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

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. (2001a) 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. (2001b) 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.