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... filaments and active contraction generated by the acto-myosin molecular motors. Internal tension is partially counterbalanced by microtubules and other intracellular compression-resistive components =-=(34)-=-. In cell monolayers, the net internal force is counterbalanced by external forces applied at the cell boundary by the adjacent cells and the substrate. Therefore, preservation of the structural integ...

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...ange but restricts movement of liquid, macromolecules, and cells into the alveoli. A key feature of acute lung injury is alveolar flooding and infiltration of leukocytes into the alveolar compartment =-=(22, 36)-=-. Enhanced permeability of the alveolar barrier in acute lung injury has been associated with disruption of the cell monolayer by formation of gaps between adjacent cells (22, 36). Therefore, preserva...

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...ine the effect of thrombin in the force balance that regulates alveolar barrier permeability in lung inflammation. Traction microscopy (TM) is a recently developed technique to probe cell contraction =-=(7, 11)-=-. TM allows us to measure the regional distribution of contraction forces and the time course of the contractile response to pharmacological stimuli at the single-cell level. Address for reprint reque...

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... bronchoalveolar cell carcinoma. These cells are widely used as a model of alveolar epithelial cells in vitro because they are readily cultured and retain important features of type II alveolar cells =-=(20)-=-. In particular, it has been recently shown that this cell type forms a tight monolayer when grown to confluence and that the barrier permeability is modulated by thrombin (16). Moreover, A549 cells d...

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...meter coverslips 24 h before experiments (15,000 cells/coverslip). TM Polyacrylamide gels. Preparation of thin collagen-coated polyacrylamide gel disks was carried out as described by Pelham and Wang =-=(25)-=-. Green fluorescent latex beads 0.2 m in diameter were mixed with 2% acrylamide and 0.3% bis-acrylamide solution (1:125 vol/vol bead solution volume of acrylamide mixture). Gel disks 70 m thick and...

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...hysical integrity of the cell monolayer is governed by a dynamic force balance at the cell-cell and cell-matrix attachments between centripetal cell mechanical tension and centrifugal adhesive forces =-=(12, 24)-=-. Mechanical tension arises from both active contraction generated by the actomyosin machinery and passive elastic recoil caused by cyclic stretching due to breathing or mechanical ventilation. Mainte...

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...ange but restricts movement of liquid, macromolecules, and cells into the alveoli. A key feature of acute lung injury is alveolar flooding and infiltration of leukocytes into the alveolar compartment =-=(22, 36)-=-. Enhanced permeability of the alveolar barrier in acute lung injury has been associated with disruption of the cell monolayer by formation of gaps between adjacent cells (22, 36). Therefore, preserva...

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...a pyramidal tip (Mikromasch, Tallin, Estonia) as previously described (1, 27). The spring constant of the atomic force microscopy cantilever was calibrated by the thermal fluctuations method in water =-=(6)-=-. Force-displacement curves (1-m indentation at 1Hz) were recorded at four distant points on the surface of four gel samples. E was computed from the force-displacement curves (4) using nonlinear lea...

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...ed to the surface of the gel at the end of the experiment, the relaxed position of the beads in unstrained gel is easily determined. Moreover, in contrast with microfabricated pillar-array techniques =-=(28)-=-, the stiffness of the substrate and the spatial force resolution can be easily tuned to each particular cell type. Thus TM enabled measurement of the time course of the absolute magnitude and regiona...

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... substrate was first determined from the stored fluorescent bead images. The displacement field between each fluorescence image and the reference image was computed using the Image Correlation Method =-=(30)-=-. Images were iteratively divided into smaller windows, and the displacement field between a pair of images was obtained by identifying the coordinates of the peak of the cross-correlation function be...

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...net contractile moment (M) was computed as defined by Butler and coworkers (7). M is a measure of the cell contractile strength that can be used as an index of cytoskeleton tensile stress (prestress) =-=(35)-=-. The polarity of cell contraction was defined as Mxx/M, where Mxx is the contractile moment along the principal axis of contraction (7). For F-actin-to-G-actin fluorescence ratio quantification, back...

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...ine the effect of thrombin in the force balance that regulates alveolar barrier permeability in lung inflammation. Traction microscopy (TM) is a recently developed technique to probe cell contraction =-=(7, 11)-=-. TM allows us to measure the regional distribution of contraction forces and the time course of the contractile response to pharmacological stimuli at the single-cell level. Address for reprint reque...

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...ntilation. Maintenance of the cell monolayer requires tethering adhesive forces to withstand active and passive tension. Thrombin is a serine protease that plays a key role in the coagulation cascade =-=(21)-=-. In addition, thrombin has been associated with endothelial barrier dysfunction (5, 12). Cultured endothelial cell monolayers show increased permeability after thrombin addition, which is indicative ...

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...the gels. Young’s modulus (E) of polyacrylamide gels was measured with atomic force microscopy using a triangular cantilever with a pyramidal tip (Mikromasch, Tallin, Estonia) as previously described =-=(1, 27)-=-. The spring constant of the atomic force microscopy cantilever was calibrated by the thermal fluctuations method in water (6). Force-displacement curves (1-m indentation at 1Hz) were recorded at fou...

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...ening response recently reported in confluent monolayers of the same cell type (31). Although alveolar epithelial cell stiffening could be a result of actin polymerization and cytoskeleton remodeling =-=(17)-=-, the comparable increase and temporal correlation between stiffening and contractile responses suggest that the thrombin stiffening effect is dominated by the increase in cell prestress. It has been ...

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...the gels. Young’s modulus (E) of polyacrylamide gels was measured with atomic force microscopy using a triangular cantilever with a pyramidal tip (Mikromasch, Tallin, Estonia) as previously described =-=(1, 27)-=-. The spring constant of the atomic force microscopy cantilever was calibrated by the thermal fluctuations method in water (6). Force-displacement curves (1-m indentation at 1Hz) were recorded at fou...

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... basal contractile state of the cell and the thrombin-induced contractile response. Cell contraction can be readily probed by plating cells on top of a soft elastic gel or casting them inside the gel =-=(13, 18, 23, 33)-=-. Contractile cell response to pharmacological stimulation can be assessed with force transducers attached to the gel (18, 24) or by measuring the reduction in the gel area (13, 33). These approaches ...

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...titial space, stimulating alveolar epithelial cells at the sites of lung inflammation. Nevertheless, the effect of thrombin in epithelial barrier function remains unclear. Kawkitinarong and coworkers =-=(16)-=- recently found increased transepithelial resistance (TER) of alveolar epithelial cell monolayers in response to thrombin. These authors suggested a barrier protective effect of thrombin in alveolar e...

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...hysical integrity of the cell monolayer is governed by a dynamic force balance at the cell-cell and cell-matrix attachments between centripetal cell mechanical tension and centrifugal adhesive forces =-=(12, 24)-=-. Mechanical tension arises from both active contraction generated by the actomyosin machinery and passive elastic recoil caused by cyclic stretching due to breathing or mechanical ventilation. Mainte...

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...sing epithelial cells to thrombin. Consistently, elevated levels of thrombin have been found in bronchoalveolar lavage fluid obtained from patients with asthma and acute respiratory distress syndrome =-=(19, 29)-=-. In contrast to the fall in TER found in endothelial cells, increased TER has been recently reported in alveolar epithelial cells exposed to thrombin, indicative of improved sealing of the cell monol...

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...ltured endothelial cell monolayers show increased permeability after thrombin addition, which is indicative of monolayer disruption with leakage of liquid and macromolecules through paracellular gaps =-=(2)-=-. The detachment between adjacent cells has been attributed to a rise in contractile activation and loss of cell-cell adhesion (5, 12, 23). Endothelial barrier disruption may allow the passage of thro...

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...sing epithelial cells to thrombin. Consistently, elevated levels of thrombin have been found in bronchoalveolar lavage fluid obtained from patients with asthma and acute respiratory distress syndrome =-=(19, 29)-=-. In contrast to the fall in TER found in endothelial cells, increased TER has been recently reported in alveolar epithelial cells exposed to thrombin, indicative of improved sealing of the cell monol...

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...oli, cell contraction polarity might be regulated by cell signaling or structural inhomogeneities. Traction exerted by alveolar epithelial cells was in the range of that reported in endothelial cells =-=(3)-=-, suggesting comparable actomyosin motor activity. On the other hand, forces reported on migrating fibroblasts were 10-fold higher (11). The propulsive forces produced during cell locomotion might ex...

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...ons method in water (6). Force-displacement curves (1-m indentation at 1Hz) were recorded at four distant points on the surface of four gel samples. E was computed from the force-displacement curves =-=(4)-=- using nonlinear least squares regression. For each gel sample, the value of E was taken as the average of the four measurements done at different surface points. The coefficient of variation (SD/mean...

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...n with leakage of liquid and macromolecules through paracellular gaps (2). The detachment between adjacent cells has been attributed to a rise in contractile activation and loss of cell-cell adhesion =-=(5, 12, 23)-=-. Endothelial barrier disruption may allow the passage of thrombin into the interstitial space, stimulating alveolar epithelial cells at the sites of lung inflammation. Nevertheless, the effect of thr...

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...d in TM experiments adopted a nonmigrating behavior. The low motility exhibited by the A549 cells could be attributed to the high concentration of type I collagen used to coat the polyacrylamide gels =-=(26)-=-. Therefore, despite the limitations of transformed cell lines, we considered A549 cells to be a suitable model to study contractile properties of nonmigrating alveolar epithelial cells. Our TM measur...

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...ombin stiffening effect is dominated by the increase in cell prestress. It has been recently reported that thrombin induces cell stiffening both in confluent and nonconfluent monolayers of A549 cells =-=(32)-=-. This suggests a thrombin contractile response of A549 cells in confluent monolayers similar to that found in isolated cells. Thrombin cleaves protease-activated receptors leading to enhanced MLC pho...

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... response found in endothelial cells. Enhancement of epithelial barrier function reflects changes in the cell force balance. We have recently observed that thrombin stiffens alveolar epithelial cells =-=(31)-=-, which could result in increased internal elastic tension. In addition, thrombin has been shown to enhance myosin light chain (MLC) phosphorylation in alveolar epithelial cells (16), which could lead...

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...halloidin-tetramethylrhodamine isothiocyanate (0.2 g/ml) and Alexa Fluor 488 DNase I conjugate (9 g/ml) were used to localize F-actin and G-actin, respectively, as described by Cramer and coworkers =-=(9)-=-. Fluorescent dyes were diluted on blocking solution (1% BSA and 0.025% saponin in PBS) and added to coverslips for 40 min at room temperature. After three washes with PBS, coverslips were mounted on ...

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...ls displayed ability similar to that of primary rat type II cells to contract collagen gels (33). Cell speed of the isolated A549 cells was 25-fold smaller than that reported for migrating cell types =-=(10)-=-. Moreover, immunofluorescence images of isolated A549 cells did not display front-rear polarity of the F-actin cytoskeleton (Fig. 5, inset). These results indicate that the A549 cells studied in TM e...

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... basal contractile state of the cell and the thrombin-induced contractile response. Cell contraction can be readily probed by plating cells on top of a soft elastic gel or casting them inside the gel =-=(13, 18, 23, 33)-=-. Contractile cell response to pharmacological stimulation can be assessed with force transducers attached to the gel (18, 24) or by measuring the reduction in the gel area (13, 33). These approaches ...

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...thstand active and passive tension. Thrombin is a serine protease that plays a key role in the coagulation cascade (21). In addition, thrombin has been associated with endothelial barrier dysfunction =-=(5, 12)-=-. Cultured endothelial cell monolayers show increased permeability after thrombin addition, which is indicative of monolayer disruption with leakage of liquid and macromolecules through paracellular g...

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...lution of the displacement and traction maps was 1.3 m. For each traction field, the total force magnitude (F) was computed by integrating the magnitude of T(x,y) over the projected area of the cell =-=(14)-=-. Although the net vectorial force over the contact area is zero, the integral of the modulus provides a useful index of the cell contractile strength. The average traction of the cell was computed as...

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... basal contractile state of the cell and the thrombin-induced contractile response. Cell contraction can be readily probed by plating cells on top of a soft elastic gel or casting them inside the gel =-=(13, 18, 23, 33)-=-. Contractile cell response to pharmacological stimulation can be assessed with force transducers attached to the gel (18, 24) or by measuring the reduction in the gel area (13, 33). These approaches ...

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...ubtracted. The sum of pixel intensities was computed for each F-actin and G-actin image, and the resultant values were used to calculate the ratio of fluorescent intensities (F/G actin) for each view =-=(15)-=-. Five pairs of images were acquired for each coverslip and averaged for a single data point. Statistics Unless stated otherwise, data are reported as means SE. For time-course experiments, baseline...