The mammalian Scribble polarity protein regulates epithelial cell adhesion and migration through E-cadherin

Scribble (Scrib) is a conserved polarity protein required in Drosophila melanogaster for synaptic function, neuroblast differentiation, and epithelial polarization. It is also a tumor suppressor. In rodents, Scrib has been implicated in receptor recycling and planar polarity but not in apical/basal polarity. We now show that knockdown of Scrib disrupts adhesion between Madin-Darby canine kidney epithelial cells. As a consequence, the cells acquire a mesenchymal appearance, migrate more rapidly, and lose directionality. Although tight junction assembly is delayed, confluent monolayers remain polarized. These effects are independent of Rac activation or Scrib binding to betaPIX. Rather, Scrib depletion disrupts E-cadherin-mediated cell-cell adhesion. The changes in morphology and migration are phenocopied by E-cadherin knockdown. Adhesion is partially rescued by expression of an E-cadherin-alpha-catenin fusion protein but not by E-cadherin-green fluorescent protein. These results suggest that Scrib stabilizes the coupling between E-cadherin and the catenins and are consistent with the idea that mammalian Scrib could behave as a tumor suppressor by regulating epithelial cell adhesion and migration.     

Truncation of the beta-catenin binding domain of E-cadherin precedes epithelial apoptosis during prostate and mammary involution

A potential target of hormone action during prostate and mammary involution is the intercellular junction of adjacent secretory epithelium. This is supported by the long-standing observation that one of the first visible stages of prostate and mammary involution is the disruption of interepithelial adhesion prior to the onset of apoptosis. In a previous study addressing this aspect of involution, we acquired compelling evidence indicating that the disruption of E-cadherin-dependent adhesion initiates apoptotic programs during prostate and mammary involution. In cultured prostate and mammary epithelial cells, inhibition of E-cadherin-dependent aggregation resulted in cell death following apoptotic stimuli. Loss of cell-cell adhesion in the nonaggregated population appeared to result from the rapid truncation within the cytosolic domain of the mature, 120-kDa species of E-cadherin (E-cad(120)). Immunoprecipitations from cell culture and involuting mammary gland demonstrated that this truncation removed the beta-catenin binding domain from the cytoplasmic tail of E-cadherin, resulting in a non-beta-catenin binding, membrane-bound 97-kDa species (E-cad(97)) and a free cytoplasmic 35-kDa form (E-cad(35)) that is bound to beta-catenin. Examination of E-cadherin expression and cellular distribution during prostate and mammary involution revealed a dramatic reduction in junctional membrane staining that correlated with a similar reduction in E-cad(120) and accumulation of E-cad(97) and E-cad(35). The observation that E-cadherin was truncated during involution suggested that hormone depletion activated the same apoptotic pathway in vivo as observed in vitro. Based on these findings, we hypothesize that truncation of E-cadherin results in the loss of beta-catenin binding and cellular dissociation that may signal epithelial apoptosis during prostate and mammary involution. Thus, E-cadherin may be central to homeostatic regulation in these tissues by coordinating adhesion-dependent survival and dissociation-induced apoptosis.     

The intracellular domain of ErbB4 induces differentiation of mammary epithelial cells

Differentiation of mammary epithelium in vivo requires signaling through prolactin- and ErbB4/HER4-dependent mechanisms; how these pathways intersect is unknown. We show herein that HC11 mouse mammary cells undergo ErbB4-dependent lactational differentiation. Prolactin and the ErbB4 ligand HB-EGF each induced STAT5A activation, expression of lactogenic differentiation markers, and lumen formation in three-dimensional Matrigel cultures in HC11 cells. ErbB4 undergoes ligand-dependent transmembrane domain cleavage at Val-675, releasing a soluble 80-kDa intracellular domain (s80(HER4)) that localizes to nuclei; the physiological relevance of s80(HER4) is unknown. A HER4(V675A) mutant abolishing transmembrane cleavage impaired STAT5A activity, lactogenic gene expression, and lumen formation. Kinase-dead HER4(KD) was neither cleaved nor able to induce differentiation of HC11 cells. Without treating HC11 cells with prolactin or HB-EGF, s80(HER4) (expressed from a cDNA construct) localized to the nucleus, activated STAT5A, and induced three-dimensional lumen formation. Nuclear localization of exogenous s80(HER4) required intact kinase activity of s80(HER4), as did activation of STAT5A. In contrast, nuclear localization of s80(HER4) and STAT5A activation did not require the 16-amino acid region of the ErbB4 intracellular domain specific to the Cyt-1 isoform of ErbB4, and absent in the Cyt-2 isoform. These results suggest that s80(HER4) formation contributes to ErbB4-dependent differentiation of mammary epithelial cells.     

A molecular mechanism directly linking E-cadherin adhesion to initiation of epithelial cell surface polarity

Mechanisms involved in maintaining plasma membrane domains in fully polarized epithelial cells are known, but when and how directed protein sorting and trafficking occur to initiate cell surface polarity are not. We tested whether establishment of the basolateral membrane domain and E-cadherin-mediated epithelial cell-cell adhesion are mechanistically linked. We show that the basolateral membrane aquaporin (AQP)-3, but not the equivalent apical membrane AQP5, is delivered in post-Golgi structures directly to forming cell-cell contacts where it co-accumulates precisely with E-cadherin. Functional disruption of individual components of a putative lateral targeting patch (e.g., microtubules, the exocyst, and soluble N-ethylmaleimide-sensitive factor attachment protein receptors) did not inhibit cell-cell adhesion or colocalization of the other components with E-cadherin, but each blocked AQP3 delivery to forming cell-cell contacts. Thus, components of the lateral targeting patch localize independently of each other to cell-cell contacts but collectively function as a holocomplex to specify basolateral vesicle delivery to nascent cell-cell contacts and immediately initiate cell surface polarity.     

Perfluorocarbon induces alveolar epithelial cell response through structural and mechanical remodeling

During total liquid ventilation, lung cells are exposed to perfluorocarbon (PFC) whose chemophysical properties highly differ from standard aqueous cell feeding medium (DMEM). We herein perform a systematic study of structural and mechanical properties of A549 alveolar epithelial cells in order to characterize their response to PFC exposure, using DMEM as control condition. Changes in F-actin structure, focal adhesion density and glycocalyx distribution are evaluated by confocal fluorescent microscopy. Changes in cell mechanics and adhesion are measured by multiscale magnetic twisting cytometry (MTC). Two different microrheological models (single Voigt and power law) are used to analyze the cell mechanics characterized by cytoskeleton (CSK) stiffness and characteristic relaxation times. Cell–matrix adhesion is analyzed using a stochastic multibond deadhesion model taking into account the non-reversible character of the cell response, allowing us to quantify the adhesion weakness and the number of associated bonds. The roles of F-actin structure and glycocalyx layer are evaluated by depolymerizing F-actin and degrading glycocalyx, respectively. Results show that PFC exposure consistently induces F-actin remodeling, CSK softening and adhesion weakening. These results demonstrate that PFC triggers an alveolar epithelial cell response herein evidenced by a decay in intracellular CSK tension, an adhesion weakening and a glycocalyx layer redistribution. These PFC-induced cell adjustments are consistent with the hypothesis that cells respond to a decrease in adhesion energy at cell surface. This adhesion energy can be even further reduced in the presence of surfactant adsorbed at the cell surface.     

Control of photoreceptor cell morphology, planar polarity and epithelial integrity during Drosophila eye development

We report that the hindsight (hnt) gene, which encodes a nuclear zinc-finger protein, regulates cell morphology, cell fate specification, planar cell polarity and epithelial integrity during Drosophila retinal development. In the third instar larval eye imaginal disc, HNT protein expression begins in the morphogenetic furrow and is refined to cells in the developing photoreceptor cell clusters just before their determination as neurons. In hnt mutant larval eye tissue, furrow markers persist abnormally posterior to the furrow, there is a delay in specification of preclusters as cells exit the furrow, there are morphological defects in the preclusters and recruitment of cells into specific R cell fates often does not occur. Additionally, genetically mosaic ommatidia with one or more hnt mutant outer photoreceptor cells, have planar polarity defects that include achirality, reversed chirality and misrotation. Mutants in the JNK pathway act as dominant suppressors of the hnt planar polarity phenotype, suggesting that HNT functions to downregulate JUN kinase (JNK) signaling during the establishment of ommatidial planar polarity. HNT expression continues in the photoreceptor cells of the pupal retina. When an ommatidium contains four or more hnt mutant photoreceptor cells, both genetically mutant and genetically wild-type photoreceptor cells fall out of the retinal epithelium, indicating a role for HNT in maintenance of epithelial integrity. In the late pupal stages, HNT regulates the morphogenesis of rhabdomeres within individual photoreceptor cells and the separation of the rhabdomeres of adjacent photoreceptor cells. Apical F-actin is depleted in hnt mutant photoreceptor cells before the observed defects in cellular morphogenesis and epithelial integrity. The analyses presented here, together with our previous studies in the embryonic amnioserosa and tracheal system, show that HNT has a general role in regulation of the F-actin-based cytoskeleton, JNK signaling, cell morphology and epithelial integrity during development.     

A role for the cleaved cytoplasmic domain of E-cadherin in the nucleus

Cell-cell contacts play a vital role in intracellular signaling, although the molecular mechanisms of these signaling pathways are not fully understood. E-cadherin, an important mediator of cell-cell adhesions, has been shown to be cleaved by gamma-secretase. This cleavage releases a fragment of E-cadherin, E-cadherin C-terminal fragment 2 (E-cad/CTF2), into the cytosol. Here, we study the fate and function of this fragment. First, we show that coexpression of the cadherin-binding protein, p120 catenin (p120), enhances the nuclear translocation of E-cad/CTF2. By knocking down p120 with short interfering RNA, we also demonstrate that p120 is necessary for the nuclear localization of E-cad/CTF2. Furthermore, p120 enhances and is required for the specific binding of E-cad/CTF2 to DNA. Finally, we show that E-cad/CTF2 can regulate the p120-Kaiso-mediated signaling pathway in the nucleus. These data indicate a novel role for cleaved E-cadherin in the nucleus.     

E-cadherin mediates aggregation-dependent survival of prostate and mammary epithelial cells through the retinoblastoma cell cycle control pathway

E-cadherin and the retinoblastoma tumor suppressor (Rb) are traditionally associated with diverse regulatory aspects of cell growth and differentiation. However, we have discovered new evidence, which suggests that these proteins are functionally linked in a physiologic pathway required for cell survival and programmed cell death. Pharmacological activation of protein kinase C (PKC) or inducible overexpression and activation of the alpha isozyme of PKC (PKCalpha) resulted in approximately 60% apoptosis of mammary and prostate epithelial cells. Interestingly, the surviving cells had undergone dramatic aggregation concurrent with increased E-cadherin expression. When aggregation was inhibited by the addition of an E-cadherin-blocking antibody, apoptosis increased synergistically. We hypothesized that survival of the aggregated population was associated with contact-inhibited growth and that apoptosis might result from aberrant growth regulatory signals in non-aggregated, cycling cells. This hypothesis was confirmed by experiments that demonstrated that E-cadherin-dependent aggregation resulted in Rb-mediated G1 arrest and survival. Immunoblot analysis and flow cytometry revealed that hypophosphorylated Rb was present in non-aggregated, S phase cultures concurrent with synergistic cell death. We have also determined that the loss of membrane E-cadherin and subsequent hypophosphorylation of Rb in luminal epithelial cells preceded apoptosis induced by castration. These findings provide compelling evidence that suggests that E-cadherin-mediated aggregation results in Rb activation and G1 arrest that is critical for survival of prostate and mammary epithelial cells. These data also indicate that Rb can initiate a fatal growth signal conflict in non-aggregated, cycling cells when the protein is hypophosphorylated as these epithelial cells enter S phase.     

Differential behavior of E-cadherin and occludin in their colocalization with ZO-1 during the establishment of epithelial cell polarity

At the initial stage of cell-cell contact of epithelial cells, primordial spot-like junctions are formed at the tips of thin cellular protrusions radiating from adjacent cells, where E-cadherin and ZO-1 are precisely coconcentrated (Yonemura et al., 1995, J. Cell Sci. 108:127-142). In fully polarized epithelial cells, E-cadherin and ZO-1 are completely sorted into belt-like adherens junctions (AJ) and tight junctions (TJ), respectively. Here we examined the behavior of occludin, an integral membrane protein consisting of TJ, during the establishment of epithelial cell polarity. Using confocal immunofluorescence microscopy, we quantitatively compared the spatial relationship of occludin/ZO-1 with that of E-cadherin/ZO-1 during epithelial cellular polarization by replating or wounding cultured mouse epithelial cells (MTD1-A). At the initial stage of cell-cell contact, E-cadherin and ZO-1 appeared to be simultaneously recruited to the primordial form of spot-like junctions at the tips of cellular processes which showed no concentration of occludin. Then, as cellular polarization proceeded, occludin was gradually accumulated at the ZO-1-positive spot-like junctions to form belt-like TJ, and in a complementary manner E-cadherin was sorted out from the ZO-1-positive spot-like junctions to form belt-like AJ. The molecular mechanism of TJ/AJ formation during epithelial cellular polarization is discussed with special reference to the roles of ZO-1.     

Sustained epithelial beta-catenin activity induces precocious hair development but disrupts hair follicle down-growth and hair shaft formation

During embryonic and postnatal development, Wnt/beta-catenin signaling is involved in several stages of hair morphogenesis from placode formation to hair shaft differentiation. Using a transgenic approach, we have investigated further the role of beta-catenin signaling in embryonic hair development. Forced epithelial stabilization of beta-catenin resulted in precocious and excessive induction of hair follicles even in the absence of Eda/Edar signaling, a pathway essential for primary hair placode formation. In addition, the spacing and size of the placodes was randomized. Surprisingly, the down-growth of follicles was suppressed and hair shaft production was severely impaired. Gene and reporter expression analyses revealed elevated mesenchymal Wnt activity, as well as increased BMP signaling, throughout the skin that was accompanied by upregulation of Sostdc1 (Wise, ectodin) expression. Our data suggest that BMPs are downstream of Wnt/beta-catenin and that their interplay may be a critical component in establishing correct patterning of hair follicles through the reaction-diffusion mechanism.     

Cell binding function of E-cadherin is regulated by the cytoplasmic domain.

Cadherins are a family of transmembrane glycoproteins responsible for Ca2+-dependent cell-cell adhesion. Their amino acid sequences are highly conserved in the cytoplasmic domain. To study the role of the cytoplasmic domain in the function of cadherins, we constructed expression vectors with cDNAs encoding the deletion mutants of E-cadherin polypeptides, in which the carboxy terminus was truncated at various lengths. These vectors were introduced into L cells by transfection, and cell lines expressing the mutant E-cadherin molecules were isolated. In all transfectants obtained, the extracellular domain of the mutant E-cadherins was exposed on the cell surface, and had normal Ca2+-sensitivity and molecular size. However, these cells did not show any Ca2+-dependent aggregation, indicating that the mutant molecules cannot mediate cell-cell binding. The mutant E-cadherin molecules could be released from cells by nonionic detergents, whereas a fraction of normal E-cadherin molecules could not be extracted with the detergent and appeared to be anchored to the cytoskeleton at cell-cell junctions. These results suggest that the cytoplasmic domain regulates the cell-cell binding function of the extracellular domain of E-cadherin, possibly through interaction with some cytoskeletal components.     

Control of epithelial cell shape and polarity

The polarised character of a cell is often obvious from its shape and is largely dependent on the actin cytoskeleton and the membrane-associated cell cortex---a dense network comprising spectrin and other related proteins. Spatially and functionally distinct protein scaffolds, assembled from transmembrane and cytoplasmic proteins, provide the cues for polarisation. Recent data have provided new insights into the molecular nature of these cues and the mechanisms by which they may be translated into a polarised phenotype.     

Bleomycin induces alveolar epithelial cell death through JNK-dependent activation of the mitochondrial death pathway

Exposure to bleomycin in rodents induces lung injury and fibrosis. Alveolar epithelial cell death has been hypothesized as an initiating mechanism underlying bleomycin-induced lung injury and fibrosis. In the present study we evaluated the contribution of mitochondrial and receptor-meditated death pathways in bleomycin-induced death of mouse alveolar epithelial cells (MLE-12 cells) and primary rat alveolar type II cells. Control MLE-12 cells and primary rat alveolar type II cells died after 48 h of exposure to bleomycin. Both MLE-12 cells and rat alveolar type II cells overexpressing Bcl-X(L) did not undergo cell death in response to bleomycin. Dominant negative Fas-associating protein with a death domain failed to prevent bleomycin-induced cell death in MLE-12 cells. Caspase-8 inhibitor CrmA did not prevent bleomycin-induced cell death in primary rat alveolar type II cells. Furthermore, fibroblast cells deficient in Bax and Bak, but not Bid, were resistant to bleomycin-induced cell death. To determine whether the stress kinase JNK was an upstream regulator of Bax activation, MLE-12 cells were exposed to bleomycin in the presence of an adenovirus encoding a dominant negative JNK. Bleomycin-induced Bax activation was prevented by the expression of a dominant negative JNK in MLE-12 cells. Dominant negative JNK prevented cell death in MLE-12 cells and in primary rat alveolar type II cells exposed to bleomycin. These data indicate that bleomycin induces cell death through a JNK-dependent mitochondrial death pathway in alveolar epithelial cells.     

Selective anchoring in the specific plasma membrane domain: a role in epithelial cell polarity

We have studied the role of restrictions to lateral mobility in the segregation of proteins to apical and basolateral domains of MDCK epithelial cells. Radioimmunoassay and semiquantitative video analysis of immunofluorescence on frozen sections showed that one apical and three basolateral glycoproteins, defined by monoclonal antibodies and binding of beta-2-microglobulin, were incompletely extracted with 0.5% Triton X-100 in a buffer that preserves the cortical cytoskeleton (Fey, E. G., K. M. Wan, and S. Penman. 1984. J. Cell Biol. 98:1973-1984; Nelson, W. T. and P. J. Veshnock. 1986. J. Cell Biol. 103:1751-1766). The marker proteins were preferentially extracted from the "incorrect" domain (i.e., the apical domain for a basolateral marker), indicating that the cytoskeletal anchoring was most effective on the "correct" domain. The two basolateral markers were unpolarized and almost completely extractable in cells prevented from establishing cell-cell contacts by incubation in low Ca++ medium, while an apical marker was only extracted from the basal surface under the same conditions. Procedures were developed to apply fluorescent probes to either the apical or the basolateral surface of live cells grown on native collagen gels. Fluorescence recovery after photobleaching of predominantly basolateral antigens showed a large percent of cells (28- 52%) with no recoverable fluorescence on the basal domain but normal fluorescence recovery on the apical surface of most cells (92-100%). Diffusion coefficients in cells with normal fluorescence recovery were in the order of 1.1 x 10(-9) cm2/s in the apical domain and 0.6-0.9 x 10(-9) cm2/s in the basal surface, but the difference was not significant. The data from both techniques indicate (a) the existence of mobile and immobile protein fractions in both plasma membrane domains, and (b) that linkage to a domain specific submembrane cytoskeleton plays an important role in the maintenance of epithelial cell surface polarity.     

Inactivation of the p19(ARF) tumor suppressor affects intestinal epithelial cell proliferation and integrity

p19(ARF) is a tumor suppressor that is frequently deleted in human cancer. It lies at chromosome 9p21 and shares exons 2 and 3 with p16(ink4a), which is also inactivated by these cancer-associated deletions. The "canonical pathway" by which p19(ARF) is thought to suppress tumorigenesis through activation of the p53 tumor suppressor. In response to hyperproliferative signals, such as expression of oncogenes, p19(ARF) is induced and binds to the MDM2 ubiquitin ligase, sequestering it in the nucleolus to allow the accumulation of p53. However, p19(ARF) also has MDM2 and p53 independent functions. In human colon cancer, p19(ARF) is only rarely deleted, but it is more frequently silenced by DNA promoter methylation. Here we show that inactivation of p19(ARF) in mice increases the number of cycling cells in the crypts of the colonic epithelium. Moreover, inactivation of p19(ARF) exacerbated the ulceration of the colonic epithelium caused by dextran sodium sulfate (DSS). These effects were similar to those observed in mice lacking myeloid translocation gene-related-1 (Mtgr1), and mice lacking both of these genes showed an even greater sensitivity to DSS. Surprisingly, inactivation of p19(ARF) restored the loss of the secretory lineage in mice deficient in Mtgr1, suggesting an additional role for p19(ARF) in the small intestinal epithelium.     

Control of mammary epithelial differentiation: basement membrane induces tissue-specific gene expression in the absence of cell-cell interaction and morphological polarity

Functional differentiation in mammary epithelia requires specific hormones and local environmental signals. The latter are provided both by extracellular matrix and by communication with adjacent cells, their action being intricately connected in what appears to be a cascade of events leading to milk production. To distinguish between the influence of basement membrane and that of cell-cell contact in this process, we developed a novel suspension culture assay in which mammary epithelial cells were embedded inside physiological substrata. Single cells, separated from each other, were able to assimilate information from a laminin-rich basement membrane substratum and were induced to express beta-casein. In contrast, a stromal environment of collagen I was not sufficient to induce milk synthesis unless accompanied by cell-cell contact. The expression of milk proteins did not depend on morphological polarity since E-cadherin and alpha 6 integrin were distributed evenly around the surface of single cells. In medium containing 5 microM Ca2+, cell-cell interactions were impaired in small clusters and E-cadherin was not detected at the cell surface, yet many cells were still able to produce beta-casein. Within the basement membrane substratum, signal transfer appeared to be mediated through integrins since a function-blocking anti-integrin antibody severely diminished the ability of suspension-cultured cells to synthesize beta-casein. These results provide evidence for a central role of basement membrane in the induction of tissue-specific gene expression.     

Functional differentiation of alveolar type II epithelial cells in vitro: effects of cell shape, cell-matrix interactions and cell-cell interactions

Alveolar type II epithelial cells rapidly lose characteristics of differentiated function when cultured on plastic dishes. We have attempted to circumvent this problem by culturing type II cells under conditions that might better reproduce their environment in vivo. Cell-matrix interactions were studied by culturing isolated adult rat type II cells on Engelbreth-Holm-Swarm (EHS) tumor basement membrane. Aggregates of type II cells formed on the surface of the matrix during 4 days in culture. Microscopic examination of these aggregates revealed cuboidal cells that retained more characteristics of differentiated type II cells than did cells cultured on plastic. Type II cells cultured on EHS matrix incorporated a higher percentage of acetate into phosphatidylcholine (PC) than did cells on plastic, and a higher percentage of this PC was saturated. Phosphatidylglycerol (PG) synthesis by these cells was no different from that seen in cells on plastic. The effects of cell-cell interactions and cell shape were evaluated by culturing type II cells on feeder layers that in turn were grown on collagen gels. The feeder layer cells included fetal rat lung fibroblasts, adult rat lung fibroblasts, fetal rat skin fibroblasts, bovine aortic endothelial cells, and rat mammary tumor epithelial cells. One-half of the gels remained attached to the culture dish and one-half of the gels were detached after 24 h and allowed to float free in the medium. Type II cells grown in association with any of the attached feeder layers became flattened and lost their differentiated phenotype. These cells incorporated no greater percentage of acetate into PC than did cells on plastic. Saturated PC synthesis was modestly increased. PG synthesis declined in parallel with that seen in cells cultured on plastic. Type II cells cultured on feeder layers that were detached assumed their native cuboidal shape and also exhibited many morphological characteristics of differentiated function. These cells incorporated a significantly greater percentage of acetate into PC compared to cells on either plastic or attached feeder layers. Saturated PC synthesis also increased markedly. These cells, however, incorporated no greater percentage of acetate into PG than did cells on plastic or attached feeder layers. These data suggest an important role for cell shape and cell-matrix interactions and maintenance of type II cell differentiation. The effects of cell-cell interactions, while beneficial, appear to be non-specific.     

Effect of stretch on structural integrity and micromechanics of human alveolar epithelial cell monolayers exposed to thrombin

Alveolar epithelial cells in patients with acute lung injury subjected to mechanical ventilation are exposed to increased procoagulant activity and mechanical strain. Thrombin induces epithelial cell stiffening, contraction, and cytoskeletal remodeling, potentially compromising the balance of forces at the alveolar epithelium during cell stretching. This balance can be further compromised by the loss of integrity of cell-cell junctions in the injured epithelium. The aim of this work was to study the effect of stretch on the structural integrity and micromechanics of human alveolar epithelial cell monolayers exposed to thrombin. Confluent and subconfluent cells (A549) were cultured on collagen-coated elastic substrates. After exposure to thrombin (0.5 U/ml), a stepwise cell stretch (20%) was applied with a vacuum-driven system mounted on an inverted microscope. The structural integrity of the cell monolayers was assessed by comparing intercellular and intracellular strains within the monolayer. Strain was measured by tracking beads tightly bound to the cell surface. Simultaneously, cell viscoelasticity was measured using optical magnetic twisting cytometry. In confluent cells, thrombin did not induce significant changes in transmission of strain from the substrate to overlying cells. By contrast, thrombin dramatically impaired the ability of subconfluent cells to follow imposed substrate deformation. Upon substrate unstretching, thrombin-treated subconfluent cells exhibited compressive strain (9%). Stretch increased stiffness (56-62%) and decreased cell hysteresivity (13-22%) of vehicle cells. By contrast, stretch did not increase stiffness of thrombin-treated cells, suggesting disruption of cytoskeletal structures. Our findings suggest that thrombin could exacerbate epithelial barrier dysfunction in injured lungs subjected to mechanical ventilation.