Filamin A Is Essential for Active Cell Stiffening but not Passive Stiffening under External Force

The material properties of a cell determine how mechanical forces are transmitted through and sensed by that cell. Some types of cells stiffen passively under large external forces, but they can also alter their own stiffness in response to the local mechanical environment or biochemical cues. Here we show that the actin-binding protein filamin A is essential for the active stiffening of cells plated on collagen-coated substrates. This appears to be due to a diminished capability to build up large internal contractile stresses in the absence of filamin A. To show this, we compare the material properties and contractility of two human melanoma cell lines that differ in filamin A expression. The filamin A-deficient M2 cells are softer than the filamin A-replete A7 cells, and exert much smaller contractile stresses on the substratum, even though the M2 cells have similar levels of phosphorylated myosin II light chain and only somewhat diminished adhesion strength. In contrast to A7 cells, the stiffness and contractility of M2 cells are insensitive to either myosin-inhibiting drugs or the stiffness of the substratum. Surprisingly, however, filamin A is not required for passive stiffening under large external forces.     

Stresses at the cell-to-substrate interface during locomotion of fibroblasts

Recent technological improvements in the elastic substrate method make it possible to produce spatially resolved measurements of the tractions exerted by single motile cells. In this study we have applied these developments to produce maps of the tractions exerted by 3T3 fibroblasts during steady locomotion. The resulting images have a spatial resolution of approximately 5 micrometers and a maximum intensity of approximately 10(2) kdyn/cm2 (10(4) pN/micrometers2). We find that the propulsive thrust for fibroblast locomotion, approximately 0.2 dyn, is imparted to the substratum within 15 micrometers of the leading edge. These observations demonstrate that the lamellipodium of the fibroblast is able to generate intense traction stress. The cell body and posterior seem to be mechanically passive structures pulled forward entirely by this action.     

Assessing adhesion forces of type I and type IV pili of Xylella fastidiosa bacteria by use of a microfluidic flow chamber

Xylella fastidiosa, a bacterium responsible for Pierce's disease in grapevines, possesses both type I and type IV pili at the same cell pole. Type IV pili facilitate twitching motility, and type I pili are involved in biofilm development. The adhesiveness of the bacteria and the roles of the two pili types in attachment to a glass substratum were evaluated using a microfluidic flow chamber in conjunction with pilus-defective mutants. The average adhesion force necessary to detach wild-type X. fastidiosa cells was 147 +/- 11 pN. Mutant cells possessing only type I pili required a force of 204 +/- 22 pN for removal, whereas cells possessing only type IV pili required 119 +/- 8 pN to dislodge these cells. The experimental results demonstrate that microfluidic flow chambers are useful and convenient tools for assessing the drag forces necessary for detaching bacterial cells and that with specific pilus mutants, the role of the pilus type can be further assessed.     

Forces exerted by locomoting cells

The movement of cells along substrata is a complex phenomenon involving cell extension and retraction, and cell-substratum adhesion. Knowledge is beginning to accumulate about the forces required for cell protrusion and retraction. Both of these processes also require traction to be exerted on the substratum, and new assays for these forces are under development. This review briefly discusses the forces that locomotory cells exert, in terms of both recent force measurements and possible mechanisms for their generation.     

Three-dimensional cellular deformation analysis with a two-photon magnetic manipulator workstation

The ability to apply quantifiable mechanical stresses at the microscopic scale is critical for studying cellular responses to mechanical forces. This necessitates the use of force transducers that can apply precisely controlled forces to cells while monitoring the responses noninvasively. This paper describes the development of a micromanipulation workstation integrating two-photon, three-dimensional imaging with a high-force, uniform-gradient magnetic manipulator. The uniform-gradient magnetic field applies nearly uniform forces to a large cell population, permitting statistical quantification of select molecular responses to mechanical stresses. The magnetic transducer design is capable of exerting over 200 pN of force on 4.5-microm-diameter paramagnetic particles and over 800 pN on 5.0-microm ferromagnetic particles. These forces vary within +/-10% over an area 500 x 500 microm2. The compatibility with the use of high numerical aperture (approximately 1.0) objectives is an integral part of the workstation design allowing submicron-resolution, three-dimensional, two-photon imaging. Three-dimensional analyses of cellular deformation under localized mechanical strain are reported. These measurements indicate that the response of cells to large focal stresses may contain three-dimensional global deformations and show the suitability of this workstation to further studying cellular response to mechanical stresses.     

Traction forces generated by locomoting keratocytes

Traction forces produced by moving fibroblasts have been observed as distortions in flexible substrata including wrinkling of thin, silicone rubber films. Traction forces generated by fibroblast lamellae were thought to represent the forces required to move the cell forwards. However, traction forces could not be detected with faster moving cell types such as leukocytes and growth cones (Harris, A. K., D. Stopak, and P. Wild. 1981. Nature (Lond.). 290:249-251). We have developed a new assay in which traction forces produced by rapidly locomoting fish keratocytes can be detected by the two-dimensional displacements of small beads embedded in the plane of an elastic substratum. Traction forces were not detected at the rapidly extending front edge of the cell. Instead the largest traction forces were exerted perpendicular to the left and right cell margins. The maximum traction forces exerted by keratocytes were estimated to be approximately 2 x 10(-8) N. The pattern of traction forces can be related to the locomotion of a single keratocyte in terms of lamellar contractility and area of close cell- substratum contact.     

Assessing Adhesion Forces of Type I and Type IV Pili of Xylella fastidiosa Bacteria by Use of a Microfluidic Flow Chamber

Xylella fastidiosa, a bacterium responsible for Pierce's disease in grapevines, possesses both type I and type IV pili at the same cell pole. Type IV pili facilitate twitching motility, and type I pili are involved in biofilm development. The adhesiveness of the bacteria and the roles of the two pili types in attachment to a glass substratum were evaluated using a microfluidic flow chamber in conjunction with pilus-defective mutants. The average adhesion force necessary to detach wild-type X. fastidiosa cells was 147 ± 11 pN. Mutant cells possessing only type I pili required a force of 204 ± 22 pN for removal, whereas cells possessing only type IV pili required 119 ± 8 pN to dislodge these cells. The experimental results demonstrate that microfluidic flow chambers are useful and convenient tools for assessing the drag forces necessary for detaching bacterial cells and that with specific pilus mutants, the role of the pilus type can be further assessed.     

Micro magnetic tweezers for nanomanipulation inside live cells

This study reports the design, realization, and characterization of a multi-pole magnetic tweezers that enables us to maneuver small magnetic probes inside living cells. So far, magnetic tweezers can be divided into two categories: I), tweezers that allow the exertion of high forces but consist of only one or two poles and therefore are capable of only exerting forces in one direction; and II), tweezers that consist of multiple poles and allow exertion of forces in multiple directions but at very low forces. The magnetic tweezers described here combines both aspects in a single apparatus: high forces in a controllable direction. To this end, micron scale magnetic structures are fabricated using cleanroom technologies. With these tweezers, magnetic flux gradients of nablaB = 8 x 10(3) T m(-1) can be achieved over the dimensions of a single cell. This allows exertion of forces up to 12 pN on paramagnetic probes with a diameter of 350 nm, enabling us to maneuver them through the cytoplasm of a living cell. It is expected that with the current tweezers, picoNewton forces can be exerted on beads as small as 100 nm.     

Mechanics of cellular adhesion to artificial artery templates

We are using polymer templates to grow artificial artery grafts in vivo for the replacement of diseased blood vessels. We have previously shown that adhesion of macrophages to the template starts the graft formation. We present a study of the mechanics of macrophage adhesion to these templates on a single cell and single bond level with optical tweezers. For whole cells, in vitro cell adhesion densities decreased significantly from polymer templates polyethylene to silicone to Tygon (167, 135, and 65 cells/mm(2)). These cell densities were correlated with the graft formation success rate (50%, 25%, and 0%). Single-bond rupture forces at a loading rate of 450 pN/s were quantified by adhesion of trapped 2-microm spheres to macrophages. Rupture force distributions were dominated by nonspecific adhesion (forces <40 pN). On polystyrene, preadsorption of fibronectin or presence of serum proteins in the cell medium significantly enhanced adhesion strength from a mean rupture force of 20 pN to 28 pN or 33 pN, respectively. The enhancement of adhesion by fibronectin and serum is additive (mean rupture force of 43 pN). The fraction of specific binding forces in the presence of serum was similar for polystyrene and polymethyl-methacrylate, but specific binding forces were not observed for silica. Again, we found correlation to in vivo experiments, where the density of adherent cells is higher on polystyrene than on silica templates, and can be further enhanced by fibronectin adsorption. These findings show that in vitro adhesion testing can be used for template optimization and to substitute for in-vivo experiments.     

Cytoskeletal forces produced by extremely low‐frequency electric fields acting on extracellular glycoproteins

The physical mechanism by which cells transduce an applied electric field is not well understood. This article establishes for the first time a direct, quantitative model that links the field to cytoskeletal forces. In a previous article, applied electric fields of physiological strength were shown to produce significant mechanical torques at the cellular level. In this article, the corresponding forces exerted on the cytoskeleton are computed and found to be comparable in magnitude to mechanical forces known to produce physiological effects. In addition to the electrical force, the viscous drag force exerted by the surrounding medium and the restoring force exerted by the neighboring structures are considered in the analysis. For an applied electric field of 10 V/m, the force transmitted to the CD44 receptor of a hyaluronan chain in cartilage is about 1 pN at 10 Hz and 7 pN at 1 Hz. For an applied electric field of 100 V/m, the force transmitted to the cytoskeleton at one focus of the glycocalyx is about 0.5 pN at 10 Hz and 1.3 pN at 1 Hz. Mechanical forces of similar magnitude have been observed to produce physiological effects. Hence, this electromechanical transduction process is a plausible mechanism for the production of physiological effects by such electric fields. Bioelectromagnetics 31:77–84, 2010. © 2009 Wiley‐Liss, Inc.     

Molecular dynamics and forces of a motile cell simultaneously visualized by TIRF and force microscopies

Cells must exert traction forces onto the substratum for continuous migration. Molecular dynamics such as actin polymerization at the front of the cell and myosin II accumulation at the rear should play important roles in the exertion of forces required for migration. Therefore, it is important to reveal the relationships between the traction forces and molecular dynamics. Traction forces can be calculated from the deformation of the elastic substratum under a migrating cell. A transparent and colorless elastic substratum with a high refractive index (1.40) and a low Young's modulus (1.0 kPa) were made from a pair of platinum-catalyzed silicones. We used this substratum to develop a new method for simultaneous recording of molecular dynamics and traction forces under a migrating cell in which total internal refractive fluorescence (TIRF) and force microscopies were combined. This new method allows the detection of the spatiotemporal distribution of traction forces produced by individual filopodia in migrating Dictyostelium cells, as well as simultaneous visualization of these traction forces and the dynamics of filamentous myosin II.     

Mechanical Forces Impeding Exocytotic Surfactant Release Revealed by Optical Tweezers

The release of surfactant from alveolar type II cells is essential to lower the surface tension in the lung and to facilitate inspiration. However, the factors controlling dispersal and diffusion of this hydrophobic material are still poorly understood. Here we report that release of surfactant from the fused vesicle, termed lamellar body (LB), resisted mechanical forces applied by optical tweezers: At constant trapping force, the probability to expand LB contents, i.e., to “pull” surfactant into the extracellular fluid, increased with time after LB fusion with the plasma membrane, consistent with slow fusion pore expansion in these cells. Elevations of the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_c) had a similar effect. Inasmuch as surfactant did not disintegrate in the extracellular space, this method permitted for the first time the determination of elastic and recoil properties of the macromolecular complex, yielding a spring constant of ~12.5 pN/μm. This is the first functional evidence that release of hydrophobic material is mechanically impeded and occurs in an “all-or-none” fashion. This mode of release is most probably the result of cohesive forces of surfactant, combined with adhesive forces and/or retaining forces exerted by a constrictive fusion pore acting as a regulated mechanical barrier, withstanding forces up to 160 pN. In independent experiments equiaxial strain was exerted on cells without optical tweezers. Strain facilitated surfactant release from preexisting fused vesicles, consistent with the view of mechanical impediments during the release process, which can be overcome by cell strain.     

Quantitative Modeling and Optimization of Magnetic Tweezers

Magnetic tweezers are a powerful tool to manipulate single DNA or RNA molecules and to study nucleic acid-protein interactions in real time. Here, we have modeled the magnetic fields of permanent magnets in magnetic tweezers and computed the forces exerted on superparamagnetic beads from first principles. For simple, symmetric geometries the magnetic fields can be calculated semianalytically using the Biot-Savart law. For complicated geometries and in the presence of an iron yoke, we employ a finite-element three-dimensional PDE solver to numerically solve the magnetostatic problem. The theoretical predictions are in quantitative agreement with direct Hall-probe measurements of the magnetic field and with measurements of the force exerted on DNA-tethered beads. Using these predictive theories, we systematically explore the effects of magnet alignment, magnet spacing, magnet size, and of adding an iron yoke to the magnets on the forces that can be exerted on tethered particles. We find that the optimal configuration for maximal stretching forces is a vertically aligned pair of magnets, with a minimal gap between the magnets and minimal flow cell thickness. Following these principles, we present a configuration that allows one to apply ≥40 pN stretching forces on ≈1-μm tethered beads.     

Cyclic AMP-dependent cell shape changes induced by mechanical forces

Biomaterials used in some biomedical devices are exposed to flow of physiological fluids. The flow-induced forces may influence the morphological and the biochemical responses of adhering cells. The objective of this work is to examine the capacity of a mechanical stress to cause changes in cell/substratum and cell/cell interactions via the second messenger cAMP pathway (cyclic Adenosine Monophosphate). Cyclic AMP is known to modulate cell shape, cell adhesion and intercellular communication in static conditions. A specially designed flow chamber was used to analyze the responses of mouse 3T3 fibroblasts spread on biocompatible substrata and submitted to controlled shear stresses. A 1.1-Pa shear stress induced: cell rounding, disruption of vitronectin receptors clusters and clustering of connexins 43 at cell-cell apposition points. These cell responses were cAMP-dependent. These investigations should help provide a better understanding of the early biochemical events triggered by mechanical forces.     

High-resolution micromechanical measurement in real time of forces exerted by living cells

The aim of this study was to compare uniaxial traction forces exerted by different cell types using a novel sensor design and to test the dependence of measured forces on cytoskeletal integrity. The sensor design detects forces generated between 2 contact points by cells spanning a gap. The magnitude of these forces varied according to cell type and were dependent on cytoskeletal integrity. The response time for drug-induced cytoskeletal disruption also varied between cell types: dermal fibroblasts exerted the greatest forces and had the slowest drug response times; EBV-transformed epithelial cells also had slow cytoskeletal depolymerisation times but exerted the lowest forces overall. Conversely, lung epithelial tumor cells exerted low forces but had the fastest depolymerisation drug response. These results provide proof of principle for a new design of force-measurement sensor based on optical interferometry, an approach that can be used to study cytoskeletal dynamics in real time.     

Properties of the force exerted by filopodia and lamellipodia and the involvement of cytoskeletal components

During neuronal differentiation, lamellipodia and filopodia explore the environment in search for the correct path to the axon's final destination. Although the motion of lamellipodia and filopodia has been characterized to an extent, little is known about the force they exert. In this study, we used optical tweezers to measure the force exerted by filopodia and lamellipodia with a millisecond temporal resolution. We found that a single filopodium exerts a force not exceeding 3 pN, whereas lamellipodia can exert a force up to 20 pN. Using metabolic inhibitors, we showed that no force is produced in the absence of actin polymerization and that development of forces larger than 3 pN requires microtubule polymerization. These results show that actin polymerization is necessary for force production and demonstrate that not only do neurons process information, but they also act on their environment exerting forces varying from tenths pN to tens of pN.     

Membrane tether extraction from human umbilical vein endothelial cells and its implication in leukocyte rolling

During the rolling of human neutrophils on the endothelium, tethers (cylindrical membrane tubes) are likely extracted from the neutrophil. Tether extraction reduces the force imposed on the adhesive bond between the neutrophil and endothelium, thereby facilitating the rolling. However, whether tethers can be extracted from the endothelium is still unknown. Here, with the micropipette-aspiration technique, we show that tethers can be extracted from either suspended or attached human umbilical vein endothelial cells. We also show that a linear relationship between the pulling force and tether growth velocity exists and this relationship does not depend on the receptor type (used to impose point forces), tumor necrosis factor-alpha stimulation, or cell attachment state. With linear regression, we determined that the threshold force was 50 pN and the effective viscosity was 0.50 pN.s/microm. Therefore, tethers might be simultaneously extracted from the neutrophil and endothelial cell during the rolling and, more importantly, the endothelial cell might contribute much more to the total composite tether length than the neutrophil. Compared with tether extraction from the neutrophil alone, simultaneous tether extraction results in a larger increase in the lifetime of the adhesive bond, and thus further stabilizes the rolling of neutrophils under high physiological shear stresses.     

On the electrophysiological response of bone cells using a Stokesian fluid stimulus probe for delivery of quantifiable localized picoNewton level forces

A Stokesian fluid stimulus probe (SFSP), capable of delivering quantifiable pN level hydrodynamic forces, is developed to distinguish the electrophysiological response of the cell process and cell body of osteocyte-like MLO-Y4 cells without touching the cell or its substrate. The hydrodynamic disturbance is a short lived (100 ms), constant strength pressure pulse that propagates nearly instantaneously through the medium creating a nearly spherical expanding fluid bolus surrounding a 0.8 μm micropipette tip. Laboratory model experiments show that the growth of the bolus and the pressure field can be closely modeled by quasi-steady Stokes flow through a circular orifice provided the tip Reynolds number, Re(t)<0.03. By measuring the deflection of the dendritic processes between discrete attachment sites, and applying a detailed ultrastructural model for the central actin filament bundle within the process, one is able to calculate the forces produced by the probe using elastic beam theory. One finds that forces between 1 and 2.3 pN are sufficient to initiate electrical signaling when applied to the cell process, but not the much softer cell body. Even more significantly, cellular excitation by the process only occurs when the probe is directed at discrete focal attachment sites along the cell process. This suggests that electrical signaling is initiated at discrete focal attachments along the cell process and that these sites are likely integrin-mediated complexes associated with stretch-activated ion channels though their molecular structure is unknown.     

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.     

The effect of tenascin and embryonic basal lamina on the behavior and morphology of neural crest cells in vitro

We have investigated the morphology and migratory behavior of quail neural crest cells on isolated embryonic basal laminae or substrata coated with fibronectin or tenascin. Each of these substrata have been implicated in directing neural crest cell migration in situ. We also observed the altered behavior of cells in response to the addition of tenascin to the culture medium independent of its effect as a migratory substratum. On tenascin-coated substrata, the rate of neural crest cell migration from neural tube explants was significantly greater than on uncoated tissue culture plastic, on fibronectin-coated plastic, or on basal lamina isolated from embryonic chick retinae. Neural crest cells on tenascin were rounded and lacked lamellipodia, in contrast to the flattened cells seen on basal lamina and fibronectin-coated plastic. In contrast, when tenascin was added to the culture medium of neural crest cells migrating on isolated basal lamina, a significant reduction in the rate of cell migration was observed. To study the nature of this effect, we used human melanoma cells, which have a number of characteristics in common with quail neural crest cells though they would be expected to have a distinct family of integrin receptors. A dose-dependent reduction in the rate of translocation was observed when tenascin was added to the culture medium of the human melanoma cell line plated on isolated basal laminae, indicating that the inhibitory effect of tenascin bound to the quail neural crest surface is probably not solely the result of competitive inhibition by tenascin for the integrin receptor. Our results show that tenascin can be used as a migratory substratum by avian neural crest cells and that tenascin as a substratum can stimulate neural crest cell migration, probably by permitting rapid detachment. Tenascin in the medium, on the other hand, inhibits both the migration rates and spreading of motile cells on basal lamina because it binds only the cell surface and not the underlying basal lamina. Cell surface-bound tenascin may decrease cell-substratum interactions and thus weaken the tractional forces generated by migrating cells. This is in contrast to the action of fibronectin, which when added to the medium stimulates cell migration by binding both to neural crest cells and the basal lamina, thus providing a bridge between the motile cells and the substratum.