These are the most recent papers from the lab. We also offer older papersfrom '83-'92, and some really early ones.

Cook, B., R. W. Hardy, W. B. McConnaughey and C. S. Zuker (2008). Preserving cell shape under environmental stress. Nature 452: 361-4.

Maintaining cell shape and tone is essential for the function and survival of cells and tissues. Mechanotransduction relies on the transformation of minuscule mechanical forces into high-fidelity electrical responses. When mechanoreceptors are stimulated, mechanically sensitive cation channels open and produce an inward transduction current that depolarizes the cell. For this process to operate effectiely, the transduction machinery has to retain integrity and remain unfailingly independent of environmental changes. This is particularly challenging for poikilothermic organisms, where changes in temperature in the environment may impact the function of mechanoreceptor neurons. Thus, we wondered how insects whose habitat might quickly vary over several tens of degrees of temperature manage to maintain highly effective mechanical senses. We screened for Drosophila mutants with defective mechanical responses at elevated ambient temperatures, and identified a gene, spam, whose role is to protect the mechanosensory organ from massive cellular deformation caused by heat-induced osmotic imbalance. Here we show that Spam protein forms an extracellular shield that guards mechanosensory neurons from environmental insult. Remarkably, heterologously expressed Spam protein also endowed other cells with superb defence against physically and chemically induced deformation. We studied the mechanical impact of Spam coating and show that spam-coated cells are up to ten times stiffer than uncoated controls. Together, these results help explain how poikilothermic organisms preserve the architecture of critical cells during environmental stress, and illustrate an elegant and simple solution to such challenge.

Nekouzadeh, A., K. M. Pryse, E. L. Elson and G. M. Genin (2008). Stretch-activated force shedding, force recovery, and cytoskeletal remodeling in contractile fibroblasts. J Biomech 41(14): 2964-71.

The stress fiber network within contractile fibroblasts structurally reinforces and provides tension, or "tone", to tissues such as those found in healing wounds. Stress fibers have previously been observed to polymerize in response to mechanical forces. We observed that, when stretched sufficiently, contractile fibroblasts diminished the mechanical tractions they exert on their environment through depolymerization of actin filaments then restored tissue tension and rebuilt actin stress fibers through staged Ca(++)-dependent processes. These staged Ca(++)-modulated contractions consisted of a rapid phase that ended less than a minute after stretching, a plateau of inactivity, and a final gradual phase that required several minutes to complete. Active contractile forces during recovery scaled with the degree of rebuilding of the actin cytoskeleton. This complementary action demonstrates a programmed regulatory mechanism that protects cells from excessive stretch through choreographed active mechanical and biochemical healing responses.

Nekouzadeh, A., K. M. Pryse, E. L. Elson and G. M. Genin (2007). A simplified approach to quasi-linear viscoelastic modeling. J Biomech 40(14): 3070-8.

The fitting of quasi-linear viscoelastic (QLV) constitutive models to material data often involves somewhat cumbersome numerical convolution. A new approach to treating quasi-linearity in 1-D is described and applied to characterize the behavior of reconstituted collagen. This approach is based on a new principle for including nonlinearity and requires considerably less computation than other comparable models for both model calibration and response prediction, especially for smoothly applied stretching. Additionally, the approach allows relaxation to adapt with the strain history. The modeling approach is demonstrated through tests on pure reconstituted collagen. Sequences of "ramp-and-hold" stretching tests were applied to rectangular collagen specimens. The relaxation force data from the "hold" was used to calibrate a new "adaptive QLV model" and several models from literature, and the force data from the "ramp" was used to check the accuracy of model predictions. Additionally, the ability of the models to predict the force response on a reloading of the specimen was assessed. The "adaptive QLV model" based on this new approach predicts collagen behavior comparably to or better than existing models, with much less computation.

Saffarian, S., Y. Li, E. L. Elson and L. J. Pike (2007). Oligomerization of the EGF receptor investigated by live cell fluorescence intensity distribution analysis. Biophys J 93(3): 1021-31.

Recent evidence suggests that the EGF receptor oligomerizes or clusters in cells even in the absence of agonist ligand. To assess the status of EGF receptors in live cells, an EGF receptor fused to eGFP was stably expressed in CHO cells and studied using fluorescence correlation spectroscopy and fluorescent brightness analysis. By modifying FIDA for use in a two-dimensional system with quantal brightnesses, a method was developed to quantify the degree of clustering of the receptors on the cell surface. The analysis demonstrates that under physiological conditions, the EGF receptor exists in a complex equilibrium involving single molecules and clusters of two or more receptors. Acute depletion of cellular cholesterol enhanced EGF receptor clustering whereas cholesterol loading decreased receptor clustering, indicating that receptor aggregation is sensitive to the lipid composition of the membrane.

Asnes, C. F., J. P. Marquez, E. L. Elson and T. Wakatsuki (2006). Reconstitution of the Frank-Starling mechanism in engineered heart tissues. Biophys J 91(5): 1800-10.

According to the Frank-Starling mechanism, as the heart is stretched, it increases its contraction force. Reconstitution of the Frank-Starling mechanism is an important milestone for producing functional heart tissue constructs. Spontaneously contracting engineered heart tissues (EHTs) were reconstituted by growing dissociated chicken embryo cardiomyocytes in collagen matrices. Twitch and baseline tensions were recorded at precisely controlled levels of tissue strain. The EHTs showed a steep increase in twitch tension from 0.47 +/- 0.02 to 0.91 +/- 0.02 mN/mm2 as they were stretched at a constant rate (2.67% per min) from 86% to 100% of the length at which maximum twitch force was exerted. In response to a sudden stretch (3.3%), the twitch tension increased gradually (approximately 60 s) in a Gd3+-sensitive manner, suggesting the presence of stretch-activated Ca2+ channels. A large difference in baseline tension between lengthening (loading) and shortening (unloading) was also recorded. Disruption of nonsarcomeric actin filaments by cytochalasin D and latrunculin B decreased this difference. A simple mechanical model interprets these results in terms of mechanical connections between myocytes and nonmuscle cells. The experimental results strongly suggest that regulation of twitch tension in EHTs is similar to that of natural myocardium.

Marquez, J. P., G. M. Genin, K. M. Pryse and E. L. Elson (2006). Cellular and matrix contributions to tissue construct stiffness increase with cellular concentration. Ann Biomed Eng 34(9): 1475-82.

The mechanics of bio-artificial tissue constructs result from active and passive contributions of cells and extracellular matrix (ECM). We delineated these for a fibroblast-populated matrix (FPM) consisting of chick embryo fibroblast cells in a type I collagen ECM through mechanical testing, mechanical modeling, and selective biochemical elimination of tissue components. From a series of relaxation tests, we found that contributions to overall tissue mechanics from both cells and ECM increase exponentially with the cell concentration. The force responses in these relaxation tests exhibited a logarithmic decay over the 3600 second test duration. The amplitudes of these responses were nearly linear with the amplitude of the applied stretch. The active component of cellular forces rose dramatically for FPMs containing higher cell concentrations.

Pablo Marquez, J., G. M. Genin and E. L. Elson (2006). On the application of strain factors for approximation of the contribution of anisotropic cells to the mechanics of a tissue construct. J Biomech 39(11): 2145-51.

Bio-artificial tissue constructs consisting of fibroblast cells embedded in a collagenous matrix are valuable in vitro systems in which to study cellular mechanics. Deriving cellular mechanics from the results of experimentation on tissue constructs requires a mathematical relationship that delineates amongst the contributions of the constituents of a tissue construct. A scaling between the average strain in a uniformly stretched tissue and the axial strain in isotropic cells was used in earlier work to study relations between cell mechanics and the overall mechanics of a tissue construct. That work showed that a scaling factor called a "strain factor" provided an accurate representation of the average axial strain in isotropic cells. The present study analyzes such relationships for anisotropic cells. We incorporate Eshelby's (1957; Proceedings of the Royal Society of London A 241, 376; 1959; Proceedings of the Royal Society of London A 252, 561) exact solution for the strain field in isolated ellipsoidal inclusions into the Zahalak (Biophysical journal 79, 2369) constitutive model for tissue constructs. Results showed that, for the case of prolate cells, the strain along the major cell axis is mostly influenced by the remote strain projected along that axis; off-axis cell mechanics plays only a small role in most tissues. The strain factor approximation is shown to be accurate for anisotropic cells to within a few percent for the vast majority of tissues. The results presented in this paper provide an explicit measure of the effects of cellular anisotropy, and a mechanism for calculating the contributions of these effects to overall tissue mechanics when these effects are important.

Remond, M. C., J. A. Fee, E. L. Elson and L. A. Taber (2006). Myosin-based contraction is not necessary for cardiac c-looping in the chick embryo. Anat Embryol (Berl) 211(5): 443-54.

During the initial phase of cardiac looping, known as c-looping, the heart bends and twists into a c-shaped tube with the convex outer curvature normally directed toward the right side of the embryo. Despite intensive study for more than 80 years, the biophysical mechanisms that drive and regulate looping remain poorly understood, although some investigators have speculated that differential cytoskeletal contraction supplies the driving force for c-looping. The purpose of this investigation was to test this hypothesis. To inhibit contraction, embryonic chick hearts at stages 10-12 (10-16 somites, 33-48 h) were exposed to the myosin inhibitors 2,3-butanedione monoxime (BDM), ML-7, Y-27632, and blebbistatin. Experiments were conducted in both whole embryo culture and, to focus on bending alone, isolated heart culture. Measurements of heart stiffness and phosphorylation of the myosin regulatory light chains showed that BDM, Y-27632, and blebbistatin significantly reduced myocardial contractility, while ML-7 had a lesser effect. None of these drugs significantly affected looping during the studied stages. These results suggest that active contraction is not required for normal c-looping of the embryonic chick heart between stages 10 and 12.

Wille, J. J., E. L. Elson and R. J. Okamoto (2006). Cellular and matrix mechanics of bioartificial tissues during continuous cyclic stretch. Ann Biomed Eng 34(11): 1678-90.

Bioartificial tissues are useful model systems for studying cell and extra-cellular matrix mechanics. These tissues provide a 3D environment for cells and allow tissue components to be easily modified and quantified. In this study, we fabricated bioartificial tissue rings from a 1 ml solution containing one million cardiac fibroblasts and 1 mg collagen. After 8 days, rings compacted to <1% of original volume and cell number increased 2.4 fold. We initiated continuous cyclic stretching of the rings after 2, 4, or 8 days of incubation, while monitoring the tissue forces. Peak tissue force during each cycle decreased rapidly after initiating stretch, followed by further slow decline. We added 2 microM Cytochalasin-D to some rings prior to initiation of stretch to determine the force contributed by the matrix. Cell force was estimated by subtracting matrix force from tissue force. After 12 h, matrix force-strain curves were highly nonlinear. Cell force-strain curves were linear during loading and showed hysteresis indicating viscoelastic behavior. Cell stiffness increased with stretching frequency from 0.001-0.25 Hz. Cell stiffness decreased with stretch amplitude (5-25%) at 0.1 Hz. The trends in cell stiffness do not fit simple viscoelastic models previously proposed, and suggest possible strain-amplitude related changes during cyclic stretch.

Chattopadhyay, K., E. L. Elson and C. Frieden (2005). The kinetics of conformational fluctuations in an unfolded protein measured by fluorescence methods. Proc Natl Acad Sci U S A 102(7): 2385-9.

The simplest dynamic model for an unfolded protein is a statistical coil that continually undergoes substantial conformational fluctuations. A growing number of studies indicate that the unfolded protein is not a simple random coil but rather forms transient structures. We have directly measured the rate of conformational fluctuations of unfolded intestinal fatty acid binding protein (131 aa, 15 kDa) by using fluorescence self-quenching in combination with fluorescence correlation spectroscopy. The conformational fluctuations in this state have an apparent relaxation time, tauR, of 1.6 microsec in 3 M guanidine-HCl at pH 7 and 20 degrees C. The value of tauR increases with increasing solution viscosity, suggesting a diffusive process. In the molten globule state at pH 2, tauR is 2.5 microsec, increasing further with the formation of salt-induced secondary structure. These measurements, which should be widely applicable to other systems, can provide important information about the still incompletely understood conformational properties of unfolded proteins and the mechanism of protein folding.

Chattopadhyay, K., S. Saffarian, E. L. Elson and C. Frieden (2005). Measuring unfolding of proteins in the presence of denaturant using fluorescence correlation spectroscopy. Biophys J 88(2): 1413-22.

IFABP is a small (15 kDa) protein consisting mostly of antiparallel beta-strands that surround a large cavity into which ligands bind. We have previously used FCS to show that the native protein, labeled with fluorescein, exhibits dynamic fluctuation with a relaxation time of 35 micros. Here we report the use of FCS to study the unfolding of the protein induced by guanidine hydrochloride. Although the application of this technique to measure diffusion coefficients and molecular dynamics is straightforward, the FCS results need to be corrected for both viscosity and refractive index changes as the guanidine hydrochloride concentration increases. We present here a detailed study of the effects of viscosity and refractive index of guanidine hydrochloride solutions to calibrate FCS data. After correction, the increase in the diffusion time of IFABP corresponds well with the unfolding transition monitored by far ultraviolet circular dichroism. We also show that the magnitude of the 35 micros phase, reflecting the conformational fluctuation in the native state, decreases sharply as the concentration of denaturant increases and the protein unfolds. Although FCS experiments indicate that the unfolded state at pH 2 is rather compact and native-like, the radius in the presence of guanidine hydrochloride falls well within the range expected for a random coil.

Latacha, K. S., M. C. Remond, A. Ramasubramanian, A. Y. Chen, et al. (2005). Role of actin polymerization in bending of the early heart tube. Dev Dyn 233(4): 1272-86.

During cardiac c-looping, the heart transforms from a straight tube into a c-shaped tube, presenting the first evidence of left-right asymmetry in the embryo. C-looping consists of two primary deformation components: ventral bending and dextral rotation. This study examines the role of actin polymerization in bending of the heart tube. Exposure of stage 9-11 chick embryos to low concentrations of the actin polymerization inhibitors cytochalasin D (5 nM-2.0 microM) and latrunculin A (LA; 25 nM-2.0 microM) suppressed looping in a stage- and concentration-dependent manner in both whole embryos and isolated hearts. Local exposure of either the dorsal or ventral sides of isolated hearts to LA also inhibited looping, but less than global exposure, indicating that both sides contribute to the bending mechanism. Taken together, these data suggest that ongoing actin polymerization is required for the bending component of cardiac c-looping, and we speculate that polymerization-driven myocardial cell shape changes cause this deformation.

Marquez, J. P., G. M. Genin, G. I. Zahalak and E. L. Elson (2005). Thin bio-artificial tissues in plane stress: the relationship between cell and tissue strain, and an improved constitutive model. Biophys J 88(2): 765-77.

Constitutive models are needed to relate the active and passive mechanical properties of cells to the overall mechanical response of bio-artificial tissues. The Zahalak model attempts to explicitly describe this link for a class of bio-artificial tissues. A fundamental assumption made by Zahalak is that cells stretch in perfect registry with a tissue. We show this assumption to be valid only for special cases, and we correct the Zahalak model accordingly. We focus on short-term and very long-term behavior, and therefore consider tissue constituents that are linear in their loading response (although not necessarily linear in unloading). In such cases, the average strain in a cell is related to the macroscopic tissue strain by a scalar we call the "strain factor". We incorporate a model predicting the strain factor into the Zahalak model, and then reinterpret experiments reported by Zahalak and co-workers to determine the in situ stiffness of cells in a tissue construct. We find that, without the modification in this article, the Zahalak model can underpredict cell stiffness by an order of magnitude.

Marquez, J. P., G. M. Genin, G. I. Zahalak and E. L. Elson (2005). The relationship between cell and tissue strain in three-dimensional bio-artificial tissues. Biophys J 88(2): 778-89.

Continuum constitutive laws are needed to ensure that bio-artificial tissue constructs replicate the mechanical response of the tissues they replace, and to understand how the constituents of these constructs contribute to their overall mechanical response. One model designed to achieve both of these aims is the Zahalak model, which was modified by Marquez and co-workers to incorporate inhomogeneous strain fields within very thin tissues. When applied to reinterpret previous measurements, the modified Zahalak model predicted higher values of the continuum stiffness of fibroblasts than earlier estimates. In this work, we further modify the Zahalak model to account for inhomogeneous strain fields in constructs whose cell orientations have a significant out-of-plane component. When applied to reinterpret results from the literature, the new model shows that estimates of continuum cell stiffness might need to be revised upward. As in this article's companion, we updated the average cell strain by defining a correction factor ("strain factor"), based upon the elastic response. Three different cell orientation distributions were studied. We derived an approximate scaling model for the strain factor, and validated it against exact and self-consistent (mean-field) solutions from the literature for dilute cell concentrations, and Monte Carlo simulations involving three-dimensional finite element analyses for high cell concentrations.

Elson, E. L. (2004). Quick tour of fluorescence correlation spectroscopy from its inception. J Biomed Opt 9(5): 857-64.

Fluorescence correlation spectroscopy (FCS) was originally developed in the early 1970s as a way to measure the kinetics of chemical reactions under zero perturbation conditions. At its inception, the measurement was difficult due to experimental limitations and was primarily used during the 1970s and 1980s to characterize diffusion. More recently, as a result of technological advances, FCS measurements have become easier and more versatile. In addition to measurements of diffusion both in solution and in cells, FCS is now also used to measure not only chemical reaction kinetics but also extents of molecular aggregation, the dynamics of photophysical processes, conformational fluctuations, molecular interactions in solution and in cells, and has even found application as a pharmaceutical screening method. From its inception to the present, the contributions of Webb and his coworkers have had a central and defining role in the development and applications of FCS.

Emmert, D. A., J. A. Fee, Z. M. Goeckeler, J. M. Grojean, et al. (2004). Rho-kinase-mediated Ca2+-independent contraction in rat embryo fibroblasts. Am J Physiol Cell Physiol 286(1): C8-21.

Thus far, determining the relative contribution of Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) and Ca2+-independent Rho-kinase pathways to myosin II activation and contraction has been difficult. In this study, we characterize the role of Rho-kinase in a rat embryo fibroblast cell line (REF-52), which contains no detectable MLCK. No endogenous MLCK could be detected in REF-52 cells by either Western or Northern blot analysis. In the presence or absence of Ca2+, thrombin or lysophosphatidic acid (LPA) increased RhoA activity and Rhokinase activity, correlating with isometric tension development and myosin II regulatory light chain (RLC) phosphorylation. Resting tension is associated with a basal phosphorylation of 0.31 +/- 0.02 mol PO4/mol RLC, whereas upon LPA or thrombin treatment myosin II RLC phosphorylation increases to 1.08 +/- 0.05 and 0.82 +/- 0.05 mol PO4/mol RLC, respectively, within 2.5 min. Ca2+ chelation has minimal effect on the kinetics and magnitude of isometric tension development and RLC phosphorylation. Treatment of REF-52 cells with the Rho-kinase-specific inhibitor Y-27632 abolished thrombin- and LPA-stimulated contraction and RLC phosphorylation. These results suggest that Rho-kinase is sufficient to activate myosin II motor activity and contraction in REF-52 cells.

Qian, H. and E. L. Elson (2004). Fluorescence correlation spectroscopy with high-order and dual-color correlation to probe nonequilibrium steady states. Proc Natl Acad Sci U S A 101(9): 2828-33.

In living cells, biochemical reaction networks often function in nonequilibrium steady states. Under these conditions, the networks necessarily have cyclic reaction kinetics that are maintained by sustained constant input and output, i.e., pumping. To differentiate this state from an equilibrium state without flux, we propose a microscopic method based on concentration fluctuation measurements, via fluorescence correlation spectroscopy, and statistical analyses of high-order correlations and cross correlations beyond the standard fluorescence correlation spectroscopy autocorrelation. We show that, for equilibrium systems with time reversibility, the correlation functions possess certain symmetries, the violation of which is a measure of steady-state fluxes in reaction cycles. This result demonstrates the theoretical basis for experimentally measuring reaction fluxes in a biochemical network in situ and the importance of single-molecule measurements in providing fundamental information on nonequilibrium steady-states in biochemistry.

Saffarian, S., I. E. Collier, B. L. Marmer, E. L. Elson, et al. (2004). Interstitial collagenase is a Brownian ratchet driven by proteolysis of collagen. Science 306(5693): 108-11.

We show that activated collagenase (MMP-1) moves processively on the collagen fibril. The mechanism of movement is a biased diffusion with the bias component dependent on the proteolysis of its substrate, not adenosine triphosphate (ATP) hydrolysis. Inactivation of the enzyme by a single amino acid residue substitution in the active center eliminates the bias without noticeable effect on rate of diffusion. Monte Carlo simulations using a model similar to a "burnt bridge" Brownian ratchet accurately describe our experimental results and previous observations on kinetics of collagen digestion. The biological implications of MMP-1 acting as a molecular ratchet tethered to the cell surface suggest new mechanisms for its role in tissue remodeling and cell-matrix interaction.

Wagenseil, J. E., E. L. Elson and R. J. Okamoto (2004). Cell orientation influences the biaxial mechanical properties of fibroblast populated collagen vessels. Ann Biomed Eng 32(5): 720-31.

Bioartificial tissues, composed of cells in a collagen matrix, can be fabricated with preferred cell orientations to mimic the histologic arrangement of biologic tissues. The influence of preferred cell orientations on the biaxial mechanical behavior of bioartificial tissues is unknown. Characterizing the biaxial mechanical behavior is necessary for better predicting the in vivo behavior of bioartificial tissues. Fibroblast populated collagen vessels (FPCVs) were fabricated with two different cell orientations by controlling the mechanical constraints during incubation. The cell orientation was verified by confocal microscopy and the collagen fiber organization was examined by confocal reflection and scanning electron microscopy (SEM). Pressure-diameter, force-length tests were performed to determine the influence of cell orientation on the biaxial mechanical behavior. FPCVs were more extensible in the direction perpendicular to the preferred cell orientation, than in the direction parallel to the cell orientation. Biaxial tests were also performed in the presence of Cytochalasin D (Cyto D) to minimize the mechanical contribution of the cells. After Cyto D treatment, the FPCVs remained more extensible in the direction perpendicular to the cell orientation, even though a preferred collagen fiber orientation was not observed in the microscopy images.

Wakatsuki, T., J. A. Fee and E. L. Elson (2004). Phenotypic screening for pharmaceuticals using tissue constructs. Curr Pharm Biotechnol 5(2): 181-9.

Compounds can be screened for pharmaceutical activity either by detecting interactions with specified target molecules such as receptors or enzymes (molecular screening) or observing effects on the structure or physiological activities of cells or tissues (phenotypic screening). Screening at the molecular level has been greatly enhanced by fluorescence methods. Especially the combination of confocal detection with measurements of the amplitudes and time courses of fluorescence fluctuations have reduced sample volumes to < microliters and have increased throughputs to >100000 compounds per day. Screening at the molecular level, however, does not provide information about the effects of test compounds on cellular functions. Phenotypic screening, although much slower than molecular screening, does provide information about effects on cell or tissue structure or function and therefore can be used to eliminate at an early stage compounds that are toxic or do not produce the desired cellular response. Tissue constructs reconstituted using cells of specified types and defined extracellular matrix components provide test systems for detecting the effects of test compounds on cellular mechanical functions such as the development of contractile force and on cell and matrix structure and stiffness. For example, constructs based on vascular smooth muscle cells provide information about effects on cellular contractile force that can be used to identify agents that control blood pressure. Tissue constructs that mimic skeletal, smooth and heart muscles and connective tissues have been produced and can be used to study mechanical and structural responses to active compounds.

Wakatsuki, T., J. Schlessinger and E. L. Elson (2004). The biochemical response of the heart to hypertension and exercise. Trends Biochem Sci 29(11): 609-17.

Mechanical stress on the heart can lead to crucially different outcomes. Exercise is beneficial because it causes heart muscle cells to enlarge (hypertrophy). Chronic hypertension also causes hypertrophy, but in addition it causes an excessive increase in fibroblasts and extracellular matrix (fibrosis), death of cardiomyocytes and ultimately heart failure. Recent research shows that stimulation of physiological (beneficial) hypertrophy involves several signaling pathways, including those mediated by protein kinase B (also known as Akt) and the extracellular-signal-regulated kinases 1 and 2 (ERK1/2). Hypertension, beta-adrenergic stimulation and agonists such as angiotensin II (Ang II) activate not only ERK1/2 but also p38 and the Jun N-terminal kinase (JNK), leading to pathological heart remodeling. Despite this progress, the mechanisms that activate fibroblasts to cause fibrosis and those that differentiate between exercise and hypertension to produce physiological and pathological responses, respectively, remain to be established.

Pryse, K. M., A. Nekouzadeh, G. M. Genin, E. L. Elson, et al. (2003). Incremental mechanics of collagen gels: new experiments and a new viscoelastic model. Ann Biomed Eng 31(10): 1287-96.

Paired incremental uniaxial step (i.e., relaxation) and ramp tests were conducted simultaneously on four (nominally) identical samples of type I collagen gel, over a direct strain range 0 < epsilon < 0.2. The paired step and ramp responses could not both be predicted by a simple viscoelastic constitutive relation (either linear or Fung-type), but could be predicted reasonably accurately by a general nonlinear viscoelastic relation with a strain-dependent relaxation spectrum, of the form sigma(t) = f(t)-infinity g(t-tau,epsilon)[d(epsilon)(tau)/d(tau)]d(tau). Based on a four-term exponential-series approximation, we measured the stiffness moduli and time constants of the relaxation function, g(t,epsilon), for the four gel samples that we tested, and found that the time constants were independent of strain but the moduli increased strongly with strain. Further, we found that the time constants did not vary across the four gels, but the moduli varied by a factor of about 2 across the gels. Some additional tests show features of the response of collagen gels to cycles of application and removal of loading.

Wagenseil, J. E., T. Wakatsuki, R. J. Okamoto, G. I. Zahalak, et al. (2003). One-dimensional viscoelastic behavior of fibroblast populated collagen matrices. J Biomech Eng 125(5): 719-25.

Bio-artificial tissues are being developed as replacements for damaged biologic tissues. Their mechanical properties are critical for load bearing applications. Current testing protocols for bio-artificial tissues vary widely and often do not consider viscoelasticity. Uniaxial stretch tests were performed on fibroblast populated collagen matrices (FPCMs) to determine the influence of specific test protocols on the mechanical behavior. The peak force, hysteresis and shape of the force-stretch curve are affected by the stretch rate, rest period, stretch amplitude and the number and magnitude of preconditioning cycles.

Wakatsuki, T., Elson, E. L. (2003) Reciprocal interactions between cells and extracellular matrix during remodeling of tissue constructs. Biophys. Chem. 100(1-3):593-605.

Cells remodel extracellular matrix during tissue development and wound healing. Similar processes occur when cells compress and stiffen collagen gels. An important task for cell biologists, biophysicists, and tissue engineers is to guide these remodeling processes to produce tissue constructs that mimic the structure and mechanical properties of natural tissues. This requires an understanding of the mechanisms by which this remodeling occurs. Quantitative measurements of the contractile force developed by cells and the extent of compression and stiffening of the matrix describe the results of the remodeling processes. Not only do forces exerted by cells influence the structure of the matrix but also external forces exerted on the matrix can modulate the structure and orientation of the cells. The mechanisms of these processes remain largely unknown, but recent studies of the regulation of myosin-dependent contractile force and of cell protrusion driven by actin polymerization provide clues about the regulation of cellular functions during remodeling.

Wakatsuki, T., Wysolmerski, R. B., Elson, E. L. (2003) Mechanics of cell spreading: role of myosin II. J. Cell Sci. 116(Pt 8):1617-25.

As it migrates over a substratum, a cell must exert different kinds of forces that act at various cellular locations and at specific times. These forces must therefore be coordinately regulated. The Rho-family GTPases Rac1 and Cdc42 promote actin polymerization that drives extension of the leading cell edge. Subsequently, RhoA regulates myosin- dependent contractile force, which is required for formation of adhesive contacts and stress fibers. During cell spreading, however, the activity of RhoA is reduced by a mechanism involving the tyrosine kinases c-Src and focal adhesion kinase (FAK), and the p190RhoGAP. It has been proposed that this reduction of RhoA activity facilitates edge extension by reducing myosin-dependent contractile forces that could resist this process. We have directly tested this hypothesis by correlating myosin activity with the rate of cell spreading on a substratum. The rate of spreading is inversely related to the myosin activity. Furthermore, spreading is inhibited by low concentrations of cytochalasin D, as expected for a process that depends on the growth of uncapped actin filaments. Cell indentation measurements show that a myosin-dependent viscoelastic force resists cell deformation.

Saffarian, S., Elson, E. L. (2003) Statistical analysis of fluorescence correlation spectroscopy: the standard deviation and bias. Biophys. J. 84(3):2030-42.

We present a detailed statistical analysis of fluorescence correlation spectroscopy for a wide range of timescales. The derivation is completely analytical and can provide an excellent tool for planning and analysis of FCS experiments. The dependence of the signal-to-noise ratio on different measurement conditions is extensively studied. We find that in addition to the shot noise and the noise associated with correlated molecular dynamics there is another source of noise that appears at very large lag times. We call this the "particle noise," as its behavior is governed by the number of particles that have entered and left the laser beam sample volume during large dwell times. The standard deviations of all the points on the correlation function are calculated analytically and shown to be in good agreement with experiments. We have also investigated the bias associated with experimental correlation function measurements. A "phase diagram" for FCS experiments is constructed that demonstrates the significance of the bias for any given experiment. We demonstrate that the value of the bias can be calculated and added back as a first-order correction to the experimental correlation function.

Qian, H., Saffarian, S., Elson, E. L. (2002) Concentration fluctuations in a mesoscopic oscillating chemical reaction system. Proc. Natl. Acad. Sci. U. S. A. 99(16):10376-81.

Under sustained pumping, kinetics of macroscopic nonlinear biochemical reaction systems far from equilibrium either can be in a stationary steady state or can execute sustained oscillations about a fixed mean. For a system of two dynamic species X and Y, the concentrations n(x) and n(y) will be constant or will repetitively trace a closed loop in the (n(x), n(y)) phase plane, respectively. We study a mesoscopic system with n(x) and n(y) very small; hence the occurrence of random fluctuations modifies the deterministic behavior and the law of mass action is replaced by a stochastic model. We show that n(x) and n(y) execute cyclic random walks in the (n(x), n(y)) plane whether or not the deterministic kinetics for the corresponding macroscopic system represents a steady or an oscillating state. Probability distributions and correlation functions for n(x)(t) and n(y)(t) show quantitative but not qualitative differences between states that would appear as either oscillating or steady in the corresponding macroscopic systems. A diffusion-like equation for probability P(n(x), n(y), t) is obtained for the two-dimensional Brownian motion in the (n(x), n(y)) phase plane. In the limit of large n(x), n(y), the deterministic nonlinear kinetics derived from mass action is recovered. The nature of large fluctuations in an oscillating nonequilibrium system and the conceptual difference between "thermal stochasticity" and "temporal complexity" are clarified by this analysis. This result is relevant to fluorescence correlation spectroscopy and metabolic reaction networks.

Frieden, C., Chattopadhyay, K., Elson, E. L. (2002) What fluorescence correlation spectroscopy can tell us about unfolded proteins. Adv. Protein Chem. 62:91-109.

Chattopadhyay, K., Saffarian, S., Elson, E. L., Frieden, C. (2002) Measurement of microsecond dynamic motion in the intestinal fatty acid binding protein by using fluorescence correlation spectroscopy. Proc. Natl. Acad. Sci. U. S. A. 99(22):14171-6.

Fluorescence correlation spectroscopy (FCS) measurements have been carried out on the intestinal fatty acid binding protein (IFABP) to study microsecond dynamics of the protein in its native state as well as in pH-induced intermediates. IFABP is a small (15 kDa) protein that consists mostly of antiparallel beta-strands enclosing a large central cavity into which the ligand binds. Because this protein does not contain cysteine, two cysteine mutants (Val60Cys and Phe62Cys) have been prepared and covalently modified with fluorescein. Based on fluorescence measurements, one of the mutants (Val60Flu) has the fluorescein moiety inside the cavity of the protein, whereas the fluorescein is exposed to solvent in the other (Phe62Flu). The protein modified at position 60 demonstrates the presence of a conformational event on the order of 35 microsec, which is not seen in the other mutant (Phe62Flu). The amplitude of this fast conformational event decreases sharply at low pH as the protein unfolds. Experiments measuring the diffusion as a function of pH indicate the formation of a compact state distinct from the native state at about pH 3.5. Steady state fluorescence and far-UV CD indicates that unfolding occurs at pH values below pH 3.

Allen, F. D., Asnes, C. F., Chang, P., Elson, E. L., Lauffenburger, D. A., Wells, A. (2002) Epidermal growth factor induces acute matrix contraction and subsequent calpain-modulated relaxation. Wound Repair Regen. 10(1):67-76.

During wound healing, dermal fibroblasts switch from a migratory, repopulating phenotype to a contractile, matrix-reassembling phenotype. The mechanisms controlling this switch are unknown. A possible explanation is suggested by the finding that chemokines that appear late in wound repair prevent growth factor-induced cell-substratum de- adhesion by blocking calpain activation. In this study, we tested the specific hypothesis that fibroblast contraction of the matrix is promoted by a pro-repair growth factor, epidermal growth factor, and is modulated by calpain-mediated release of adhesions. We employed an isometric force transduction system designed to measure the contraction of a collagen matrix under tension by a population of NR6 fibroblasts transfected with the human epidermal growth factor receptor. By maintaining a fixed level of strain, we could monitor both the initial contraction and subsequent relaxation of the matrix. Epidermal growth factor stimulated a transient, dose-dependent increase in matrix contraction that peaked within 60 minutes and then decayed over the ensuing 3 to 6 hours. Calpain inhibitor I (ALLN) prevented epidermal growth factor-stimulated cell de-adhesion and resulted in a significantly slower decay of matrix contraction, with only a slight decrease of the peak magnitude of contraction. The mitogen-activated protein kinase kinase-1-selective inhibitor PD 98059 that blocks signaling through the extracellular signal-regulated kinase/mitogen- activated protein kinase pathway, required for epidermal growth factor receptor-mediated activation of calpain and de-adhesion, does not significantly affect the magnitude of matrix contraction within minutes of epidermal growth factor addition, but slows the decay similarly to calpain inhibition. Epidermal growth factor receptor signaling thus stimulates the complementary mechanisms of intracellular contractile force generation and calpain-mediated de-adhesion, which are known to coordinately facilitate cell migration. These findings suggest that calpain can act as a functional switch for transmission of intracellular contractile force to the surrounding matrix, with calpain- mediated de-adhesion reducing this transmission and corresponding matrix contraction. Countervailing processes that down-regulate calpain activation can, accordingly, direct the transition of cell function from locomotion to matrix contraction.

Wakatsuki, T., Schwab, B., Thompson, N. C., Elson, E. L. (2001) Effects of cytochalasin D and latrunculin B on mechanical properties of cells. J. Cell Sci. 114(Pt 5):1025-36.

Actin microfilaments transmit traction and contraction forces generated within a cell to the extracellular matrix during embryonic development, wound healing and cell motility, and to maintain tissue structure and tone. Therefore, the state of the actin cytoskeleton strongly influences the mechanical properties of cells and tissues. Cytochalasin D and Latrunculin are commonly used reagents that, by different mechanisms, alter the state of actin polymerization or the organization of actin filaments. We have investigated the effect of a wide range of Cytochalasin D and Latrunculin B concentrations (from 40 pM to 10 microM) on the mechanical properties of the cells within fibroblast populated collagen matrices. Contractile force and dynamic stiffness were measured by uniaxial stress-strain testing. The range of effective concentrations of Cytochalasin D (200 pM-2 microM) was broader than that of Latrunculin B (20 nM-200 nM). Activating the cells by serum did not change the effective range of Cytochalasin D concentrations but shifted that of Latrunculin B upward by tenfold. Simple mathematical binding models based on the presumed mechanisms of action of Cytochalasin D and Latrunculin B simulated the concentration-dependent mechanical changes reasonably well. This study shows a strong dependence of the mechanical properties of cells and tissues on the organization and degree of polymerization of actin filaments.

Elson, E. (2001) Fluorescence correlation spectroscopy. Springer Series in Chemical Physics 65(Fluorescence Correlation Spectroscopy):1-6.

A review with 17 refs. Recent upsurge of interest to Fluorescence Correlation Spectroscopy (FCS) is related to the fact that FCS offers several advantages for measuring mol. transport and chem. kinetics. FCS is closely related to an earlier optical fluctuation method, quasi-classic light scattering, or dynamic light scattering (DLS).

Elson, E. L. (2001) Fluorescence correlation spectroscopy measures molecular transport in cells. Traffic 2(11):789-96.

Fluorescence correlation spectroscopy (FCS) can measure dynamics of fluorescent molecules in cells. FCS measures the fluctuations in the number of fluorescent molecules in a small volume illuminated by a thin beam of excitation light. These fluctuations are processed statistically to yield an autocorrelation function from which rates of diffusion, convection, chemical reaction, and other processes can be extracted. The advantages of this approach include the ability to measure the mobility of a very small number of molecules, even down to the single molecule level, over a wide range of rates in very small regions of a cell. In addition to rates of diffusion and convection, FCS also provides unique information about the local concentration, states of aggregation and molecular interaction using fluctuation amplitude and cross-correlation methods. Recent advances in technology have rendered these once difficult measurements accessible to routine use in cell biology and biochemistry. This review provides a summary of the FCS method and describes current areas in which the FCS approach is being extended beyond its original scope.

Collier, I. E., Saffarian, S., Marmer, B. L., Elson, E. L., Goldberg, G. (2001) Substrate recognition by gelatinase A: the C-terminal domain facilitates surface diffusion. Biophys. J. 81(4):2370-7.

An investigation of gelatinase A binding to gelatin produced results that are inconsistent with a traditional bimolecular Michaelis-Menten formalism but are effectively accounted for by a power law characteristic of fractal kinetics. The main reason for this inconsistency is that the bulk of the gelatinase A binding depends on its ability to diffuse laterally on the gelatin surface. Most interestingly, we show that the anomalous lateral diffusion and, consequently, the binding to gelatin is greatly facilitated by the C-terminal hemopexin-like domain of the enzyme whereas the specificity of binding resides with the fibronectin-like gelatin-binding domain.

Zahalak, G. I., Wagenseil, J. E., Wakatsuki, T., Elson, E. L. (2000) A cell-based constitutive relation for bio-artificial tissues. Biophys. J. 79(5):2369-81.

By using a combination of continuum and statistical mechanics we derive an integral constitutive relation for bio-artificial tissue models consisting of a monodisperse population of cells in a uniform collagenous matrix. This constitutive relation quantitatively models the dependence of tissue stress on deformation history, and makes explicit the separate contribution of cells and matrix to the mechanical behavior of the composite tissue. Thus microscopic cell mechanical properties can be deduced via this theory from measurements of macroscopic tissue properties. A central feature of the constitutive relation is the appearance of "anisotropy tensors" that embody the effects of cell orientation on tissue mechanics. The theory assumes that the tissues are stable over the observation time, and does not in its present form allow for cell migration, reorientation, or internal remodeling. We have compared the predictions of the theory to uniaxial relaxation tests on fibroblast-populated collagen matrices (FPMs) and find that the experimental results generally support the theory and yield values of fibroblast contractile force and stiffness roughly an order of magnitude smaller than, and viscosity comparable to, the corresponding properties of active skeletal muscle. The method used here to derive the tissue constitutive equation permits more sophisticated cell models to be used in developing more accurate representations of tissue properties.

Wakatsuki, T., Kolodney, M. S., Zahalak, G. I., Elson, E. L. (2000) Cell mechanics studied by a reconstituted model tissue. Biophys. J. 79(5):2353-68.

Tissue models reconstituted from cells and extracellular matrix (ECM) simulate natural tissues. Cytoskeletal and matrix proteins govern the force exerted by a tissue and its stiffness. Cells regulate cytoskeletal structure and remodel ECM to produce mechanical changes during tissue development and wound healing. Characterization and control of mechanical properties of reconstituted tissues are essential for tissue engineering applications. We have quantitatively characterized mechanical properties of connective tissue models, fibroblast-populated matrices (FPMs), via uniaxial stretch measurements. FPMs resemble natural tissues in their exponential dependence of stress on strain and linear dependence of stiffness on force at a given strain. Activating cellular contractile forces by calf serum and disrupting F-actin by cytochalasin D yield "active" and "passive" components, which respectively emphasize cellular and matrix mechanical contributions. The strain-dependent stress and elastic modulus of the active component were independent of cell density above a threshold density. The same quantities for the passive component increased with cell number due to compression and reorganization of the matrix by the cells.

Eschenhagen, T., Fink, C., Rau, T., Remmers, U., Weil, J., Zimmermann, W. H., Aigner, S., Eppenberger, H. M., Wakatsuki, T., Elson, E. L. (2000) Transfection studies using a new cardiac 3D gel system. Molecular Approaches to Heart Failure Therapy:144-156.

A review with 23 refs. This paper summarizes recent progress in reconstituting embryonic chick or neonatal rat cardiac myocytes to a 3-dimensional heart tissue-like structure (\"engineered heart tissue\", EHT). The EHT is anchored to Velcro-coated silicone tubes, by which it can be attached to an isometric force transducer to measure steady (diastolic) and twitch (systolic) forces generated by the myocytes. EHTs exhibit a highly organized network of elongated, mostly longitudinally aligned cardiac myocytes with well-developed myofilaments, cross-striation, and cell-to-cell contacts, which cause EHT to beat coherently. This allows for simple measurement of the isometric force of contraction (0.1-0.5 mN) in std. organ baths. We show that cardiac myocytes within EHTs are easily and effectively transduced with adenovirus coding for .beta.-galactosidase or green fluorescence protein. Adenovirus-mediated gene transfer does not affect basal, calcium, nor isoprenaline-stimulated force of contraction. Thus, EHTs are a suitable model to study the impact of short-term genetic manipulation on force of contraction.

Zutter, M. M., Santoro, S. A., Wu, J. E., Wakatsuki, T., Dickeson, S. K., Elson, E. L. (1999) Collagen receptor control of epithelial morphogenesis and cell cycle progression. Am. J. Pathol. 155(3):927-40.

To define the unique contributions of the alpha subunit cytoplasmic tails of the alpha(1)beta(1) and alpha(2)beta(1) integrin to epithelial differentiation and branching morphogenesis, a variant NMuMG cell line lacking alpha(1)beta(1) and alpha(2)beta(1) integrin expression was stably transfected with the full-length alpha(2) integrin subunit cDNA (X2C2), chimeric cDNA consisting of the extracellular and transmembrane domains of the alpha(2) subunit and the cytoplasmic domain of the alpha(1) subunit (X2C1), or alpha(2) cDNA truncated after the GFFKR sequence (X2C0). The X2C2 and X2C1 transfectants effectively adhered, spread, and formed focal adhesion complexes on type I collagen matrices. The X2C0 transfectants were less adherent to low concentrations of type I collagen, spread less well, and formed poorly defined focal adhesion complexes in comparison to the X2C2 and X2C1 transfectants. The X2C2 and X2C1 transfectants but not the X2C0 transfectants proliferated on collagen substrates. Only the X2C2 transfectants developed elongate branches and tubules in three-dimensional collagen gels and migrated on type I collagen. These findings suggest a unique role for the alpha(2) integrin cytoplasmic domain in postligand binding events and cooperative interactions with growth factors that mediate epithelial differentiation and branching morphogenesis. Either intact alpha(1) or alpha(2) integrin subunit cytoplasmic domain can promote cell cycle progression.

Qian, H., Elson, E. L. (1999) Quantitative study of polymer conformation and dynamics by single-particle tracking. Biophys. J. 76(3):1598-605.

We present a new method for analyzing the dynamics of conformational fluctuations of individual flexible polymer molecules. In single-particle tracking (SPT), one end of the polymer molecule is tethered to an immobile substratum. A microsphere attached to the other end serves as an optical marker. The conformational fluctuations of the polymer molecule can be measured by optical microscopy via the motion of the microsphere. The bead-and-spring theory for polymer dynamics is further developed to account for the microsphere, and together the measurement and the theory yield quantitative information about molecular conformations and dynamics under nonperturbing conditions. Applying the method to measurements carried out on DNA molecules provides information complementary to recent studies of single DNA molecules under extensional force. Combining high precision measurements with the theoretical analysis presented here creates a powerful tool for studying conformational dynamics of biological and synthetic macromolecules at the single-molecule level.

Kucik, D. F., Elson, E. L., Sheetz, M. P. (1999) Weak dependence of mobility of membrane protein aggregates on aggregate size supports a viscous model of retardation of diffusion. Biophys. J. 76(1 Pt 1):314-22.

Proteins in plasma membranes diffuse more slowly than proteins inserted into artificial lipid bilayers. On a long-range scale (>250 nm), submembrane barriers, or skeleton fences that hinder long-range diffusion and create confinement zones, have been described. Even within such confinement zones, however, diffusion of proteins is much slower than predicted by the viscosity of the lipid. The cause of this slowing of diffusion on the micro scale has not been determined and is the focus of this paper. One way to approach this question is to determine the dependence of particle motion on particle size. Some current models predict that the diffusion coefficient of a membrane protein aggregate will depend strongly on its size, while others do not. We have measured the diffusion coefficients of membrane glycoprotein aggregates linked together by concanavalin A molecules bound to beads of various sizes, and also the diffusion coefficients of individual concanavalin A binding proteins. The measurements demonstrate at most a weak dependence of diffusion coefficient on aggregate size. This finding supports retardation by viscous effects, and is not consistent with models involving direct interaction of diffusing proteins with cytoskeletal elements.

Elson, E. L., Felder, S. F., Jay, P. Y., Kolodney, M. S., Pasternak, C. (1999) Forces in cell locomotion. Biochem. Soc. Symp. 65:299-314.

The molecular mechanisms that drive animal cell locomotion are partially characterized, but not definitively understood. It seems likely that actin polymerization contributes to the forward protrusion of the leading edge of a migrating cell. Both myosin-dependent contractile forces and selective detachment of adhesive interactions with the substratum seem to contribute to release of the posterior of an extended cell. It is probable, but not certain, that a separate 'traction' force advances the cell body towards the forward anchorage sites formed by the advancing lamellipodium. The molecular mechanism of this force is unknown. Determining the role of traction forces in migrating fibroblasts and keratocytes is complicated by the fact that the primary functions of the relatively strong forces exerted on the substratum by these cells may be to establish tissue 'tone' and to remodel tissue matrices, rather than to drive locomotion. In accordance with this notion, rapidly moving cells such as neutrophils and Dictyostelium amoebae exert weaker forces on the substratum as they migrate. The traction force in cell migration may be distinct from traction forces with tissue functions. Ultimately, the mechanism may be revealed by using molecular genetics to disrupt the motors that provide this force. Reconstituted tissues provide systems in which to investigate the regulation of cell forces and their contribution to tissue mechanical properties and development.

Ulfendahl, M., Chan, E., McConnaughey, W. B., Prost-Domasky, S., Elson, E. L. (1998) Axial and transverse stiffness measures of cochlear outer hair cells suggest a common mechanical basis. Pflugers Arch. 436(1):9-15.

The function of the hearing organ is based on mechanical processes occurring at the cellular level. The mechanical properties of guinea-pig isolated sensory cells were investigated using two different techniques. The stiffness of the outer hair cells along the longitudinal axis was measured by compressing the cell body using stiffness-calibrated quartz fibres. For cells with a mean length of 69 micron, the mean axial compression stiffness was 1. 1+/-0.8 mN/m (+/-SD). There was an inverse relation between stiffness and cell length. The stiffness of the cell membrane perpendicular to the longitudinal axis of the sensory cell was measured by indenting the cell membrane with a known force. The mean lateral indentation stiffness was 3.3+/-1.5 mN/m (+/-SD) for cells with a mean length of 64 microm. Longer cells were less stiff than short cells. Modelling the hair cell as a shell with bending resistance, finite element calculations demonstrated that the axial compression stiffness correlated well with the lateral indentation stiffness, and that a simple isotropic model is sufficient to explain the experimental observations despite the different stress strain states produced by the two techniques. The results imply that the two different stiffness properties may originate from the same cytoskeletal structures. It is suggested that the mechanical properties of the outer hair cells are designed to influence the sound-induced motion of the reticular lamina. In such a system, stiffness changes of the outer hair cell bodies could actively control the efficiency of the mechanical coupling between the basilar membrane and the important mechanoelectrical transduction sites at the surface of the hearing organ.

Luna, E. J., Hitt, A. L., Shutt, D., Wessels, D., Soll, D., Jay, P., Hug, C., Elson, E. L., Vesley, A., Downey, G. P. and others. (1998) Role of ponticulin in pseudopod dynamics, cell-cell adhesion, and mechanical stability of an amoeboid membrane skeleton. Biol. Bull. 194(3):345-6; discussion 346-7.

Cai, S., Pestic-Dragovich, L., O'Donnell, M. E., Wang, N., Ingber, D., Elson, E., De Lanerolle, P. (1998) Regulation of cytoskeletal mechanics and cell growth by myosin light chain phosphorylation. Am. J. Physiol. 275(5 Pt 1):C1349-56.

The role of myosin light chain phosphorylation in regulating the mechanical properties of the cytoskeleton was studied in NIH/3T3 fibroblasts expressing a truncated, constitutively active form of smooth muscle myosin light chain kinase (tMK). Cytoskeletal stiffness determined by quantifying the force required to indent the apical surface of adherent cells showed that stiffness was increased twofold in tMK cells compared with control cells expressing the empty plasmid (Neo cells). Cytoskeletal stiffness quantified using magnetic twisting cytometry showed an approximately 1.5-fold increase in stiffness in tMK cells compared with Neo cells. Electronic volume measurements on cells in suspension revealed that tMK cells had a smaller volume and are more resistant to osmotic swelling than Neo cells. tMK cells also have smaller nuclei, and activation of mitogen-activated protein kinase (MAP kinase) and translocation of MAP kinase to the nucleus are slower in tMK cells than in control cells. In tMK cells, there is also less bromodeoxyuridine incorporation, and the doubling time is increased. These data demonstrate that increased myosin light chain phosphorylation correlates with increased cytoskeletal stiffness and suggest that changing the mechanical characteristics of the cytoskeleton alters the intracellular signaling pathways that regulate cell growth and division.

Eschenhagen, T., Fink, C., Remmers, U., Scholz, H., Wattchow, J., Weil, J., Zimmermann, W., Dohmen, H. H., Schafer, H., Bishopric, N. and others. (1997) Three-dimensional reconstitution of embryonic cardiomyocytes in a collagen matrix: a new heart muscle model system. FASEB J. 11(8):683-94.

A method has been developed for culturing cardiac myocytes in a collagen matrix to produce a coherently contracting 3-dimensional model heart tissue that allows direct measurement of isometric contractile force. Embryonic chick cardiomyocytes were mixed with collagen solution and allowed to gel between two Velcro-coated glass tubes. During culture, the cardiomyocytes formed spontaneously beating cardiac myocyte-populated matrices (CMPMs) anchored at opposite ends to the Velcro-covered tubes through which they could be attached to a force measuring system. Immunohistochemistry and electron microscopy revealed a highly organized tissue-like structure of alpha-actin and alpha-tropomyosin-positive cardiac myocytes exhibiting typical cross-striation, sarcomeric myofilaments, intercalated discs, desmosomes, and tight junctions. Force measurements of paced or unpaced CMPMs were performed in organ baths after 6-11 days of cultivation and were stable for up to 24 h. Force increased with frequency between 0.8 and 2.0 Hz (positive "staircase"), increasing rest length (Starling mechanism), and increasing extracellular calcium. The utility of this system as a test bed for genetic manipulation was demonstrated by infecting the CMPMs with a recombinant beta-galactosidase-carrying adenovirus. Transduction efficiency increased from about 5% (MOI 0.1) to about 50% (MOI 100). CMPMs display more physiological characteristics of intact heart tissue than monolayer cultures. This approach, simpler and faster than generation of transgenic animals, should allow functional consequences of genetic or pharmacological manipulation of cardiomyocytes in vitro to be studied under highly controlled conditions.

Hecht, G., Pestic, L., Nikcevic, G., Koutsouris, A., Tripuraneni, J., Lorimer, D. D., Nowak, G., Guerriero, V., Jr., Elson, E. L., Lanerolle, P. D. (1996) Expression of the catalytic domain of myosin light chain kinase increases paracellular permeability. Am. J. Physiol. 271(5 Pt 1):C1678-84.

Contractile events resulting from phosphorylation of the 20-kDa myosin light chain (MLC20) have been implicated in the regulation of epithelial tight junction permeability. To address this question, Madin-Darby canine kidney cells were transfected with a murine leukemia retroviral vector containing DNA encoding either the catalytic domain of myosin light chain kinase (tMK) or the beta-galactosidase gene (beta-gal). Autoradiograms of sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of myosin immunoprecipitated from 32Pi-labeled transfected cells demonstrated that MLC20 phosphorylation was increased 3.1 +/- 0.9-fold in cells expressing tMK compared with cells expressing beta-gal. Phosphopeptide mapping confirmed that myosin light chain kinase was responsible for the increased MLC20 phosphorylation. Transepithelial electrical resistance, a measurement of barrier function, of tMK cell monolayers was consistently < 10% (123 +/- 20 omega.cm2) of that of monolayers comprised of wild-type cells (1,456 +/- 178 omega.cm2) or cells expressing beta-gal (1,452 +/- 174 omega.cm2). Dual 22Na+ and [3H]mannitol flux studies indicated that the decrease in resistance in tMK cells was attributable to increased paracellular flow. These data support the idea that MLC20 phosphorylation by myosin light chain kinase is involved in regulating epithelial tight junction permeability.

Pasternak, C., Wong, S., Elson, E. L. (1995) Mechanical function of dystrophin in muscle cells. J. Cell Biol. 128(3):355-61.

We have directly measured the contribution of dystrophin to the cortical stiffness of living muscle cells and have demonstrated that lack of dystrophin causes a substantial reduction in stiffness. The inferred molecular structure of dystrophin, its preferential localization underlying the cell surface, and the apparent fragility of muscle cells which lack this protein suggest that dystrophin stabilizes the sarcolemma and protects the myofiber from disruption during contraction. Lacking dystrophin, the muscle cells of persons with Duchenne muscular dystrophy (DMD) are abnormally vulnerable. These facts suggest that muscle cells with dystrophin should be stiffer than similar cells which lack this protein. We have tested this hypothesis by measuring the local stiffness of the membrane skeleton of myotubes cultured from mdx mice and normal controls. Like humans with DMD mdx mice lack dystrophin due to an x-linked mutation and provide a good model for the human disease. Deformability was measured as the resistance to indentation of a small area of the cell surface (to a depth of 1 micron) by a glass probe 1 micron in radius. The stiffness of the membrane skeleton was evaluated as the increment of force (mdyne) per micron of indentation. Normal myotubes with an average stiffness value of 1.23 +/- 0.04 (SE) mdyne/micron were about fourfold stiffer than myotubes cultured from mdx mice (0.34 +/- 0.014 mdyne/micron). We verified by immunofluorescence that both normal and mdx myotubes, which were at a similar developmental stage, expressed sarcomeric myosin, and that dystrophin was detected, diffusely distributed, only in normal, not in mdx myotubes. These results confirm that dystrophin and its associated proteins can reinforce the myotube membrane skeleton by increasing its stiffness and that dystrophin function and, therefore, the efficiency of therapeutic restoration of dystrophin can be assayed through its mechanical effects on muscle cells.

Obara, K., Nikcevic, G., Pestic, L., Nowak, G., Lorimer, D. D., Guerriero, V., Jr., Elson, E. L., Paul, R. J., de Lanerolle, P. (1995) Fibroblast contractility without an increase in basal myosin light chain phosphorylation in wild type cells and cells expressing the catalytic domain of myosin light chain kinase. J. Biol. Chem. 270(32):18734-7.

We investigated the role of myosin light chain (MLC20) phosphorylation (MLC-P) in non-muscle contractility by comparing MLC-P and the contractile properties of wild type 3T3 fibroblasts and 3T3 fibroblasts expressing the catalytic domain of myosin light chain kinase (tMK). MLC-P is 0.96 MOL of PO4/mol of MOL20 in cell expressing tMK compared to 0.20 mol of PO4/mol of MLC20 in control cells. Expressing tMK also results in a 2-fold increase in cortical stiffness compared to control cells. Contractile properties were quantified by growing wild type and transfected fibroblasts in collagen and attaching the ensuing fibers to an apparatus for performing mechanical measurements. Serum stimulation resulted in a dose-dependent increase in force with maximal force generated in the presence of 30% (v/v) serum. Surprisingly, MLC-P did not increase in wild type cells following stimulation with 30% serum, and tMK expression did not affect the contractile properties of fibers made from these cells. Moreover, the dose responses to serum, maximal force, force-velocity relationships, and dynamic stiffness were similar in the wild type cells and fibroblasts expressing tMK. These data demonstrate that non-muscle cells can generate force without an increase in MLC-P, and that an increase in MLC-P does not affect the contractile properties of fibroblast fibers.

Kolodney, M. S., Elson, E. L. (1995) Contraction due to microtubule disruption is associated with increased phosphorylation of myosin regulatory light chain. Proc. Natl. Acad. Sci. U. S. A. 92(22):10252-6.

Microtubules have been proposed to function as rigid struts which oppose cellular contraction. Consistent with this hypothesis, microtubule disruption strengthens the contractile force exerted by many cell types. We have investigated alternative explanation for the mechanical effects of microtubule disruption: that microtubules modulate the mechanochemical activity of myosin by influencing phosphorylation of the myosin regulatory light chain (LC20). We measured the force produced by a population of fibroblasts within a collagen lattice attached to an isometric force transducer. Treatment of cells with nocodazole, an inhibitor of microtubule polymerization, stimulated an isometric contraction that reached its peak level within 30 min and was typically 30-45% of the force increase following maximal stimulation with 30% fetal bovine serum. The contraction following nocodazole treatment was associated with a 2- to 4-fold increase in LC20 phosphorylation. The increases in both force and LC20 phosphorylation, after addition of nocodazole, could be blocked or reversed by stabilizing the microtubules with paclitaxel (former generic name, taxol). Increasing force and LC20 phosphorylation by pretreatment with fetal bovine serum decreased the subsequent additional contraction upon microtubule disruption, a finding that appears inconsistent with a load-shifting mechanism. Our results suggest that phosphorylation of LC20 is a common mechanism for the contractions stimulated both by microtubule poisons and receptor-mediated agonists. The modulation of myosin activity by alterations in microtubule assembly may coordinate the physiological functions of these cytoskeletal components.

Jay, P. Y., Pham, P. A., Wong, S. A., Elson, E. L. (1995) A mechanical function of myosin II in cell motility. J. Cell Sci. 108 ( Pt 1):387-93.

Myosin II mutant Dictyostelium amoebae crawl more slowly than wild-type cells. Thus, myosin II must contribute to amoeboid locomotion. We propose that contractile forces generated by myosin II help the cell's rear edge to detach from the substratum and retract, allowing the cell to continue forward. To test this hypothesis, we measured the speed of wild-type and myosin II null mutant Dictyostelium cells on surfaces of varying adhesivity. As substratum adhesivity increased, the speed of myosin II null mutant cells decreased substantially compared to wild-type cells, suggesting that the mutant is less able to retract from sticky surfaces. Furthermore, interference reflection microscopy revealed a myosin-II-dependent contraction in wild-type but not null mutant cells that is consistent with a balance of adhesive and contractile forces in retraction. Although myosin II null mutant cells have a defect in retraction, pseudopod extension does not cause the cells to become elongated on sticky surfaces. This suggests a mechanism, based possibly on cytoskeletal tension, for regulating cell shape in locomotion. The tension would result from the transmission of tractional forces through the cytoskeletal network, providing the myosin II null mutant with a limited means of retraction and cell division on a surface.

Hug, C., Jay, P. Y., Reddy, I., McNally, J. G., Bridgman, P. C., Elson, E. L., Cooper, J. A. (1995) Capping protein levels influence actin assembly and cell motility in dictyostelium. Cell 81(4):591-600.

Actin assembly is important for cell motility, but the mechanism of assembly and how it relates to motility in vivo is largely unknown. In vitro, actin assembly can be controlled by proteins, such as capping protein, that bind filament ends. To investigate the function of actin assembly in vivo, we altered the levels of capping protein in Dictyostelium cells and found changes in resting and chemoattractant-induced actin assembly that were consistent with the in vitro properties of capping protein in capping but not nucleation. Significantly, overexpressers moved faster and underexpressers moved slower than control cells. Mutants also exhibited changes in cytoskeleton architecture. These results provide insights into in vivo actin assembly and the role of the actin cytoskeleton in motility.

Doherty, D. E., Downey, G. P., Schwab, B., 3rd, Elson, E., Worthen, G. S. (1994) Lipolysaccharide-induced monocyte retention in the lung. Role of monocyte stiffness, actin assembly, and CD18-dependent adherence. J. Immunol. 153(1):241-55.

Blood monocytes and monocyte-derived macrophages accumulate in the lungs and can modulate pulmonary inflammatory and reparative processes through their elaboration of cytokines and growth factors. Endotoxemia, often a prelude to acute lung injury, induces a monocytopenia, likely resulting from monocyte accumulation in the lung. We hypothesized that LPS would induce monocyte lung retention by increasing monocyte stiffness and thereby diminishing the cell's ability to deform and transit the narrow pulmonary capillary network, and that LPS would induce CD18-dependent adhesion of monocytes to endothelium, prolonging their retention. LPS induced a rapid and concentration-dependent increase in human monocyte stiffness, net filamentous actin assembly, and retention in a filtration model of pulmonary capillaries. These LPS-induced responses were dependent on the integrity of actin filaments in that cytochalasin D, an agent that disrupts filamentous actin assembly, attenuated each of these processes. LPS induced CD18-dependent and -independent human monocyte adhesion to unstimulated human endothelial cell monolayers. In vivo, rabbit monocytes were retained in the lungs of animals rendered endotoxemic. Pretreatment of monocytes ex vivo with LPS enhanced their lung retention suggesting that LPS was acting directly on monocytes. Initial lung retention during endotoxemia was attenuated by inhibiting monocyte F-actin assembly with cytochalasin D. Anti-CD18 Abs caused a slight decrease in initial retention of monocytes, but led to a 90% inhibition of retention by 2 h. Control IgG had no effect. These data suggest that the initial retention of monocytes in the lung during endotoxemia is dependent on alterations in their stiffness and assembly/organization of F-actin, and that CD18-dependent adhesive mechanisms prolong monocyte retention in the lung during this process.

Kolodney, M. S., Elson, E. L. (1993) Correlation of myosin light chain phosphorylation with isometric contraction of fibroblasts. J. Biol. Chem. 268(32):23850-5.

In vitro studies have indicated that the enzymatic activity of myosin II from non-muscle cells is controlled by phosphorylation of its regulatory light chain (LC20). We have studied one likely functional consequence of phosphorylating LC20 in living chick embryo fibroblasts (CEF) by measuring contractile force developed by these cells. Using a recently developed method, we recorded quantitative changes in isometric force generated by a population of cells following mitogenic stimulation. Fetal bovine serum, thrombin, and lysophosphatidic acid stimulate rapid isometric contraction of CEF. Cells stimulated with thrombin develop maximal force within 5-10 min. Force development correlates temporally with a 3-5-fold increase in the overall fraction of LC20 phosphorylated and with the fractions of LC20 in both the monophosphorylated and diphosphorylated states. Unloaded shortening velocity also increases after thrombin stimulation. Although both force and phosphorylation begin to decline 10 min after stimulation, the level of phosphorylation declined more rapidly than the force. These results suggest that the role of LC20 phosphorylation in regulating fibroblast contractility is analogous to its well established role in regulating smooth muscle contraction and that quantitative measurements of the force developed by populations of fibroblasts (or other cultured cells) can be used to study the regulation of non-sarcomeric myosin at the molecular level in vivo.

Jay, P. Y., Pasternak, C., Elson, E. L. (1993) Studies of mechanical aspects of amoeboid locomotion. Blood Cells 19(2):375-86; discussion 386-8.

When a cell crawls over a surface, it exerts forces which both change its shape and deformability and propel it forward. The mechanisms involved are poorly understood. They can best be studied by combining biochemical and molecular genetic methods with direct, quantitative measurements of mechanical properties. Measurements of cellular deformability provide indications of contractile tension developed within the cell and of cytoskeletal reorganizations which influence local cellular viscoelasticity. An example is the capping of cross-linked cell surface proteins, which occurs on cells as diverse as mammalian lymphocytes and the unicellular amoeba, Dictyostelium discoideum. Deformability measurements show that cells stiffen as they cap. Measurements on wild-type Dictyostelium cells and on cells engineered to lack conventional myosin (myosin II) demonstrate that capping requires myosin II and that the concurrent cellular stiffening results from a myosin-II-dependent contractile force. Measurements of the systematic transport of beads rearward over the surfaces of cells characterize a mechanism of movement which could be used to drive the cell forward. Capping is one such mechanism. A distinct myosin-II-independent form of rearward transport is revealed in measurements of fluorescent beads on the Dictyostelium cells which lack this protein. In addition to studies of cell locomotion, measurements of cellular mechanical properties can provide quantitative assays of the functions of cytoskeletal components. Such studies are motivated by the nature of cytoskeletal proteins whose function, in contrast to enzymes, are mechanical rather that catalytic.

Elson, E. (1993) Barriers to diffusion. Curr. Biol. 3(3):152-4.

A review, with 20 refs., on cell membrane domain formation and maintenance. The targeting, trapping and barrier-limited diffusion of membrane components (proteins, ion channels, receptors etc.) in polarized epithelium, neuromuscular junction, and neuron, resp., are discussed.