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EXTRACELLULAR MATRIX, CELL INTERACTIONS
,11Department of Chemical Engineering and 2Department of Physics, Princeton University, Princeton; 3Department of Molecular Genetics and Microbiology, State University of New York Stony Brook, Stony Brook, New York; and 4Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, New Jersey
Submitted 12 August 2005 ; accepted in final form 19 February 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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ERK; reactive oxygen species; coordinated cell sheet migration; cell communication; multicellular dynamics; surface patterning
In embryogenesis, a significant fraction of the cell-cell coordination in epithelial sheet dynamics may be derived from the spatial patterns of gene expression imposed at earlier stages of development (16). In wound healing, the location of cells with respect to the wound can provide positional information for their collective behavior during tissue repair (9, 16). Experiments with epithelial monolayers of Madin-Darby canine kidney (MDCK) cells showed that wounding in this model system is accompanied by two traveling waves of MAPK activation that propagate from the wound edge into the bulk of the monolayer (18). These dynamic signaling patterns were found to be essential for coordinated cell migration during wound healing, which involves spreading of the monolayer, with essentially no cell division. Furthermore, it has been proposed that MAPK participates in a positive feedback loop (Fig. 1A) composed of cell spreading, MAPK activation, and cell migration, which drives the spreading of a wounded MDCK monolayer (18).
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By manipulating the initial and boundary conditions of an MDCK monolayer, we show that an injury is essential for coordinated behavior of cells in the epithelial sheet in this system. We find that the first wave of MAPK activation is induced by injury and that unconstraining of the sheet without injury induces only the second, slower wave of MAPK activation. We show that, immediately following injury, reactive oxygen species (ROS) are generated at the wound interface, and demonstrate that ROS are essential for the generation of the first (fast) wave of MAPK activation that spreads through the layer. We present the results of cell tracking analysis in the spreading MDCK epithelium and discuss the correlation between MAPK signaling and the dynamics of cell sheet migration. While the origin of the MAPK waves in MDCK epithelial wound healing remains to be elucidated by further experiments, our analysis should enable the formulation of increasingly quantitative models of epithelial sheet dynamics in wound healing and embryogenesis.
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MATERIALS AND METHODS |
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Wound closure and cell tracking.
MDCK cells were plated in a tissue culture 24-well plate at an initial density of 1.5x105 cells/cm2. A uniform monolayer formed in 
2–3 days. All wounding assays were performed in a serum-free medium by first preincubating the cells in the serum-free medium for 30 min before the start of the experiments. A micropipette was used to create a wound in the monolayer by scraping. Wound closure was followed for 16 h by phase-contrast microscopy on a Zeiss Axiovert 200M microscope and digital images of the wound area were taken once every 5 min with a Hamamatsu Orka-ER charge-coupled device (CCD) camera. Image analysis and cell tracking were done manually, by selecting individual cells with IPLab software (Scanalytics, Fairfax, VA) and extracting the centroid position of a cell.
Immunofluorescence. Cells were fixed with freshly prepared 4% formaldehyde solution (Sigma). After permeabilizing with 5% Triton X-100 (Sigma) in PBS, and blocking with 10% horse serum (Sigma) in 3% BSA (Sigma) in PBS, cells were incubated in rabbit antiphosphoERK1/2 antibody (Cell Signaling Technologies, Beverly, MA) overnight at 4°C, followed by 1 h incubation with anti-rabbit Alexa 568 secondary antibody (Molecular Probes, Portland, OR). The primary antibody (concentration of 0.2 mg/ml) was diluted at 50:1 and the secondary (concentration of 2 mg/ml) at 400:1, both in 3% BSA solution in PBS (Invitrogen). The surfaces were mounted with antifading Fluoroguard (Bio-Rad Laboratories, Hercules, CA) and digital pictures were taken on an inverted Nikon Eclipse TE300 microscope with a Photometrics CoolSnap FX CCD camera (Roper Scientific, Tucson, AZ). The images were analyzed with IPLab software. For the analysis of the spatiotemporal patterns of MAPK activation, 10 lines were drawn across each image at random and the fluorescence intensity across each of the lines averaged. The average intensity across these 10 slices was then examined and a noticeable drop-off in the intensity of fluorescence was taken as an indication of the ending point the signal reached. The intensity plots shown are the normalized values of intensity to the maximal signal in each image. All optical and camera settings were kept constant throughout image acquisition.
Inhibition of ERK1/2. To inhibit MAPK ERK1/2, UO126 (Promega, Madison, WI) was added to the culture at the concentration of 20 µg/ml (as described in Ref. 18) in anhydrous DMSO (Sigma) at the same time when serum was removed.
Manipulation of boundary conditions for model wounds.
Slabs of polydimethylsiloxane (PDMS; Dow Corning, Midland, MI) were created by modifying a previously published procedure for cell patterning with polymeric membranes (12, 21). Briefly, PDMS was mixed at 10:1 precursor/curer ratio, poured over a silicon wafer in a petri dish to 7 mm height, degassed in a desiccator for at least 1 h, and baked for at least 2 h in a 65°C. Blocks of PDMS roughly 5 mm x 5 mm (already 7 mm high) were cut with a razor, extracted in dichloromethane (Sigma) for 24 h, dried overnight, sterilized with ethanol and then blocked in 3% heat denatured BSA in PBS for 1 h. The slabs were then placed onto a tissue culture surface allowing for a conformal contact with the surface and seeded with MDCK cells at the same density as when no PDMS was present. The hydrophobic to hydrophobic interactions between the PDMS slab and the tissue culture surface resulting from the conformal contact (complete contact between surfaces) prevented the contact of medium and cells with the tissue culture surface under the PDMS slab (21). Once a uniform epithelial monolayer formed, the slab was lifted with sterile tweezers to create an empty space next to a noninjured uniform epithelial monolayer, effectively unconstraining the cells. PDMS membranes (very thin, 30 µm, layers of PDMS) were created as published previously (12, 21). Briefly, PDMS was mixed as stated above and then dissolved in a 1:1 wt:wt ratio in toluene (Sigma). One milliliter of 1,1,1,3,3,3-hexamethyldisilazane (Sigma) was spin coated (a process that uses the centripetal force to coat a spinning surface) onto a 2-in. silicon wafer (Universitywafer.com, South Boston, MA) at 3,000 rpm for 40 s, and then about a quarter coin size of POMS-toluene solution was poured onto the wafer and spin-coated at 900 rpm for 60 s. For easier handling of the membranes, the edges were built up with an addition of another PDMS layer. The membranes were then baked for at least 2 h in a 65°C oven. Additional layers of PDMS on the edges could be added once the previous layer had solidified. The membranes were cut with a razor, peeled off, extracted in dichloromethane for 24 h, dried overnight, sterilized with ethanol, and then placed on a tissue culture surface allowing for a conformal contact with the surface and seeded with MDCK cells. Once a uniform monolayer was formed, the membrane was peeled off with sterile tweezers ripping the epithelial sheet that had partly grown over it, forming an injured monolayer next to the plain tissue culture surface.
Data analysis. At least 45 cells were tracked for each of the 4 treatments. The displacement of a cell was calculated as the Euclidean distance between the n + 1 and n positions in time. Total displacement was calculated as the Euclidean distance between nfinal and ninitial positions of a cell. The correlation coefficient was calculated in MATLAB (Mathworks, Natick, MA) using the corrcoef.m function. Confidence intervals for correlation coefficients were computed with the use of the Bootstrap method (25). All experiments were replicated at least three times, and the data presented here are from a representative experiment.
Visualization and inhibition of ROS. The dye solution 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA; Invitrogen) was used to visualize the formation of ROS. The dye was first diluted to a stock solution of 1,000 µM in anhydrous DMSO immediately before use. Phenol red-free DMEM (Invitrogen) was used as a loading solution, into which the dye was diluted to a working concentration of 5 µM immediately before use. The working concentration was empirically obtained from the suggested guidelines from the manufacturer. The cells were incubated in this loading solution for 30 min before wounding the monolayer. The fluorescent signal used to detect the ROS results from the intracellular removal of the acetate groups of CM-H2DCFDA. Because the fluorescent species is removed relatively quickly from cells by an active efflux (as stated by the manufacturer), we implemented extensive controls to obtain the correct duration of the ROS wave presence in the cells. CM-H2DCFDA was added at different times after the injury. This allowed us to test for the presence of ROS at different times (from minutes to hours) after the injury. The digital pictures were taken on an inverted Nikon Eclipse TE300 microscope with a Photometrics CoolSnap FX CCD camera. The images were analyzed with IPLab software. To inhibit the formation of ROS, N-acetyl-L-cysteine (Sigma) was added to the culture at the same time when the serum was removed at a concentration of 5 µM (29).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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3 min after wounding. This wave reached 500 µm into the cell sheet from the wound interface (Fig. 3); it was receding by 5 min after wounding and was confined only to the first row of cells at the wound interface by 10 min after wounding. After 30 min, the second wave of MAPK phosphorylation initiated close to the wound boundary; by 4 h after wounding, this wave propagated 500 µm into the cell sheet (Fig. 3). At the level of individual cells, the second wave led to sustained MAPK phosphorylation (measurement were stopped after 18 h, results not shown).
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A nonadhesive elastomeric PDMS slab was placed on a tissue culture surface where cells were subsequently seeded. After a monolayer was formed, the slab was lifted, not damaging the cells but removing a physical barrier for sheet migration (12, 21). When the MDCK monolayer was unconstrained in this manner, the spatiotemporal pattern of MAPK phosphorylation was different from the one seen in a wounding assay: only one wave of signal propagation was observed. This wave coincided with the second wave observed after scratching, both temporally and spatially, reaching 500 µm 4 h after unconstraining (Fig. 3). This experiment allowed us to dissect the two waves of MAPK phosphorylation observed in the classical wounding experiment and demonstrate that only the slow wave of MAPK activation is induced in the absence of injury.
In these two cases, however, the surfaces on which the epithelial sheet was migrating were different (Fig. 1B). In the case of an injury created by scratching, cells were scraped off the tissue culture surface, leaving behind the remnants of cell-deposited extracellular matrix, cell debris, and other proteins that might have adsorbed from serum during the time that the cell sheet was grown to confluency. On the other hand, when empty space was induced into a monolayer via removal of a PDMS slab, the surface underlying the slab had not come into contact with any proteins or cell debris, thus presenting the monolayer with a plain tissue culture surface onto which to migrate. The presence of the proteins in the first case and their absence in the second was verified by immunofluorescence staining for fibronectin (results not shown), a major extracellular matrix protein that is also present in the serum, and ELISA for fibronectin as well (results not shown). In the latter case, PDMS interacts with the tissue culture surface via hydrophobic forces, acting as a cover that prevents proteins from adsorbing onto the surface, maintaining it denuded. The presence of fibronectin on the surface after the scraping of the cells and its absence in the case of PDMS slab removal are a clear indication of the distinct differences between the surfaces in these two treatments. We have observed reproducible reduction of fibronectin levels in PDMS-covered wells; furthermore, the extent of reduction correlated with the fractional area of the surface covered with PDMS (results not shown).
The type, quantity, and the spatial distribution of adhesive ligands on the surface can greatly affect cell migration (15). We therefore wanted to devise a method that would allow us to study just the effects of initial mechanical injury, with the cell monolayers migrating on the same kind of surface. The peeling off of a PDMS membrane over which a monolayer had partially grown (Fig. 1B) allowed us to do that. In this case, as in the case of a "scratch" assay, the two waves of MAPK activation were observed, with similar characteristics to the MAPK waves observed after a scratch (Fig. 3), suggesting a similar kind of signaling response to these two kinds of injury. On the basis of these experiments, we conclude that the primary wave of MAPK phosphorylation is induced by injury alone. Finally, the coincidence of the secondary MAPK phosphorylation waves in all three experimental designs (injury, PDMS slab removal, and membrane peel-off) suggests that the two phases of MAPK phosphorylation can be decoupled.
Displacement of individual cells in a spreading epithelial sheet. To gain further insight into the epithelial sheet dynamics after wounding, we followed cell migration by video time-lapse microscopy. Images were taken every 5 min, a time interval small enough for a detailed tracking analysis of a cell’s path. Figure 4 shows positions of 45 cells in the epithelial sheet wounded by scratching. We found that cells in the epithelial sheet move in a highly coordinated manner: at any given moment in time, the distance traveled by the cell correlates with the initial distance of the cell from the wound. For example, Fig. 5A shows the distances traveled by individual cells at different times, as a function of the initial distance of the cell from the wound interface. Figure 6A shows the correlation between the total displacements of cells (>16 h after wounding) with their initial coordinates. These measurements suggest that, following wounding, the epithelial sheet moves in a quasi one-dimensional way, like a rubber band "pulled" into the denuded area. This is clearly supported by Fig. 7A, which shows the coordinates of a group of cells aligned normal to the wound interface.
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To dissect the effects of surface properties and cell injury on the overall sheet migration, we followed migration of individual cells in the membrane peel-off experiments, a treatment in which the cells are injured, but migrate on a plain tissue culture surface. If the injury alone is sufficient to initiate the coordinated migration of cells in the epithelial layer, then we should expect behavior similar to that observed with the scratch-wounded monolayer. Consistent with this prediction, we find that the epithelial sheet dynamics in the peel-off experiment qualitatively resembles the sheet dynamics in the scratch-wounding experiment. Specifically, there is a clear correlation between the displacements of cells and their location within the sheet (see Fig. 6B). The smaller overall displacements (Fig. 5B) can be attributed to the differences in the composition of the surfaces on which the cells are migrating. On the basis of these experiments, we conclude that mechanical injury provides the signals and stimuli necessary for the induction of the two waves of MAPK phosphorylation, and the coordination of cell dynamics necessary for epithelial cell sheet migration after wounding. We have confirmed that the MAPK phosphorylation patterns and sheet migration dynamics could be abolished by the addition of MAPK inhibitor (UO126), again in agreement with the results of Matsubayashi et al. (Figs. 5D and 6D).
Formation of ROS follows an injury and is responsible for initiating wound healing-like response. The well-organized spatiotemporal patterns of MAPK activation in wounded MDCK monolayers suggest an extensive degree of cell-cell communication. Among the numerous molecules that can mediate intercellular signaling are the extracellular calcium secreted growth factor and ROS. Furthermore, ROS have been shown to directly affect intracellular signaling, cytoskeletal dynamics, cell-cell adhesion, and cell motility (11, 20, 22, 23, 26). It is established that ROS, transiently generated under a variety of stress conditions, can couple to intracellular signaling pathways (24). We have hypothesized that ROS are involved in cell-cell coordination required for the generation of the traveling waves of MAPK activity. In one possible mechanism, ROS generated by a mechanical injury can be used as a fast way to propagate the information about the injury both within the cells and across the layer.
We used CM-H2DCFDA as a reagent to visualize the spatiotemporal pattern of ROS distribution across the monolayer and found that, immediately after injury (on the time scale of seconds), a wave of ROS was formed. The ROS wave was confined to the first few layers of cells immediately next to the wound interface and remained present for up to 10 min after the injury (Fig. 8A).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Taken together, our signaling data and our tracking analysis suggest the following rudimentary sequence of events after an epithelial layer is mechanically injured: immediate formation of ROS, which then initiates the fast, transient ERK1/2 wave resulting in the initiation of the coordinated directed sheet migration. We can further speculate that it is the locomotion of individual cells in the sheet that leads to the activation of the slower wave of ERK1/2, consistent with the observation that the transient wave is present only in the case of an injury, whereas the slower, sustained wave is present in both the injury and the unconstrained case, both conditions under which cell locomotion is present (Fig. 8D).
The experimental system presented here allowed us to assess the relative contributions of the initial injury, MAPK signaling pathway, and the surface properties on the coordinated migration of an epithelial sheet. Our approach is well suited for further experimental studies of cell-cell coordination in the dynamics of epithelial sheets. Future work is required to investigate the biochemical and cellular origin of the waves of MAPK activation in wounded MDCK epithelial layers. While the original paper by Matsubayashi et al. proposed that these waves might be driven by local (mechanical) cell-cell interactions, several independent experiments point to several diffusible signals that might contribute to the observed spatiotemporal patterns of MAPK activation. Among these are extracellular Ca2+, ATP, and peptide growth factors (5, 13, 14, 21a, 28). Furthermore, other signaling pathways, such as the JNK MAPK, are crucial for wound closure in the MDCK monolayers and several in vivo systems (1, 9). These signals can form a highly dynamic and spatially distributed network that regulates cell shape changes and cell-cell and cell-substrate interactions in spreading of the wounded epithelial sheet.
Experiments with MDCK epithelial sheets and our methods for systematic manipulation of initial and boundary conditions are ideally suited for the analysis of emerging collective behavior in wounded epithelial layers. When coupled with the analysis of mechanical behavior of cells (their morphology and forces exerted on the substrate and one another) and cell-cell signaling these studies can lead to the formulation of mechanistic descriptions of coordinated cell dynamics in epithelial layers (8, 18, 21b). The advanced models for migration of individual cells can form the starting point for these models (6, 7, 10).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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