Stretch of Septic Monolayers Figure 2. Phosphorylation of MAPk signaling pathways (ERK and JNK) following cyclic stretch for 1 hour to 12% DSA in sham and 2CLP cell monolayers. No increases in MAPk phosphorylation were observed in either cell population when stretched to 12% DSA for 10 or 60 minutes. (mean 6 SE, N$6 isolations). doi:10.1371/journal.pone.0038748.g002 Actin Staining Degradation of the actin cytoskeleton has been shown to negatively affect barrier function through alterations in the tight junction-actin associations [23]. Therefore, we labeled actin with phalloidin to visualize its localization within 2CLP and sham monolayers (Figure 4). In unstretched 2CLP and sham mono- layers, the actin networks were similar, with diffuse staining through the center of the cell, which extended to the cell periphery in sham monolayers. However, in unstretched 2CLP monolayers, staining at the cell-cell junction was diminished, and only a thin band of actin was observed. Following 12% DSA stretch for 60 minutes, actin in sham monolayers was indistinguishable from that in unstretched sham. In 2CLP monolayers, the staining in the cytoplasm increased in intensity, and the junctional staining diminished compared to unstretched 2CLP. Interestingly, circum- ferential stress fibers became prominent in the cytoplasm of 2CLP cells. Following 25% DSA stretch for 60 minutes, cortical actin rings began to form near the cell periphery in sham monolayers; however, staining at the junction was discontinuous. In 2CLP monolayers, bright cortical rings were formed at the cell-cell junctions, while staining intensity decreases in all other areas of the cell. These differences in actin staining in 2CLP and sham monolayers following stretch indicate alterations in cytoskeletal arrangement, even at 12% DSA. Myosin Light Chain Kinase Phosphorylation We hypothesized that alterations to the actin cytoskeleton are responsible for the stretch-induced increases in 2CLP monolayer permeability at low stretch (12% DSA). Therefore we analyzed the phosphorylation of myosin light chain kinase (MLCK), a signaling molecule that directly regulates actin cytoskeletal contractility through activation of myosin motors, and a pathway we have demonstrated to be altered during stretch [24]. We found reduced levels of MLCK phosphorylation in unstretched 2CLP monolayers compared to sham monolayers (Figure 5). Stretch to 12% DSA reduced MLCK phosphorylation levels compared to unstretched controls in both sham and 2CLP monolayers; however, phos- phorylation was consistently lower in 2CLP compared to sham. Tight Junction Protein Solubility Because our data suggested that the actin cytoskeleton was altered in 2CLP monolayers, we tested the hypothesis that TJ proteins were not anchored to the cytoskeleton in 2CLP monolayers as well as they were in sham monolayers by analyzing the ratio of triton soluble to triton insoluble cytoskeletal associated protein. We found that in unstretched 2CLP monolayers, a greater fraction of the transmembrane proteins dissociated from the cytoskeleton (Figure 6). This result could be indicative of a less organized junctional complex and further studies will be required to determine the nature of the actin-tight junction protein bond in septic epithelial monolayers. Stabilization of the Cytoskeleton To test the hypothesis that actin destabilization in 2CLP monolayers was responsible for the stretch-induced permeability increases at low stretch magnitudes, we used jasplakinolide to prevent actin depolymerization. In previous studies, jasplakino- lide has been shown to prevent stretch-induced barrier dysfunction in healthy cell monolayers stretched to 37% DSA for 1 hour [12,19]. For 60 minutes prior to stretch, 2CLP monolayers were treated with jasplakinolide (1 mM) to stabilize the actin cytoskeleton, then cyclically stretched to 12% DSA for an additional 60 minutes, after which monolayer permeability was assessed (Figure 7). As shown previously (Figure 1), monolayer permeability was significantly increased in 2CLP and not in sham monolayers treated with DMSO control. Jasplakinolide treatment of 2CLP monolayers preserved barrier properties such that permeability in stretched 2CLP wells treated with jasplakinolide was not significantly increased above unstretched sham or 2CLP levels, and was significantly lower than permeability levels in 2CLP monolayers treated with DMSO. These data show that alterations in the actin cytoskeleton in 2CLP monolayers may be associated with the stretch-induced permeability observed in these cells following even low magnitudes of cyclic stretch. PLoS ONE | www.plosone.org 4 June 2012 | Volume 7 | Issue 6 | e38748