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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.