Traction in smooth muscle cells varies with cell spreading

Changes in cell shape regulate cell growth, differentiation, and apoptosis. It has been suggested that the regulation of cell function by the cell shape is a result of the tension in the cytoskeleton and the distortion of the cell. Here we explore the association between cell-generated mechanical forces and the cell morphology. We hypothesized that the cell contractile force is associated with the degree of cell spreading, in particular with the cell length. We measured traction fields of single human airway smooth muscle cells plated on a polyacrylamide gel, in which fluorescent microbeads were embedded to serve as markers of gel deformation. The traction exerted by the cells at the cell-substrate interface was determined from the measured deformation of the gel. The traction was measured before and after treatment with the contractile agonist histamine, or the relaxing agonist isoproterenol. The relative increase in traction induced by histamine was negatively correlated with the baseline traction. On the contrary, the relative decrease in traction due to isoproterenol was independent of the baseline traction, but it was associated with cell shape: traction decreased more in elongated than in round cells. Maximum cell width, mean cell width, and projected area of the cell were the parameters most tightly coupled to both baseline and histamine-induced traction in this study. Wide and well-spread cells exerted larger traction than slim cells. These results suggest that cell contractility is controlled by cell spreading.     

Extracellular matrix controls myosin light chain phosphorylation and cell contractility through modulation of cell shape and cytoskeletal prestress

The mechanism by which vascular smooth muscle (VSM) cells modulate their contractility in response to structural cues from extracellular matrix remains poorly understood. When pulmonary VSM cells were cultured on increasing densities of immobilized fibronectin (FN), cell spreading, myosin light chain (MLC) phosphorylation, cytoskeletal prestress (isometric tension in the cell before vasoagonist stimulation), and the active contractile response to the vasoconstrictor endothelin-1 all increased in parallel. In contrast, MLC phosphorylation did not increase when suspended cells were allowed to bind FN-coated microbeads (4.5-microm diameter) or cultured on micrometer-sized (30 x 30 microm) FN islands surrounded by nonadhesive regions that support integrin binding but prevent cell spreading. Cell spreading and MLC phosphorylation also both decreased in parallel when the mechanical compliance of flexible FN substrates was raised. MLC phosphorylation was inhibited independently of cell shape when cytoskeletal prestress was dissipated using a myosin ATPase inhibitor in fully spread cells, whereas it increased to maximal levels when microtubules were disrupted using nocodazole in cells adherent to FN but not in suspended cells. These data demonstrate that changes in cell-extracellular matrix (ECM) interactions modulate smooth muscle cell contractility at the level of biochemical signal transduction and suggest that the mechanism underlying this regulation may involve physical interplay between ECM and the cytoskeleton, such that cell spreading and generation of cytoskeletal tension feed back to promote MLC phosphorylation and further increase tension generation.     

The dynamics and mechanics of endothelial cell spreading

Cell adhesion to extracellular matrix is mediated by receptor-ligand interactions. When a cell first contacts a surface, it spreads, exerting traction forces against the surface and forming new bonds as its contact area expands. Here, we examined the changes in shape, actin polymerization, focal adhesion formation, and traction stress generation that accompany spreading of endothelial cells over a period of several hours. Bovine aortic endothelial cells were plated on polyacrylamide gels derivatized with a peptide containing the integrin binding sequence RGD, and changes in shape and traction force generation were measured. Notably, both the rate and extent of spreading increase with the density of substrate ligand. There are two prominent modes of spreading: at higher surface ligand densities cells tend to spread isotropically, whereas at lower densities of ligand the cells tend to spread anisotropically, by extending pseudopodia randomly distributed along the cell membrane. The extension of pseudopodia is followed by periods of growth in the cell body to interconnect these extensions. These cycles occur at very regular intervals and, furthermore, the extent of pseudopodial extension can be diminished by increasing the ligand density. Measurement of the traction forces exerted by the cell reveals that a cell is capable of exerting significant forces before either notable focal adhesion or stress fiber formation. Moreover, the total magnitude of force exerted by the cell is linearly related to the area of the cell during spreading. This study is the first to monitor the dynamic changes in the cell shape, spreading rate, and forces exerted during the early stages (first several hours) of endothelial cell adhesion.     

Cell shape regulates collagen type I expression in human tendon fibroblasts

Understanding the relationship between cell shape and cellular function is important for study of cell biology in general and for regulation of cell phenotype in tissue engineering in particular. In this study, microcontact printing technique was used to create cell-adhesive rectangular and circular islands. The rectangular islands had three aspect ratios: 19.6, 4.9, and 2.2, respectively, whereas circular islands had a diameter of 50 microm. Both rectangular and circular islands had the same area of 1960 microm(2). In culture, we found that human tendon fibroblasts (HTFs) assumed the shapes of these islands. Quantitative immunofluorescence measurement showed that more elongated cells expressed higher collagen type I than did less stretched cells even though cell spreading area was the same. This suggests that HTFs, which assume an elongated shape in vivo, have optimal morphology in terms of expression of collagen type I, which is a major component of normal tendons. Using immunohistochemistry along with cell traction force microscopy (CTFM), we further found that these HTFs with different shapes exhibited variations in actin cytoskeletal structure, spatial arrangement of focal adhesions, and spatial distribution and magnitude of cell traction forces. The changes in the actin cytoskeletal structure, focal adhesion distributions, and traction forces in cells with different shapes may be responsible for altered collagen expression, as they are known to be involved in cellular mechanotransduction.     

Geometric control of switching between growth, apoptosis, and differentiation during angiogenesis using micropatterned substrates

Summary Past studies using micropatterned substrates coated with adhesive islands of extracellular matrix revealed that capillary endothelial cells can be geometrically switched between growth and apoptosis. Endothelial cells cultured on single islands larger than 1500 μm² spread and progressed through the cell cycle, whereas cells restricted to areas less than 500 μm² failed to extend and underwent apoptosis. The present study addressed whether island geometries that constrained cell spreading to intermediate degrees, neither supporting cell growth nor inducing apoptosis, cause cells to differentiate. Endothelial cells cultured on substrates micropatterned with 10-μm-wide lines of fibronectin formed extensive cell-cell contacts and spread to approximately 1000 μm². Within 72 h, cells shut off both growth and apoptosis programs and underwent differentiation, resulting in the formation of capillary tube-like structures containing a central lumen. Accumulation of extracellular matrix tendrils containing fibronectin and laminin beneath cells and reorganization of platelet endothelial cell adhesion molecule-positive cell-cell junctions along the lengths of the tubes preceded the formation of these structures. Cells cultured on wider (30-μm) lines also formed cell-cell contacts and aligned their actin cytoskeleton, but these cells spread to larger areas (2200 μm²), proliferated, and did not form tubes. Use of micropatterned substrates revealed that altering the geometry of cell spreading can switch endothelial cells among the three major genetic programs that govern angiogenesis—growth, apoptosis and differentiation. The system presented here provides a well-defined adhesive environment in which to further investigate the steps involved in angiogenesis.     

An array of microfabricated pillars to study cell migration

Mechanical forces play an important role in various cellular functions, such as tumor metastasis, embryonic development or tissue formation. Cell migration involves dynamics of adhesive processes and cytoskeleton remodelling, leading to traction forces between the cells and their surrounding extracellular medium. To study these mechanical forces, a number of methods have been developed to calculate tractions at the interface between the cell and the substrate by tracking the displacements of beads or microfabricated markers embedded in continuous deformable gels. These studies have provided the first reliable estimation of the traction forces under individual migrating cells. We have developed a new force sensor made of a dense array of soft micron-size pillars microfabricated using microelectronics techniques. This approach uses elastomeric substrates that are micropatterned by using a combination of hard and soft lithography. Traction forces are determined in real time by analyzing the deflections of each micropillar with an optical microscope. Indeed, the deflection is directly proportional to the force in the linear regime of small deformations. Epithelial cells are cultured on our substrates coated with extracellular matrix protein. First, we have characterized temporal and spatial distributions of traction forces of a cellular assembly. Forces are found to depend on their relative position in the monolayer : the strongest deformations are always localized at the edge of the islands of cells in the active areas of cell protrusions. Consequently, these forces are quantified and correlated with the adhesion/scattering processes of the cells.     

Temporal variations in cell migration and traction during fibroblast-mediated gel compaction

Current models used in our laboratory to assess the migration and traction of a population of cells within biopolymer gels are extended to investigate temporal changes in these parameters during compaction of mechanically constrained gels. The random cell migration coefficient, micro (t) is calculated using a windowing technique by regressing the mean-squared displacement of cells tracked at high magnification in three dimensions with a generalized least squares algorithm for a subset of experimental time intervals, and then shifting the window interval-by-interval until all time points are analyzed. The cell traction parameter, tau(0)(t), is determined by optimizing the solution of our anisotropic biphasic theory to tissue equivalent compaction. The windowing technique captured simulated sinusoidal and step changes in cell migration superposed on a persistent random walk in simulated cell movement. The optimization software captured simulated time dependence of compaction on cell spreading. Employment of these techniques on experimental data using rat dermal fibroblasts (RDFs) and human foreskin fibroblasts (HFFs) demonstrated that these cells exhibit different migration-traction relationships. Rat dermal fibroblast migration was negatively correlated to traction, suggesting migration was not the driving force for compaction with these cells, whereas human foreskin fibroblast migration was positively correlated to traction.     

Micropatterning of single endothelial cell shape reveals a tight coupling between nuclear volume in G1 and proliferation

Shape-dependent local differentials in cell proliferation are considered to be a major driving mechanism of structuring processes in vivo, such as embryogenesis, wound healing, and angiogenesis. However, the specific biophysical signaling by which changes in cell shape contribute to cell cycle regulation remains poorly understood. Here, we describe our study of the roles of nuclear volume and cytoskeletal mechanics in mediating shape control of proliferation in single endothelial cells. Micropatterned adhesive islands were used to independently control cell spreading and elongation. We show that, irrespective of elongation, nuclear volume and apparent chromatin decondensation of cells in G1 systematically increased with cell spreading and highly correlated with DNA synthesis (percent of cells in the S phase). In contrast, cell elongation dramatically affected the organization of the actin cytoskeleton, markedly reduced both cytoskeletal stiffness (measured dorsally with atomic force microscopy) and contractility (measured ventrally with traction microscopy), and increased mechanical anisotropy, without affecting either DNA synthesis or nuclear volume. Our results reveal that the nuclear volume in G1 is predictive of the proliferative status of single endothelial cells within a population, whereas cell stiffness and contractility are not. These findings show that the effects of cell mechanics in shape control of proliferation are far more complex than a linear or straightforward relationship. Our data are consistent with a mechanism by which spreading of cells in G1 partially enhances proliferation by inducing nuclear swelling and decreasing chromatin condensation, thereby rendering DNA more accessible to the replication machinery.     

The relative magnitudes of endothelial force generation and matrix stiffness modulate capillary morphogenesis in vitro

When suspended in collagen gels, endothelial cells elongate and form capillary-like networks containing lumens. Human blood outgrowth endothelial cells (HBOEC) suspended in relatively rigid 3 mg/ml floating collagen gels, formed in vivo-like, thin, branched multi-cellular structures with small, thick-walled lumens, while human umbilical vein endothelial cells (HUVEC) formed fewer multi-cellular structures, had a spread appearance, and had larger lumens. HBOEC exert more traction on collagen gels than HUVEC as evidenced by greater contraction of floating gels. When the stiffness of floating gels was decreased by decreasing the collagen concentration from 3 to 1.5 mg/ml, HUVEC contracted gels more and formed thin, multi-cellular structures with small lumens, similar in appearance to HBOEC in floating 3 mg/ml gels. In contrast to floating gels, traction forces exerted by cells in mechanically constrained gels encounter considerable resistance. In constrained collagen gels (3 mg/ml), both cell types appeared spread, formed structures with fewer cells, had larger, thinner-walled lumens than in floating gels, and showed prominent actin stress fibers, not seen in floating gels. These results suggest that the relative magnitudes of cellular force generation and apparent matrix stiffness modulate capillary morphogenesis in vitro and that this balance may play a role in regulating angiogenesis in vivo.     

Regulation of vascular smooth muscle cells by micropatterning

Vascular smooth muscle cells (SMCs) undergo morphological and phenotypic changes when cultured in vitro. To investigate whether SMC morphology regulates SMC functions, bovine aortic SMCs were grown on micropatterned collagen strips (50-, 30-, and 20-microm wide). The cell shape index and proliferation rate of SMCs on 30- and 20-microm strips were significantly lower than those on non-patterned collagen (control), and the spreading area was decreased only for cells patterned on the 20-microm strips, suggesting that SMC proliferation is dependent on cell shape index. The formation of actin stress fibers and the expression of alpha-actin were decreased in SMCs on the 20- and 30-microm collagen strips. SMCs cultured on micropatterned biomaterial poly-(D,L-lactide-co-glycolide) (PLGA) with 30-microm wide grooves also showed lower proliferation rate and less stress fibers than SMCs on non-patterned PLGA. Our findings suggest that micropatterned matrix proteins and topography can be used to control SMC morphology and that elongated cell morphology decreases SMC proliferation but is not sufficient to promote contractile phenotype.     

A mechanoresponsive cadherin-keratin complex directs polarized protrusive behavior and collective cell migration

Collective cell migration requires maintenance of adhesive contacts between adjacent cells, coordination of polarized cell protrusions, and generation of propulsive traction forces. We demonstrate that mechanical force applied locally to C-cadherins on single Xenopus mesendoderm cells is sufficient to induce polarized cell protrusion and persistent migration typical of individual cells within a collectively migrating tissue. Local tension on cadherin adhesions induces reorganization of the keratin intermediate filament network toward these stressed sites. Plakoglobin, a member of the catenin family, is localized to cadherin adhesions under tension and is required for both mechanoresponsive cell behavior and assembly of the keratin cytoskeleton at the rear of these cells. Local tugging forces on cadherins occur in vivo through interactions with neighboring cells, and these forces result in coordinate changes in cell protrusive behavior. Thus, cadherin-dependent force-inducible regulation of cell polarity in single mesendoderm cells represents an emergent property of the intact tissue.     

Analysis of the topological changes induced on cells exposed to adhesive or mechanical stimuli

Fluorescent probes are widely used to study cell structure and function. However, few reports were devoted to a quantitative analysis of the intracellular distribution of fluorescent markers. In the present work, we describe the topographical changes of surface and cytoskeletal markers on individual cells subjected to adhesive or mechanical interaction. Conjugates were prepared with a cytotoxic T-lymphocyte clone and target cells. Specific antigens, membrane phospholipids, surface glycoconjugates, and polymerized actin were labeled with fluorescent antibodies or biochemical probes. The analysis of fluorescence distributions in conjugates demonstrated a selective reorganization of the plasma membrane with a gathering of some molecular species in the intercellular adhesion area. Furthermore, individual phagocytic cells were sucked into glass micropipets, then stained with fluorescent phallacidin to analyze the effect of mechanical efforts on the cytoskeleton organization. The concentration of polymerized actin was found to be similar in mechanicallyinduced protrusions and whole cells. It is concluded that adhesive interactions may result in marked cell polarization and formation of membrane zones with a particular biochemical composition. The submembranar cytoskeleton might play a role in this process.     

Analysis of the topological changes induced on cells exposed to adhesive or mechanical stimuli.

Fluorescent probes are widely used to study cell structure and function. However, few reports were devoted to a quantitative analysis of the intracellular distribution of fluorescent markers. In the present work, we describe the topographical changes of surface and cytoskeletal markers on individual cells subjected to adhesive or mechanical interaction. Conjugates were prepared with a cytotoxic T-lymphocyte clone and target cells. Specific antigens, membrane phospholipids, surface glycoconjugates, and polymerized actin were labeled with fluorescent antibodies or biochemical probes. The analysis of fluorescence distributions in conjugates demonstrated a selective reorganization of the plasma membrane with a gathering of some molecular species in the intercellular adhesion area. Furthermore, individual phagocytic cells were sucked into glass micropipets, then stained with fluorescent phallacidin to analyze the effect of mechanical efforts on the cytoskeleton organization. The concentration of polymerized actin was found to be similar in mechanically-induced protrusions and whole cells. It is concluded that adhesive interactions may result in marked cell polarization and formation of membrane zones with a particular biochemical composition. The submembranar cytoskeleton might play a role in this process.     

Determining substrate displacement and cell traction fields--a new approach

This paper presents a new approach for the traction force microscopy (TFM) method which determines traction forces exerted by adherent cells on a thin, elastic polyacrylamide gel embedded with fluorescent microbeads. In this enhanced TFM method, a pattern recognition technique is first applied to match the pair of microbead embedded images before and after deformation, which subsequently provides the displacement field of the elastic substrate. Once the displacement field is obtained, the 3-D finite element method (FEM) is used to compute cell traction forces. The new TFM has been applied to determine traction forces of human tendon fibroblasts. Compared to existing TFM methods, the present method has the following advantages: (1) its displacement field obtained is associated with microbead movements; (2) it considers the finite thickness of the thin polyacrylamide gel and is therefore free from the infinite half-space approximation adopted by existing TFM methods; and (3) its computation procedure for determining cell traction forces is fast.     

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When the substrate is coated with an extracellular matrix protein, cells adhere to the gel and apply forces, causing the gel to deform. The deformation depends on the cell traction and the elastic properties of the gel. If the deformation field of the surface is known, surface traction can be calculated using elasticity theory. Gel deformation is commonly measured by embedding fluorescent marker beads uniformly into the gel. The probes displace as the gel deforms. The probes near the surface of the gel are tracked. The displacements reported by these probes are considered as surface displacements. Their depths from the surface are ignored. This assumption introduces error in traction force evaluations. For precise measurement of cell forces, it is critical for the location of the beads to be known. We have developed a technique that utilizes simple chemistry to confine fluorescent marker beads, 0.1 and 1 µm in diameter, in PA gels, within 1.6 μm of the surface. We coat a coverslip with poly-D-lysine (PDL) and fluorescent beads. PA gel solution is then sandwiched between the coverslip and an adherent surface. The fluorescent beads transfer to the gel solution during curing. After polymerization, the PA gel contains fluorescent beads on a plane close to the gel surface.     

Spatial and temporal traction response in human airway smooth muscle cells

Tractions that cells exert on theirsubstrates are essential in cell spreading, migration, and contraction.These tractions can be determined by plating the cells on a flexiblegel and measuring the deformation of the gel by using fluorescent beadsembedded just below the surface of the gel. In this article we describe the image correlation method (ICM) optimized for determining the displacement field of the gel under a contracting cell. For the calculation of the traction field from the displacement field we usethe recently developed method of Fourier transform traction cytometry(FTTC). The ICM and FTTC methods are applied to human airway smoothmuscle cells during stimulation with the contractile agonist histamineor the relaxing agonist isoproterenol. The overall intensity of thecell contraction (the median traction magnitude, the energy transferredfrom the cell to the gel, and the net contractile moment) increasedafter activation with histamine, and decreased after treatment withisoproterenol. Cells exhibited regional differences in the time courseof traction during the treatment. Both temporal evolution and magnitudeof traction increase induced by histamine varied markedly amongdifferent cell protrusions, whereas the nuclear region showed thesmallest response. These results suggest that intracellular mediatorsof cell adhesion and contraction respond to contractile stimuli withdifferent rates and intensities in different regions of the cell.

     

Critical conditions for pattern formation and in vitro tubulogenesis driven by cellular traction fields

In vitro angiogenesis assays have shown that tubulogenesis of endothelial cells within biogels, like collagen or fibrin gels, only appears for a critical range of experimental parameter values. These experiments have enabled us to develop and validate a theoretical model in which mechanical interactions of endothelial cells with extracellular matrix influence both active cell migration--haptotaxis--and cellular traction forces. Depending on the number of cells, cell motility and biogel rheological properties, various 2D endothelial patterns can be generated, from non-connected stripe patterns to fully connected networks, which mimic the spatial organization of capillary structures. The model quantitatively and qualitatively reproduces the range of critical values of cell densities and fibrin concentrations for which these cell networks are experimentally observed. We illustrate how cell motility is associated to the self-enhancement of the local traction fields exerted within the biogel in order to produce a pre-patterning of this matrix and subsequent formation of tubular structures, above critical thresholds corresponding to bifurcation points of the mathematical model. The dynamics of this morphogenetic process is discussed in the light of videomicroscopy time lapse sequences of endothelial cells (EAhy926 line) in fibrin gels. Our modeling approach also explains how the progressive appearance and morphology of the cellular networks are modified by gradients of extracellular matrix thickness.     

The Role of Cell Contraction and Adhesion in Dictyostelium Motility

The crawling motion of Dictyostelium discoideum on substrata involves a number of coordinated events including cell contractions and cell protrusions. The mechanical forces exerted on the substratum during these contractions have recently been quantified using traction force experiments. Based on the results from these experiments, we present a biomechanical model of the contraction phase of Dictyostelium discoideum motility with an emphasis on the adhesive properties of the cell-substratum contact. Our model assumes that the cell contracts at a constant rate and is bound to the substratum by adhesive bridges that are modeled as elastic springs. These bridges are established at a spatially uniform rate while detachment occurs at a spatially varying, load-dependent rate. Using Monte Carlo simulations and assuming a rigid substratum, we find that the cell speed depends only weakly on the detachment kinetics of the cell-substratum interface, in agreement with experimental data. By varying the parameters that control the adhesive and contractile properties of the cell, we are able to make testable predictions. We also extend our model to include a flexible substrate and show that our model is able to produce substratum deformations and force patterns that are quantitatively and qualitatively in agreement with experimental data.     

Apoptosis of syncytia induced by the HIV-1-envelope glycoprotein complex: influence of cell shape and size

Cells stably transfected with a lymphotropic HIV-1 Env gene form syncytia when cocultured with CD4(+)CXCR4(+) cells. Heterokaryons then spontaneously undergo apoptosis, while manifesting signs of mitochondrial membrane pemeabilization as well as nuclear chromatin condensation. Modulation of cellular geometry was achieved by growing syncytia on self-assembled monolayers of terminally substituted alkanethiolates designed to control the adhesive properties of the substrates. Spreading of syncytia, induced by culturing them on small circular adhesive islets (diameter 5 microm), placed at a distance that cells can bridge (10 microm), inhibited spontaneous and staurosporin-induced signs of apoptosis, both at the mitochondrial and at the nuclear levels, and allowed for the generation of larger syncytia. Transient cell spreading conferred a memory of apoptosis inhibition which was conserved upon adoption of a conventional cell shape. Limiting syncytium size by culturing them on square-shaped planar adhesive islands of defined size (400 to 2500 microm(2)), separated by nonadhesive regions, enhanced the rate of apoptotic cell death, as indicated by an accelerated permeabilization of the outer mitochondrial membrane, loss of the mitochondrial inner transmembrane potential, and an increased frequency of nuclear apoptosis. In conclusion, external constraints on syncytial size and shape strongly modulate their propensity to undergo apoptosis.     

Improved-Throughput Traction Microscopy Based on Fluorescence Micropattern for Manual Microscopy

Traction force microscopy (TFM) is a quantitative technique for measuring cellular traction force, which is important in understanding cellular mechanotransduction processes. Traditional TFM has a significant limitation in that it has a low measurement throughput, commonly one per TFM dish, due to a lack of cell position information. To obtain enough cellular traction force data, an onerous workload is required including numerous TFM dish preparations and heavy cell-seeding activities, creating further difficulty in achieving identical experimental conditions among batches. In this paper, we present an improved-throughput TFM method using the well-developed microcontact printing technique and chemical modifications of linking microbeads to the gel surface to address these limitations. Chemically linking the microbeads to the gel surface has no significant influence on cell proliferation, morphology, cytoskeleton, and adhesion. Multiple pairs of force loaded and null force fluorescence images can be easily acquired by means of manual microscope with the aid of a fluorescence micropattern made by microcontact printing. Furthermore, keeping the micropattern separate from cells by using gels effectively eliminates the potential negative effect of the micropattern on the cells. This novel design greatly improves the analysis throughput of traditional TFM from one to at least twenty cells per petri dish without losing unique advantages, including a high spatial resolution of traction measurements. This newly developed method will boost the investigation of cell-matrix mechanical interactions.