell cultures rapidly agglutinate EC cells, and EC cells preferentially adhere to substrates irreversibly derivatized with protein- and lipid-free lactosaminoglycan side chains. Under identical conditions, EC cells do not adhere to either hyaluronate- or chondroitin sulfate-derivatized substrates, relative to underivatized control surfaces. EC cell adhesion to other cells and to lactosaminoglycan-derivatized surfaces can be inhibited by reagents that selectively interfere with surface galactosyltransferase activity. First, alpha-lactalbumin specifically reduces the galactosyltransferase's affinity for its lactosaminoglycan substrate and simultaneously inhibits adhesion. Similar levels of bovine serum albumin have no effect. Second, selective inhibition of surface galactosyltransferase with UDP-dialdehyde also inhibits adhesion, while similar levels of AMP-dialdehyde do not. Results show that 1 mM Ca2+ protects the surface galactosyltransferase activity from proteolysis, which suggests the galactosyltransferase is one of the Ca2+-dependent EC cell adhesion molecules. SDS-PAGE fluorography and gel chromatography analyses have determined that the principal lactosaminoglycan substrate for EC surface galactosyltransferase has an apparent molecular weight of 90K. Taken together, these results suggest that lactosaminoglycans participate in EC cell adhesion by binding to their surface galactosyltransferase receptors.     

A contractile and counterbalancing adhesion system controls the 3D shape of crawling cells

How adherent and contractile systems coordinate to promote cell shape changes is unclear. Here, we define a counterbalanced adhesion/contraction model for cell shape control. Live-cell microscopy data showed a crucial role for a contractile meshwork at the top of the cell, which is composed of actin arcs and myosin IIA filaments. The contractile actin meshwork is organized like muscle sarcomeres, with repeating myosin II filaments separated by the actin bundling protein α-actinin, and is mechanically coupled to noncontractile dorsal actin fibers that run from top to bottom in the cell. When the meshwork contracts, it pulls the dorsal fibers away from the substrate. This pulling force is counterbalanced by the dorsal fibers’ attachment to focal adhesions, causing the fibers to bend downward and flattening the cell. This model is likely to be relevant for understanding how cells configure themselves to complex surfaces, protrude into tight spaces, and generate three-dimensional forces on the growth substrate under both healthy and diseased conditions.     

A Novel Cell Traction Force Microscopy to Study Multi-Cellular System

Traction forces exerted by adherent cells on their microenvironment can mediate many critical cellular functions. Accurate quantification of these forces is essential for mechanistic understanding of mechanotransduction. However, most existing methods of quantifying cellular forces are limited to single cells in isolation, whereas most physiological processes are inherently multi-cellular in nature where cell-cell and cell-microenvironment interactions determine the emergent properties of cell clusters. In the present study, a robust finite-element-method-based cell traction force microscopy technique is developed to estimate the traction forces produced by multiple isolated cells as well as cell clusters on soft substrates. The method accounts for the finite thickness of the substrate. Hence, cell cluster size can be larger than substrate thickness. The method allows computing the traction field from the substrate displacements within the cells'' and clusters'' boundaries. The displacement data outside these boundaries are not necessary. The utility of the method is demonstrated by computing the traction generated by multiple monkey kidney fibroblasts (MKF) and human colon cancerous (HCT-8) cells in close proximity, as well as by large clusters. It is found that cells act as individual contractile groups within clusters for generating traction. There may be multiple of such groups in the cluster, or the entire cluster may behave a single group. Individual cells do not form dipoles, but serve as a conduit of force (transmission lines) over long distances in the cluster. The cell-cell force can be either tensile or compressive depending on the cell-microenvironment interactions.     

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.     

Adhesion dynamics and durotaxis in migrating cells

When tissue cells are plated on a flexible substrate, durotaxis, the directed migration of cells toward mechanically stiff regions, has been observed. Environmental mechanical signals are not only important in cell migration but also seem to influence all aspects of cell differentiation and development, including the metastatic process in cancer cells. Based on a theoretical model suggesting that this mechanosensation has a mechanical basis, we introduce a simple model of a cell by considering the contraction of F-actin bundles containing myosin motors (stress fibers) mediated by the movement of adhesions. We show that, when presented with a linear stiffness gradient, this simple model exhibits durotaxis. Interestingly, since stress fibers do not form on soft surfaces and since adhesion sliding occurs very slowly on hard surfaces, the model predicts that the expected cell velocity reaches a maximum at an intermediate stiffness. This prediction can be experimentally tested. We therefore argue that stiffness-dependent cellular adaptations (mechanosensation) and durotaxis are intimately related and may share a mechanical basis. We therefore identify the essential physical ingredients, which combined with additional biochemical mechanisms can explain durotaxis and mechanosensation in cells.     

Laminin alpha subunits and their role in C. elegans development

Laminins are heterotrimeric (alpha/beta/gamma) glycoproteins that form a major polymer within basement membranes. Different alpha, beta and gamma subunits can assemble into various laminin isoforms that have different, but often overlapping, distributions and functions. In this study, we examine the contributions of the laminin alpha subunits to the development of C. elegans. There are two alpha, one beta and one gamma laminin subunit, suggesting two laminin isoforms that differ by their alpha subunit assemble in C. elegans. We find that near the end of gastrulation and before other basement membrane components are detected, the alpha subunits are secreted between primary tissue layers and become distributed in different patterns to the surfaces of cells. Mutations in either alpha subunit gene cause missing or disrupted extracellular matrix where the protein normally localizes. Cell-cell adhesions are abnormal: in some cases essential cell-cell adhesions are lacking, while in other cases, cells inappropriately adhere to and invade neighboring tissues. Using electron microscopy, we observe adhesion complexes at improper cell surfaces and disoriented cytoskeletal filaments. Cells throughout the animal show defective differentiation, proliferation or migration, suggesting a general disruption of cell-cell signaling. The results suggest a receptor-mediated process localizes each secreted laminin to exposed cell surfaces and that laminin is crucial for organizing extracellular matrix, receptor and intracellular proteins at those surfaces. We propose this supramolecular architecture regulates adhesions and signaling between adjacent tissues.     

Epithelia migration: A spatiotemporal interplay between contraction and adhesion

Epithelial tissues represent 60% of the cells that form the human body and where more than 90% of all cancers derived. Epithelia transformation and migration involve altered cell contractile mechanics powered by an actomyosin-based cytoskeleton and influenced by cell-cell and cell-extracellular matrix interactions. A balance between contractile and adhesive forces regulates a large number of cellular and tissue properties crucial for epithelia migration and tumorigenesis. In this review, the forces driving normal epithelia transformation into highly motile and invasive cells and tissues will be discussed.     

Direct,dynamic assessment of cell-matrix interactions inside fibrillar collagen lattices

Cell mechanical behavior has traditionally been studied using 2-D planar elastic substrates. The goal of this study was to directly assess cell-matrix mechanical interactions inside more physiologic 3-D collagen matrices. Rabbit corneal fibroblasts transfected to express GFP-zyxin were plated at low density inside 100 micro m-thick type I collagen matrices. 3-D datasets of isolated cells were acquired at 1-3-min intervals for up to 5 h using fluorescent and Nomarski DIC imaging. Unlike cells on 2-D substrates, cells inside the collagen matrices had a bipolar morphology with thin pseudopodial processes, and without lamellipodia. The organization of the collagen fibrils surrounding each cell was clearly visualized using DIC. Using time-lapse color overlays of GFP and DIC images, displacement and/or realignment of collagen fibrils by focal adhesions could be directly visualized. During pseudopodial extension, new focal adhesions often formed in a line along collagen fibrils in front of the cell, while existing adhesions moved backward. This process generated tractional forces as indicated by the pulling in of collagen fibrils in front of the cell. Meanwhile, adhesions on both the dorsal and ventral surface of the cell body generally moved forward, resulting in contractile shortening along the pseudopodia and localized extracellular matrix (ECM) compression. Cytochalasin D induced rapid disassembly of focal adhesions, cell elongation, and ECM relaxation. This experimental model allows direct, dynamic assessment of cell-matrix interactions inside a 3-D fibrillar ECM. The data suggest that adhesions organize along actin-based contractile elements that are much less complex than the network of actin filaments that mechanically links lamellar adhesions on 2-D substrates.     

Mechanical force affects expression of an in vitro metastasis-like phenotype in HCT-8 cells

Cancer deaths are primarily caused by metastases, not by the parent tumor. During metastasis, malignant cells detach from the parent tumor, and spread through the circulatory system to invade new tissues and organs. The physical-chemical mechanisms and parameters within the cellular microenvironment that initiate the onset of metastasis, however, are not understood. Here we show that human colon carcinoma (HCT-8) cells can exhibit a dissociative, metastasis-like phenotype (MLP) in vitro when cultured on substrates with appropriate mechanical stiffness. This rather remarkable phenotype is observed when HCT-8 cells are cultured on gels with intermediate-stiffness (physiologically relevant 21-47 kPa), but not on very soft (1 kPa) and very stiff (3.6 GPa) substrates. The cell-cell adhesion molecule E-Cadherin, a metastasis hallmark, decreases 4.73 ± 1.43 times on cell membranes in concert with disassociation. Both specific and nonspecific cell adhesion decrease once the cells have disassociated. After reculturing the disassociated cells on fresh substrates, they retain the disassociated phenotype regardless of substrate stiffness. Inducing E-Cadherin overexpression in MLP cells only partially reverses the MLP phenotype in a minority population of the dissociated cells. This important experiment reveals that E-Cadherin does not play a significant role in the upstream regulation of the mechanosensing cascade. Our results indicate, during culture on the appropriate mechanical microenvironment, HCT-8 cells undergo a stable cell-state transition with increased in vitro metastasis-like characteristics as compared to parent cells grown on standard, very stiff tissue culture dishes. Nuclear staining reveals that a large nuclear deformation (major/minor axis ratio, 2:5) occurs in HCT-8 cells when cells are cultured on polystyrene substrates, but it is markedly reduced (ratio, 1:3) in cells grown on 21 kPa substrates, suggesting the cells are experiencing different intracellular forces when grown on stiff as compared to soft substrates. Furthermore, MLP can be inhibited by blebbistatin, which inactivates myosin II activity and relaxes intracellular forces. This novel finding suggests that the onset of metastasis may, in part, be linked to the intracellular forces and the mechanical microenvironment of the tumor.     

Distinct Roles of Frontal and Rear Cell-Substrate Adhesions in Fibroblast Migration

Cell migration involves complex physical and chemical interactions with the substrate. To probe the mechanical interactions under different regions of migrating 3T3 fibroblasts, we have disrupted cell-substrate adhesions by local application of the GRGDTP peptide, while imaging stress distribution on the substrate with traction force microscopy. Both spontaneous and GRGDTP-induced detachment of the trailing edge caused extensive cell shortening, without changing the overall level of traction forces or the direction of migration. In contrast, disruption of frontal adhesions caused dramatic, global loss of traction forces before any significant shortening of the cell. Although traction forces and cell migration recovered within 10-20 min of transient frontal treatment, persistent treatment with GRGDTP caused the cell to develop traction forces elsewhere and reorient toward a new direction. We conclude that contractile forces of a fibroblast are transmitted to the substrate through two distinct types of adhesions. Leading edge adhesions are unique in their ability to transmit active propulsive forces. Their functions cannot be transferred directly to existing adhesions upon detachment. Trailing end adhesions create passive resistance during cell migration and readily redistribute their loads upon detachment. Our results indicate the distinct nature of mechanical interactions at the leading versus trailing edges, which together generate the mechanical interactions for fibroblast migration.