Increased Cell Stiffness Research Articles

Mechanical Stiffness Grades Metastatic Potential in Patient Tumor Cells and in Cancer Cell Lines

Cancer cells are defined by their ability to invade through the basement membrane, a critical step during metastasis. While increased secretion of proteases, which facilitates degradation of the basement membrane, and alterations in the cytoskeletal architecture of cancer cells have been previously studied, the contribution of the mechanical properties of cells in invasion is unclear. Here, we applied a magnetic tweezer system to establish that stiffness of patient tumor cells and cancer cell lines inversely correlates with migration and invasion through three-dimensional basement membranes, a correlation known as a power law. We found that cancer cells with the highest migratory and invasive potential are five times less stiff than cells with the lowest migration and invasion potential. Moreover, decreasing cell stiffness by pharmacologic inhibition of myosin II increases invasiveness, whereas increasing cell stiffness by restoring expression of the metastasis suppressor TβRIII/betaglycan decreases invasiveness. These findings are the first demonstration of the power-law relation between the stiffness and the invasiveness of cancer cells and show that mechanical phenotypes can be used to grade the metastatic potential of cell populations with the potential for single cell grading. The measurement of a mechanical phenotype, taking minutes rather than hours needed for invasion assays, is promising as a quantitative diagnostic method and as a discovery tool for therapeutics. By showing that altering stiffness predictably alters invasiveness, our results indicate that pathways regulating these mechanical phenotypes are novel targets for molecular therapy of cancer.

Novel tools for investigations of cytoskeletal re‐arrangement during alveolar epithelial‐mesenchymal transition

Inherent in epithelial‐mesenchymal transition (EMT) is the establishment of an elongated cell morphology and formation of stress fibres. The influence of these cellular alterations on the biomechanics of lung parenchyma remains unclear. Alveolar A549 cells, after exposure to TGF‐β1, were studied using atomic force microscopy (AFM) and high content analysis (HCA) imaging.A549 cells were grown with or without TGF‐β1 in the presence or absence of a collagen I substrate for 72 h. Using AFM, the Young′s modulus of living cells was measured in contact mode. Surface membrane features were assessed on fixed cells using intermittent contact mode. In parallel, morphological and fluorescence staining intensity/distribution parameters were evaluated using a HCA imaging system.TGF‐β1 resulted in a greater than two‐fold increase in cell stiffness. This effect was augmented in the presence of collagen I. TGF‐β1‐treated cells also exhibiting a rougher surface profile with notable protrusions. In accordance with these findings, concurrent quantitative examination of the morphological attributes of stimulated cells using HCA, revealed dramatic alterations in cell shape, F‐actin content and distribution.These data indicate a strong correlation between the cytoskeletal‐associated cellular architecture and the mechanical dynamics of alveolar epithelial cells undergoing EMT.

Effects of Morphology vs. Cell–Cell Interactions on Endothelial Cell Stiffness

Biological processes such as atherogenesis, wound healing, cancer cell metastasis, and immune cell transmigration rely on a delicate balance between Cell-Cell and cell-substrate adhesion. Cell mechanics have been shown to depend on substrate factors such as stiffness and ligand presentation, while the effects of Cell-Cell interactions on the mechanical properties of cells has received little attention. Here, we use atomic force microscopy to measure the Young's modulus of live human umbilical vein endothelial cells (HUVECs). In varying the degree of Cell-Cell contact in HUVECs (single cells, groups, and monolayers), we observe that increased cell stiffness correlates with an increase in cell area. Further, we observe that HUVECs stiffen as they spread onto a glass substrate. When we weaken Cell-Cell junctions (i.e., through a low dose of cytochalasin B or treatment with a VE-cadherin antibody), we observe that cell-substrate adhesion increases, as measured by focal adhesion size and density, and the stiffness of cells within the monolayer approaches that of single cells. Our results suggest that while morphology can roughly be used to predict cell stiffness, Cell-Cell interactions may play a significant role in determining the mechanical properties of individual cells in tissues by careful maintenance of cell tension homeostasis.

OxLDL-induced decrease in lipid order of membrane domains is inversely correlated with endothelial stiffness and network formation

Oxidized low-density lipoprotein (oxLDL) is a major factor in development of atherosclerosis. Our earlier studies have shown that exposure of endothelial cells (EC) to oxLDL increases EC stiffness, facilitates the ability of the cells to generate force, and facilitates EC network formation in three-dimensional collagen gels. In this study, we show that oxLDL induces a decrease in lipid order of membrane domains and that this effect is inversely correlated with endothelial stiffness, contractility, and network formation. Local lipid packing of cell membrane domains was assessed by Laurdan two-photon imaging, endothelial stiffness was assessed by measuring cellular elastic modulus using atomic force microscopy, cell contractility was estimated by measuring the ability of the cells to contract collagen gels, and EC angiogenic potential was estimated by visualizing endothelial networks within the same gels. The impact of oxLDL on endothelial biomechanics and network formation is fully reversed by supplying the cells with a surplus of cholesterol. Furthermore, exposing the cells to 7-keto-cholesterol, a major oxysterol component of oxLDL, or to another cholesterol analog, androstenol, also results in disruption of lipid order of membrane domains and an increase in cell stiffness. On the basis of these observations, we suggest that disruption of lipid packing of cholesterol-rich membrane domains plays a key role in oxLDL-induced changes in endothelial biomechanics.

Modification of Cellular Cholesterol Content Affects Traction Force, Adhesion and Cell Spreading

Cellular cholesterol is a critical component of the plasma membrane, and plays a key role in determining the physical properties of the lipid bilayer, such as elasticity, viscosity, and permeability. Surprisingly, it has been shown that cholesterol depletion increases cell stiffness, not due to plasma membrane stiffening, but rather, due to the interaction between the actin cytoskeleton and the plasma membrane. This indicates that traction stresses of the acto-myosin complex likely increase during cholesterol depletion. Here we use force traction microscopy to quantify the forces individual cells are exerting on the substrate, and total internal reflection fluorescence microscopy as well as interference reflection microscopy to observe cell-substrate adhesion and spreading. We show that single cells depleted of cholesterol produce larger traction forces and have large focal adhesions compared to untreated or cholesterol-enriched cells. Cholesterol depletion also causes a decrease in adhesion area for both single cells and monolayers. Spreading experiments illustrate a decrease in spreading area for cholesterol-depleted cells, and no effect on cholesterol-enriched cells. These results demonstrate that cholesterol plays an important role in controlling and regulating the cell-substrate interactions through the actin-plasma membrane complex, cell-cell adhesion, and spreading.

Cholesterol Depletion Facilitates Recovery from Hypotonic Cell Swelling of CHO

The maintenance of cell volume homeostasis prevents pathological cell swelling that can lead to severe cellular dysfunction or death. A key step in maintaining cell volume in many cell types is activation of volume-regulated anion channels (VRAC). Our earlier studies showed that activity of VRAC is facilitated by a decrease in cellular cholesterol (Levitan et al 2000). These observations suggest that lowered cholesterol should also facilitate regulatory volume decrease (RVD), a process used by cells to recover from hypotonic swelling. The main constraint in testing this prediction, however, has been the lack of adequate methods to rapidly and reproducibly measure changes in cell volume of substrate-attached cells. In this study, we address this question using a novel microfluidic methodology from Reichert Inc. (CVC-7000), to measure cell volume response to hypotonic challenges (30% osmotic gradient) in real time. Cholesterol depletion facilitated the recovery from swelling via a more rapid onset of RVD (∼130 s vs. 215 s in control and cholesterol depleted cells, respectively) and a higher degree of volume recovery after 10 min (41%±6% vs. 65%±6% in control and cholesterol depleted cells, respectively). In contrast, enriching the cells with cholesterol had no effect on RVD. These observations are consistent with our previous studies showing that while cholesterol depletion increases cell stiffness, cholesterol enrichment has no effect (Byfield et al 2004). These observations suggest that cholesterol depletion, and the consequent increase in cell stiffness, facilitates RVD by enhancing the activity of VRAC.

Effects of the Initial Orientation of Actin Fibers on Global Tensile Properties of Cells

Tensile tests of a single cell were simulated in order to understand the effects of the initial orientation of actin fibers (AFs) on global tensile properties. The properties examined included cell deformation, stiffness, and AF behavior. In the model used, the mechanical properties of cellular components, including the cell membrane with an associated actin network, nuclear envelope, and AFs, are expressed as a result of springs that generate force as a function of their extension. Cell shape during the tensile test was determined by a quasi-static approach couched in the framework of the minimum energy concept. Cells with various initial AF orientations were prepared; in particular, AFs in four different initial orientations, namely random (mean ± SD of the initial orientation angle, 46.4 ± 26.9°), parallel to the stretched direction (3.8 ± 3.5°), perpendicular (85.9 ± 2.6°), and diagonally oriented (44.5 ± 3.6°) were examined. The results show a significant drop in initial stiffness with an increase in mean initial AF orientation angles of 0 to 45°. The initial stiffness of the cell with parallel-oriented AFs was much larger than that with perpendicularly oriented AFs. The results also demonstrate that cell elongation induces a passive reorientation of AFs in a stretched direction, thereby causing an increase in cell stiffness. When comparing the rate of change for cell stiffness of the diagonally oriented model with that of the randomly oriented model, our data reveal that the rate of change of cell stiffness is characterized not only by the mean of the initial AF orientation angle, but also by the variation of their distribution.

Aging-related differences in chondrocyte viscoelastic properties.

The biomechanical properties of articular cartilage change profoundly with aging. These changes have been linked with increased potential for cartilage degeneration and osteoarthritis. However, less is known about the change in biomechanical properties of chondrocytes with increasing age. Cell stiffness can affect mechanotransduction pathways and may alter cell function. We measured aging-related changes in the biomechanical properties of chondrocytes. Human chondrocytes were isolated from knee articular cartilage within 48 hours after death or from osteochondral specimens obtained from knee arthroplasty. Cells were divided into two age groups: between 18 and 35 years (18 - 35); and greater than 55 years (55+) of age. The 55+ group was further subdivided based on visual grade of osteoarthritis: normal (N) or osteoarthritic (OA). The viscoelastic properties of the cell were measured using the previously described micropipette cell aspiration technique. The equilibrium modulus, instantaneous modulus, and apparent viscosity were significantly higher in the 55+ year age group than in the 18 - 35 age group. On the other hand, no differences were found in the equilibrium modulus, instantaneous modulus, or apparent viscosity between the N and OA groups. The increase in cell stiffness can be attributed to altered mechanical properties of the cell membrane, the cytoplasm, or the cytoskeleton. Increased stiffness has been reported in osteoarthritic chondrocytes, which in turn has been attributed to the actin cytoskeleton. A similar mechanism may be responsible for our finding of increased stiffness in aging chondrocytes. With advancing age, changes in the biomechanical properties of the cell could alter molecular and biochemical responses.

Impact Of Oxysterols On Endothelial Elastic Properties, Contractility And Morphogenesis

Our previous studies, have shown that exposing bovine aortic endothelial cells (BAECs) to oxLDL resulted in an increase in cell stiffness, force generation, and endothelial network formation. The mechanisms responsible for these effects, however, were completely not clear. Since all three effects could be simulated by cholesterol depletion, we suggested that oxLDL may be mediated changes in the membrane cholesterol. However, the total amount of the cholesterol remains the same after exposing oxLDL to BAECs. To resolve this discrepancy, we tested whether oxLDL-induced effects can be reversed by supplying a surplus of cholesterol. To achieve this goal, we used the MBCD complexed with cholesterol, a known cholesterol donor. In all the experiments, cells were exposed first to oxLDL for one hour and then subsequently to MBCD-cholesterol for an additional 1 hour. Our observations show that after treating with MBCD -cholesterol, cell elastic modulus, force generation and network formation were back to the normal level as compared to control cells. It suggests that cholesterol plays an important role in oxLDL induced cell mechanics. Furthermore, we show that the impact of oxLDL on endothelial biomechanics and morphogenesis can be simulated by specific oxysterols, known components of oxLDL. Here, we show that specific oxysterols have distinct effects on endothelial biomechanical properties. Specifically, we tested four different oxysterol species that are present in oxLDL: 7B- hydroxycholesterol , 7-ketocholesterol , 25-hydroxycholesterol and 27-hydroxycholesterol to study their mechanic properties. The results show that 7k and 27HC are potent factors to increase cell elastic modulus. However, only 27HC raises the force generation and network formation. In contrast, exposing the cells to Bacterial SMase C to hydrolyze of sphingomyelin didn't affect neither endothelial biomechanics not morphogenesis indicating that OxLDL-induced stimulation of SMAse cannot be responsible for these effects.

Increased leukaemia cell stiffness is associated with symptoms of leucostasis in paediatric acute lymphoblastic leukaemia

Increased leukaemia cell stiffness is associated with symptoms of leucostasis in paediatric acute lymphoblastic leukaemia

Modelling of in vitro chondrocyte detachment

The experimental work of Huang et al. [Huang, W., Bahman, A., Torres, J.H., LeBaron R.G., Athanasiou, K.A., 2003. Temporal effects of cell adhesion on mechanical characteristics of the single chondrocyte. J. Orthop. Res. 21, 88–95] provides a novel experimental characterisation of the force required to detach single chondrocytes from a flat substrate with a horizontally moving probe. Measurements reveal that the force required to detach cells spread for six hours is eleven times greater than that required to detach cells spread for one hour. However, it is not clear if this phenomenon is entirely due to geometric changes of chondrocytes during spreading and the consequent increase in adhesion area between cell and substrate. The current computational study, using experimentally measured cell geometries and a cohesive zone model to simulate the cell–substrate interface, reveals that this is not the case. It is demonstrated that both an increase in cell stiffness and an increase in the cell–substrate interface strength during cell spreading are necessary to explain the experimental measurements. It is also revealed that the mechanism of cell detachment involves exclusively tangential debonding, with no lifting of the cell taking place at the cell–substrate interface. Finally, the effect of discrete focal adhesion regions on cell detachment is considered.

A theoretical model for F-actin remodeling in vascular smooth muscle cells subjected to cyclic stretch

A constrained mixture theory model was developed and used to estimate remodeling of F-actin in vascular smooth muscle cells that were subjected to 10% equibiaxial stretching for up to 30 min. The model was based on a synthesis of data on time-dependent changes in atomic force microscopy measured cell stiffness and immunofluorescence measured focal adhesion associated vinculin as well as data on stress fiber stiffness and pre-stretch. Results suggest that an observed acute (after 2 min of stretching) increase in cell stiffness is consistent with an increased stretch of the originally present F-actin plus an assembly of new F-actin having nearly homeostatic values of stretch. Moreover, the subsequent (after 30 min of stretching) decrease in cell stiffness back towards the baseline value is consistent with a replacement of the overstretched original filaments with the new (reassembled), less stretched filaments. That is, overall cell response is consistent with a recently proposed concept of “tensional homeostasis” whereby cells seek to maintain constant certain mechanical factors via a remodeling of intracellular and transmembrane proteins. Although there is a need to refine the model based on more comprehensive data sets, using multiple experimental approaches, the present results suggest that a constrained mixture theory can capture salient features of the dynamics of F-actin remodeling and that it offers some advantages over many past methods of modeling, particularly those based on classical linearized viscoelasticity.

Adaptation of cellular mechanical behavior to mechanical loading for osteoblastic cells

Numerous cellular biochemical responses to mechanical loading are transient, indicating a cell's ability to adapt its behavior to a new mechanical environment. Since load-induced cellular deformation can initiate these biochemical responses, the overall goal of this study was to investigate the adaptation of global, or whole-cell, mechanical behavior, i.e., cellular deformability, in response to mechanical loading for osteoblastic cells. Confluent cell cultures were subjected to 1 or 2 Pa flow-induced shear stress for 2 h. Whole-cell mechanical behavior was then measured for individual cells using an atomic force microscope. Compared to cells maintained under static conditions, whole-cell stiffness was 1.36-fold ( p = 0.006 ) and 1.70-fold ( p<0.001) greater for cells exposed to 1 and 2 Pa shear loading, respectively. The increase in shear stress magnitude from 1 to 2 Pa also caused a statistically significant, 1.25-fold increase in cell stiffness ( p = 0.02 ). Increases in cell stiffness were not altered in either flow group for 70 min after flow was terminated ( p = 0.15 ). Flow-induced rearrangement of the actin cytoskeleton was also maintained for at least 90 min after flow was terminated. Taken together, these findings support the hypothesis that cells become mechanically adapted to their mechanical environment via cytoskeletal modifications. Accordingly, cellular mechanical adaptation may play a key role in regulation of cellular mechanosensitivity and the related effects on tissue structure and function.

Substrate modulation of osteoblast adhesion strength, focal adhesion kinase activation, and responsiveness to mechanical stimuli.

Osteoblast interactions with extracellular matrix (ECM) proteins are known to influence many cell functions, which may ultimately affect osseointegration of implants with the host bone tissue. Some adhesion-mediated events include activation of focal adhesion kinase, and subsequent changes in the cytoskeleton and cell morphology, which may lead to changes in adhesion strength and cell responsiveness to mechanical stimuli. In this study we examined focal adhesion kinase activation (FAK), F-actin cytoskeleton reorganization, adhesion strength, and osteoblast responsiveness to fluid shear when adhered to type I collagen (ColI), glass, poly-L-lysine (PLL), fibronectin (FN), vitronectin (VN), and serum (FBS). In general, surfaces that bind cells through integrins (FN, VN, FBS) elicited the highest adhesion strength, FAK activation, and F-actin stress fiber formation after both 15 and 60 minutes of adhesion. In contrast, cells attached through non-integrin mediated means (PLL, glass) showed the lowest FAK activation, adhesion strength, and little F-actin stress fiber formation. When subjected to steady fluid shear using a parallel plate flow chamber, osteoblasts plated on FN released significantly more PGE2 compared to those on glass. In contrast, PGE2 release of osteoblasts attached to FN or glass was not different in the absence of fluid shear, suggesting that differences in binding alone are insufficient to alter PGE2 secretion. The increased adhesion strength as well as PGE2 secretion of osteoblasts adhered via integrins may be due to increased F-actin fiber formation, which leads to increased cell stiffness.

Hypoxia alters biophysical properties of endothelial cells via p38 MAPK- and Rho kinase-dependent pathways

Hypoxia alters the barrier function of the endothelial cells that line the pulmonary vasculature, but underlying biophysical mechanisms remain unclear. Using rat pulmonary microvascular endothelial cells (RPMEC) in culture, we report herein changes in biophysical properties, both in space and in time, that occur in response to hypoxia. We address also the molecular basis of these changes. At the level of the single cell, we measured cell stiffness, the distribution of traction forces exerted by the cell on its substrate, and spontaneous nanoscale motions of microbeads tightly bound to the cytoskeleton (CSK). Hypoxia increased cell stiffness and traction forces by a mechanism that was dependent on the activation of Rho kinase. These changes were followed by p38-mediated decreases in spontaneous bead motions, indicating stabilization of local cellular-extracellular matrix (ECM) tethering interactions. Cells overexpressing phospho-mimicking small heat shock protein (HSP27-PM), a downstream effector of p38, exhibited decreases in spontaneous bead motions that correlated with increases in actin polymerization in these cells. Together, these findings suggest that hypoxia differentially regulates endothelial cell contraction and cellular-ECM adhesion.

Localized mechanical stress induces time-dependent actin cytoskeletal remodeling and stiffening in cultured airway smooth muscle cells.

Mechanical stress (MS) causes cytoskeletal (CSK) and phenotypic changes in cells. Such changes in airway smooth muscle (ASM) cells might contribute to the pathophysiology of asthma. We have shown that periodic mechanical strain applied to cultured ASM cells alters the structure and expression of CSK proteins and increases cell stiffness and contractility (Smith PG, Moreno R, and Ikebe M. Am J Physiol Lung Cell Mol Physiol 272: L20-L27, 1997; and Smith PG, Deng L, Fredberg JJ, and Maksym GN. Am J Physiol Lung Cell Mol Physiol 285: L456-L463, 2003). However, the mechanically induced CSK changes, altered cell function, and their time courses are not well understood. Here we applied MS to the CSK by magnetically oscillating ferrimagnetic beads bound to the CSK. We quantified CSK remodeling by measuring actin accumulation at the sites of applied MS using fluorescence microscopy. We also measured CSK stiffness using optical magnetic twisting cytometry. We found that, during MS of up to 120 min, the percentage of beads associated with actin structures increased with time. At 60 min, 68.1 +/- 1.6% of the beads were associated with actin structures compared with only 6.7 +/- 2.8% before MS and 38.4 +/- 5.5% in time-matched controls (P < 0.05). Similarly, CSK stiffness increased more than twofold in response to the MS compared with time-matched controls. These changes were more pronounced than observed with contractile stimulation by 80 mM KCl or 10(-4) M acetylcholine. Together, these findings imply that MS is a potent stimulus to enhance stiffness and contractility of ASM cells through CSK remodeling, which may have important implications in airway narrowing and dilation in asthma.

Phorbol ester-mediated pulmonary artery endothelial barrier dysfunction through regulation of actin cytoskeletal mechanics.

The mechanisms of phorbol ester- and thrombin-mediated pulmonary artery endothelial barrier dysfunction were compared. Phorbol ester dibutyrate (PDBU) mediated slow force velocity and less force than thrombin. Taxol did not attenuate PDBU-mediated tension, while it reversed nocodazole-mediated tension. PDBU-mediated tension was not affected by acrylamide; PDBU increased cell stiffness and produced greater declines in transendothelial resistance (TER) than acrylamide. Thus PDBU caused a net increase in tension and did not unload microtubule or intermediate filaments. Microfilament remodeling, determined on the basis of immunocytochemistry and actin solubility, lacked the sensitivity and specificity to predict actin-dependent mechanical properties. Thrombin increased myosin light chain (MLC) kinase site-specific MLC phosphorylation, according to peptide map analysis, whereas PDBU did not increase PKC-specific MLC phosphorylation. The initial PDBU-mediated tension development temporally correlated with PDBU-mediated decline in TER and increased low-molecular-weight caldesmon (l-CaD) phosphorylation. PDBU-mediated tension development and decreases in TER were associated with a temporal loss of endothelial cell-matrix adhesion, based on a numerical model of TER. Although, on the basis of immunocytochemistry, thrombin-mediated tension was associated with actin insolubility, actin reorganization, and gap formation, these changes did not predict thrombin-mediated gap formation, based on TER and time-lapse differential interference contrast microscopy. These data suggest that PDBU may disrupt endothelial barrier function through loss of cell-matrix adhesion through l-CaD-dependent actin contraction.

388 Lung tumor formation in rats after intratracheal instillation of fine and ultrafine particles

The role of the different cytoskeletal structures like microfilaments (MF), microtubuli (MT), and intermediate filaments (IF) in phagosome motion is unclear. These cytoskeletal units play an important role in macrophage function (migration, phagocytosis, phagosome transport). We investigated ferromagnetic phagosome motions by cell magnetometry. J774A.1 macrophages were incubated with 1.3-μm spherical magnetite particles for 24 h, after which more than 90% of the particles had been phagocytized. Phagosome motions can be caused either by the cell itself (relaxation) or by applying magnetic twisting forces, yielding cell stiffness and viscoelastic properties of the cytoskeleton. Apparent viscosity of the cytoplasm was non-Newtonian and showed a shear-rate-dependent power law behavior. Elastically stored energy does not force the magnetic phagosomes back to their initial orientation: 57% of the twisting shear was not recoverable. Cytoskeletal drugs, like Cytochalasin D (CyD, 2 - 4 μM), Colchicine (CoL, 10 μM), or Acrylamide (AcL, 40 mM) were added in order to disturb the different cytoskeletal structures. AcL disintegrates IF, but affected neither stochastic (relaxation) nor directed phagosome motions. CyD disrupts MF, resulting in a retarded stochastic phagosome motion (relative decay 0.53 ± 0.01 after 5 min versus 0.34 ± 0.01 in control), whereas phagosome twisting shows only a small response with a 9% increase of stiffness and a small reduction of recoverable strain. CoL depolymerizes the MT, inducing a moderately accelerated relaxation (relative decay 0.28 ± 0.01 after 5 min) and a 10% increase of cell stiffness, where the pure viscous shear is increased and the viscoelastic recoil is inhibited by 40%. Combining the two drugs conserves both effects. After disintegrating either MF or MT, phagosome motion and cytoskeletal stiffness reflect the behavior of either MT or MF, respectively. The results verify that the dominant phagosome transport mechanism is MF-associated. MT depolymerization by CoL induces an activation of the F-actin synthesis, which may induce an accelerated relaxation and an increase of stiffness. Cell mechanical properties are not modulated by MF depolymerization, whereas MT depolymerization causes a loss of viscous resistance and a loss of cell elasticity. The mean energy for stochastic phagosome transport is 5*10−18 Joules and corresponds to a force of 7 pN on a single 1.3-μm phagosome.

Mechanical strain increases cell stiffness through cytoskeletal filament reorganization.

We tested the hypothesis that cytoskeletal reorganization induced by cyclic strain increases cytoskeletal stiffness (G'). G' was measured by optical magnetic twisting cytometry in control cells and cells that had received mechanical strain for 10-12 days. G' was measured before and after both contractile and relaxant agonists, and in the strained cells both parallel (Para) and perpendicular (Perp) to the aligned cytoskeleton. Before activation, G' Para was 24 +/- 5% (+/- SE) greater compared with Perp (P < 0.05), and 35% +/- 6 greater compared with control (Cont, P < 0.01). The difference between strained and control cells was enhanced by KCl, increasing G' 171 +/- 7% Para compared with 125 +/- 6% Perp and 129 +/- 8% Cont (P < 10-5 both cases). The decrease in G' from baseline due to relaxant agonists isoproterenol and dibutyryl cAMP was similar in all groups. Long-term oscillatory loading of airway smooth muscle (ASM) cells caused stiffness to increase and become anisotropic. These findings are consistent with the hypothesis that cytoskeletal reorganization can enhance ASM stiffness and contractility. They imply, furthermore, that oscillatory loading of ASM may contribute to airway narrowing and failure of airway dilation in asthma.

Stiffness changes in cultured airway smooth muscle cells.

Airway smooth muscle (ASM) cells in culture stiffen when exposed to contractile agonists. Such cell stiffening may reflect activation of the contractile apparatus as well as polymerization of cytoskeletal biopolymers. Here we have assessed the relative contribution of these mechanisms in cultured ASM cells stimulated with serotonin (5-hydroxytryptamine; 5-HT) in the presence or absence of drugs that inhibit either myosin-based contraction or polymerization of filamentous (F) actin. Magnetic twisting cytometry was used to measure cell stiffness, and associated changes in structural organization of actin cytoskeleton were evaluated by confocal microscopy. We found that 5-HT increased cell stiffness in a dose-dependent fashion and also elicited rapid formation of F-actin as marked by increased intensity of FITC-phalloidin staining in these cells. A calmodulin antagonist (W-7), a myosin light chain kinase inhibitor (ML-7) and a myosin ATPase inhibitor (BDM) each ablated the stiffening response but not the F-actin polymerization induced by 5-HT. Agents that inhibited the formation of F-actin (cytochalasin D, latrunculin A, C3 exoenzyme, and Y-27632) attenuated both baseline stiffness and the extent of cell stiffening in response to 5-HT. Together, these data suggest that agonist-evoked stiffening of cultured ASM cells requires actin polymerization as well as myosin activation and that neither actin polymerization nor myosin activation by itself is sufficient to account for the cell stiffening response.