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Single-Cell Defects Cause a Long-Range Mechanical Response in a Confluent Epithelial Cell Layer
Please cite this article in press as: Karsch et al., Single-Cell Defects Cause a Long-Range Mechanical Response in a Confluent Epithelial Cell Layer, Biophysical Journal (2017), https://doi.org/10.1016/j.bpj.2017.10.025
Biophysical Letter
Single-Cell Defects Cause a Long-Range Mechanical Response in a Confluent Epithelial Cell Layer Susanne Karsch,1 Deqing Kong,2 Jo¨rg Großhans,2 and Andreas Janshoff1,* 1 Institute for Physical Chemistry, University of Go¨ttingen, Go¨ttingen, Germany and 2Institute for Developmental Biochemistry, Medical School, University of Go¨ttingen, Go¨ttingen, Germany
ABSTRACT Epithelial cells are responsible for tissue homeostasis and form a barrier to maintain chemical gradients and mechanical integrity. Therefore, rapid wound closure is crucial for proper tissue function and restoring homeostasis. In this study, the mechanical properties of cells surrounding a single-cell wound are investigated during closure of the defect. The single-cell wound is induced in an intact layer using micropipette action and responses in neighboring cells are monitored with atomic force microscopy. Direct neighbors reveal a rise in the apparent pretension, which is dominated by cortical tension. The same effect was observed for a single-cell wound induced by laser ablation and during closure of a not fully confluent layer. Moreover, changes in the apparent pretension are far reaching and persist even in cells separated by three cell widths from the defect. This shows that epithelial cells respond to minimal wounds in a collective fashion by increased contractility with substantial reach.
INTRODUCTION Wound repair and closure is a very important physiological process in living organisms (1), which is why a lot of tissue types share similarities (2). Especially for epithelial cells, which form a tight barrier that prevents molecules from crossing the epithelium and provide mechanical stability (3), the integrity and tight regulation of the cell layer are indispensable (4). Therefore, proper wound closure must be ensured to restore tissue homeostasis, but parameters like wound size (5,6) and wound geometry (7) can influence the closing. For closing larger wounds, migration, and hence formation of lamellipodia and leader cells, is important (6,8–10). Contrastingly, for small wounds of the size of one or only a few cells, the wound is mostly closed by a multistep mechanism relying on the action of an actomyosin purse string (11–15). Depending also on the method of wounding, both mechanisms can be present to different extents (16). In this context, Trepat and co-workers (17) used traction force microscopy for wounds the size of about 20 cells and thereby found a force pattern that can be explained by a two-stage process including both mechanisms. At an early stage, leading actin protrusions from cells adjacent to the
Submitted June 16, 2017, and accepted for publication October 16, 2017. *Correspondence: [email protected] Editor: Jeffrey Fredberg. https://doi.org/10.1016/j.bpj.2017.10.025
wound generate traction forces pointing away from the defect, indicating that wound closure is driven by cell migration. At a later stage, traction forces also point inward due to the action of the actomyosin ring lining the wound and generating tension via focal adhesions to the underlying substrate, which deforms and drags the cell sheet inward. However, for wounding of a single cell in a confluent layer, as performed in this study, it was found that an explicit multistep mechanism takes place: initially, the dying cell provides a signal for the adjacent cells by exerting tension on the neighboring cells through a contractile apical F-actin ring (18,19). Then, Rho and Rho-kinase localize at the wound margin and a multicellular actomyosin purse string is established at the wound margin (20,21). In a next step, myosin light chain kinase is activated and the actomyosin ring starts to contract while moving in an apical to basal direction (19,21). Lastly, F-actin protrusions from the neighbors become visible at the basal plane, which leads to the final cell extrusion (19,21). Caspase activity and the sphingosine 1-phosphate pathway are essential for the final extrusion process (22,23). Here, the aim is to enlarge the known parameter space by mapping the apical mechanical properties of cells neighboring a wound to address the impact of wounding on cellular elasticity and cortex tension. The above-mentioned multistep purse-string mechanism was investigated and
Ó 2017 Biophysical Society.
Biophysical Journal 113, 1–8, December 19, 2017 1
Please cite this article in press as: Karsch et al., Single-Cell Defects Cause a Long-Range Mechanical Response in a Confluent Epithelial Cell Layer, Biophysical Journal (2017), https://doi.org/10.1016/j.bpj.2017.10.025
Karsch et al.
compared to a large cell-free wound devoid of a multicellular purse string. With site-specific force-indentation experiments, we can show that in both cases the cell cortex stiffens over supracellular length scales, pointing to a collective mechanical behavior when layer integrity is impaired. MATERIALS AND METHODS Cell culture Madin-Darby canine kidney cells (strain II) (MDCKII cells) were obtained from the Health Protection Agency (Salisbury, United Kingdom) and cultivated in Earl’s minimum essential medium (without L-glutamine; Lonza, Basel, Switzerland) supplemented with 4 mM glutamine (Biochrom, Berlin, Germany) and 10% (v/v) fetal calf serum (BioWest, Nuaille, France) at 37 C in a 5% CO2 humidified incubator. Cells were subcultured every 2–3 days after reaching confluency via trypsinization (trypsin/EDTA 0.5%/0.2%; Biochrom). For experiments, penicillin-streptomycin (0.2 mg/mL; PAA, Pasching, Germany) and HEPES (10 mM; Biochrom) were added to the culture medium.
(UPLFLN100xO2PH, NA 1.3; Olympus). Phase-contrast images were taken with an inverted microscope (IX 81; Olympus) and a 40 air objective. Geometrical parameters were measured in ImageJ by manually selecting the cell boundaries.
Atomic force microscopy Force indentation experiments were performed with a NanoWizard II/IV atomic force microscope (AFM) (JPK Instruments). The thermal-noise method (24) was applied to calculate the sensitivity and measure the spring constant of the pyramidal-tip cantilever. C levers (nominal spring constant of 10 pN/nm) from MLCT-Cantilevers (Bruker AFM Probes, Camarillo, CA) were used. Before each experiment, the cantilevers were cleaned with isopropanol and demineralized water. To facilitate tether pulling, the tips were incubated for 90 min in a 2.5 mg/mL concanavalin A-FITC conjugate (Sigma-Aldrich, Steinheim, Germany) in PBS, as described earlier (25). Force curves were taken in force-mapping mode with a resolution of 2 or 4 mm/pixel. A tip velocity of 3 mm/s and a set point of 1 nN was chosen. After reaching the set point, a dwell time of 0.5 s at constant height was employed to increase the likelihood of tether pulling upon retraction. Before analysis, the force curves were baseline-corrected and the contact point was assigned by visual inspection. Then, a polynomial (Eq. 1)),
Cell manipulation For single-cell manipulation cells were grown for 2 days to confluency using a gridded petri dish (m-Dish, 35 mm, grid-500; ibidi, Martinsried, Germany) and mounted on an inverted microscope (Olympus IX 81; Olympus, Tokyo, Japan) equipped with a heater (set to 37 C; JPK Instruments, Berlin, Germany). The cells were mechanically manipulated via a microinjection system (Femtojet and InjectMan NI2, both from Eppendorf, Hamburg, Germany). Glass capillaries (Femtotip; Eppendorf) were mounted on the micromanipulator and moved carefully across a cell cortex for a few seconds until a change in the phase-contrast image was visible, indicative of successful wounding. As a control, single cells were destroyed using a laser ablation setup (DSPL-355/14, 355 nm, 70 mJ/pulse; Rapp Optoelectronic, Wedel, Germany) with 40 pulses within 200 ms and a laser power of 2–3%. For the ablation, a 40 oil objective (NA 1.3) was used. For the incomplete cell monolayer, cells were seeded in a slightly lower density and also grown for 2 days using the same culture conditions.
Cell labeling At the desired time lag after manipulation, cells were washed with phosphate-buffered saline (PBS) without Ca2þ and Mg2þ (Biochrom) and fixed with 4% paraformaldehyde in PBS for 20 min. For blocking and permeabilization, the cells were treated for 30 min with blocking buffer (5% (w/v) bovine serum albumin and 0.3% (w/v) Triton-X 100 in PBS). To stain actin, 165 nM Alexa Fluor 546-phalloidin (Invitrogen, Darmstadt, Germany) was added to the cells for 1 h. Nuclei were labeled via a 15 min incubation with 50 ng/mL 40 ,6-diamino-2-phenylindole (stock 500 ng/mL in methanol). All dyes were diluted in dilution buffer (1% (w/v) bovine serum albumin and 0.3% (w/v) Triton-X 100 in PBS) to the desired concentration. Between labeling steps, cells were rinsed three times with PBS for 5 min and placed on a shaker.
Optical microscopy Fluorescence images were taken with a confocal laser scanning microscope (CLSM FluoView1200; Olympus) using an oil immersion objective
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~0d þ K ~ A d3 ; FðdÞ ¼ T
(1)
was fitted to the contact regime of the force (F)-indentation (d) curve with ~ A as the two fit parameters, which represent the mechanical propT~ 0 and K erties of the cell (vide infra). All these steps were performed with homewritten MATLAB (The MathWorks, Natick, MA) scripts. Force curves for which the contact point could not be unequivocally assigned or a drop in the force signal was found in the indentation part (‘‘breakthrough event’’) were excluded from analysis. Tether forces were obtained from measuring the final rupture force at the end of a force plateau during the retraction of the cantilever. For analysis of force curves according to the Sneddon model (26), baseline correction and definition of contact region were the same as described above. The resulting force distance curve was fitted with Eq. 2:
FðdÞ ¼
2 tan q E 2 d : p ð1 y2 Þ
(2)
y is the Poisson ratio ðy ¼ 0:5Þ , q the half-opening angle of the conical indenter, and E the Young’s modulus. Statistical analysis was done with the Wilcoxon rank sum test.
RESULTS Wounding of a single cell Single cells as part of a confluent epithelial cell layer are mechanically compromised using either micropipette action or laser ablation (Fig. 1 A). The wounded cell is extruded from the cell layer (Fig. 1 B) by contraction of the supracellular actomyosin ring acting essentially as a purse string (Fig. 1 C). Finally, the wound is closed after 100 min (Fig. 1 D). During the first 10 min after wounding, the wound size stays roughly constant, which is in accordance with findings from others showing that during this time
Please cite this article in press as: Karsch et al., Single-Cell Defects Cause a Long-Range Mechanical Response in a Confluent Epithelial Cell Layer, Biophysical Journal (2017), https://doi.org/10.1016/j.bpj.2017.10.025
Biophysical Letter FIGURE 1 (A) Schematics (top) and phase-contrast image (bottom) of singlecell wounding with a micropipette. (B) Confocal image including xz and yz projections of a wounded cell (arrows). The cell shows an increase in cell height before extrusion (red, actin; blue, nucleus). (C) Fluorescence micrographs 30 and 105 min after wounding show an accumulation of actin filaments (red) surrounding the wounded cell. The filaments remain visible even after the wound is closed. (blue, nucleus). (D) Typical time trace of the normalized wound size (normalized to the initial area of the cell) during closure. Inset: example of cell shapes before (thin black line) and 50 min after wounding (thick red line) of the center cell, showing the typical rosette formation. (E) Example of an AFM indentation experiment where the apical cell membrane is deformed (dark gray curve). The force (F)-distance (d) curve is subject to fitting with Eq. 1. During cantilever retraction (light gray curve), tethers were extracted from the cell, and the associated rupture forces provide a means to calculate the membrane tension. Polynomial fit and tether-force measurement are indicated in red. All scale bars, 20 mm.
frame Rho-mediated actomyosin ring assembly takes place before wound shrinkage (20). Mechanical parameters obtained from AFM force cycles AFM force indentation cycles (Fig. 1 E) were carried out to monitor changes in mechanical properties during wound closure. Force-indentation curves were analyzed by means of a tension-based model that was previously introduced to describe the elasticity of epithelial cells (25,27,28). The tension model treats the cell as a pre-stressed composite shell with lateral tension being the prime source of restoring force to deformation. The constant isotropic tension, T, resisting apical indentation is composed of a pretension, T0, and a term representing linear area dilatation of the bilayer, becoming important especially at larger indentations (i.e., the smaller the area compressibility modulus, KA, the easier stretching is accomplished) (see Eq. 3): DA T ¼ T0 þ KA : A0
(3)
Here, DA denotes the area change compared to the initial area, A0. As a lipid bilayer is only laterally extendable up to 3% before it ruptures (29), large expansion of the membrane cortex shell can only be accomplished by recruiting membrane from reservoirs like membrane protrusions or caveolae.
Assuming conformal contact with the indenter and a locally planar geometry of the apical cell membrane, indentation data were fitted with a simple polynomial (see Eq. 1). Derivation of the model and exact calculation of the ~ 0 , and the apparent area compressapparent pretension, T ~ ibility modulus, K A , can be found in the Supporting Material (Indentation theory according to the tension model; Fig. S1). The apical dominant pretension, T0 is a sum of the membrane tension and tension due to actomyosin contraction. To separate these contributions, we determine membrane tension, Tm, independently from tether pulling during retraction of the cantilever. The rupture force of individual tethers, FTether , directly provides a measure of the local membrane tension with Tm f F2Tether if the contribution from membrane viscosity is neglected. This tension originates in the membrane in-plane tension and is predominantly governed by the adhesion of the membrane to the cytoskeleton (30). Mechanical properties of cells neighboring a single-cell defect In the following, we recorded force cycles on cells adjacent to the wound (Fig. 2 A) and farther away to find out whether cells adjacent to a wound display altered mechanical properties. For this purpose, we focused on the three above ~0, K ~ A , and Tm . mentioned parameters, T ~ A , of cells The apparent area compressibility modulus, K did not show a dependency on cell position (namely,
Biophysical Journal 113, 1–8, December 19, 2017 3
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Karsch et al.
FIGURE 2 (A) AFM indentation maps allow measurement of cell mechanics during wound closure. (Left) Phase-contrast image. (Right) AFM force map overlaid with height reconstruction. The scale bar represents 20 mm. (B) Apparent pretension, T~0 , of cells next to a micropipette-induced single-cell wound (dashed red line) compared to control cells in an undisturbed cell layer (solid black line) (p < 0.001). Shown is the cumulative probability of T~0 and the corresponding histogram (inset). (C) Comparing the same cells before any disturbance (dashed blue line) and during closure of a neighboring defect (solid orange line) led to a significant increase (p ¼ 0.034). (D) Measuring undisturbed cells twice with the same time delay as needed for wounding did not change the apparent pretension (p ¼ 0.28). (E) Defect induction with a laser ablation setup led to the same effect as observed for mechanical micropipette action: cells neighboring the defect (dashed red line) display a higher apparent pretension than control cells in an undisturbed layer (solid black line) (p < 0.001). To see this figure in color, go online.
neighboring the defect or in an intact cell layer) during seven independently performed experiments (Fig. S2). Hence, the regulation of recruiting membrane reservoirs is not inherently altered by the closing process. Here, one also has to consider that the values vary over four orders ~ A , is dominantly of magnitude. As the measured value, K measurable at high strain, i.e., in large indentation depths, the forces recorded there might also be influenced by cell organelles, such as the nucleus, and hence depend on the position of the indentation on the cell. ~ 0 , is significantly In contrast, the apparent pretension, T different in cells neighboring a wound compared to cells of an intact cell monolayer (Fig. S3). Combining all independent experiments (N ¼ 7), a significant increase in the apparent pretension is visible for cells directly adjacent to a defect compared to control cells in a homogeneous layer (Fig. 2 B). This result was also reproducible when the same cells were measured two times, first undisturbed and then after inducing a single-cell wound in the center of the measurement area (Fig. 2 C). To rule out that the in~ 0 at the border of the wound is due to perturbacrease in T tions by the cantilever indentation, exactly the same spot was measured two times with the same time delay needed for a wounding process but without inducing any defect in the cell layer. No significant shift was measurable leading to the conclusion that wound induction is responsible for
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an increase in apparent pretension in cells neighboring and hence closing the single-cell wound (Fig. 2 D). To verify that the effect is independent of the way the wound was generated, we also used laser ablation to generate single-cell wounds. Energies of 80 mJ per cell led to the same closure process observed for the mechanical ablation. Also, after induction of the single-cell defect, an increase in the apparent pretension of neighbors is measurable (Fig. 2 E). This showed that the stiffening is an intrinsic process generally occurring in single-cell wound closure. The question now was whether the increase in pretension is limited to single-cell wounds where the purse-string mechanism prevails or whether closing of a larger cellfree wound shows the same general trend. Therefore, the front cells of an incomplete cell layer exhibiting a clear border were mapped (Fig. 3 A) and an even larger increase in the apparent pretension was detected in cells to the border (Fig. 3 B). This result shows that even in larger cell-free wounds the pretension increases in adjacent cells, most likely due to cells close to the wound crawling and dragging neighbors toward the defect in the cell layer. This is in accordance with findings that cells undergoing the epithelial-to-mesenchymal transition display higher cortical tension of the mesenchymal phenotype (31). As stated above, the cells’ pretension, T0 compiles contributions from membrane tension and actomyosin
Please cite this article in press as: Karsch et al., Single-Cell Defects Cause a Long-Range Mechanical Response in a Confluent Epithelial Cell Layer, Biophysical Journal (2017), https://doi.org/10.1016/j.bpj.2017.10.025
Biophysical Letter
most of the force curves were not accurately described by the model, which already raises questions as to the fulfillment of the underlying assumptions of this model. However, with this method, a stiffening of cells neighboring a defect is also detectable (Fig. 3 C), indicating that cell mechanics are affected by wounding independent of the exact model assumptions. Spatial extension of altered mechanics in the cell monolayer
FIGURE 3 (A) Mechanical investigation of the migration front of an incomplete cell layer with a clear border. (Left) Phasecontrast image. (Right) AFM force map (height reconstruction). The scale bar represents 20 mm. (B) Apparent pretension in cells at the migration front (dashed red line) compared to bulk cells far away from a border (solid black line) (p < 0.001). Shown is the cumulative probability of T~0 and the corresponding histogram (inset). (C) Generic Sneddon model analysis reveals an increase in the Young’s modulus in cells neighboring a defect (dashed red line) compared to cells in an intact cell layer (solid black line) (p < 0.001). Shown is the cumulative probability of the Young’s modulus and the corresponding histogram (inset). To see this figure in color, go online.
contractility of the cortex attached to the plasma membrane. To disentangle the two contributions and to see which one is responsible for the increase in pretension, tether forces were measured upon retraction of the cantilever giving access to the membrane tension. Tether rupture forces showed no difference in resilience between cells neighboring a wound and control cells surrounded by an intact cell layer (Fig. S4). As a consequence, ~ 0 , can the measured increase of the apparent pretension, T only originate from changes in cortical tension. We suggest that the increase of the apical pretension must be evoked by increased actomyosin contractility in the cell cortex of cells responding to the existence of a neighboring wound. Thus, generation of contractility happens not only in the purse string but also in the apical cell cortex during closure. As this increase is also seen in front cells of a not fully confluent layer, where the purse string is not the dominant closure mechanism, we do not state that the purse string directly induces the apical cortex stiffening. However, for single-cell wounding, both effects seem to coincide. At this point, the advantages of the tension model come to light, as it allows for distinguishing different contributions to the overall cell mechanics. Nonetheless, a generic set of force curves (experiment G) was additionally fitted with the traditional Sneddon model. As only one fit parameter exists,
Spatz and co-workers (32) investigated the force correlation of cells as a function of distance from a larger wound and found that up to 10 cells away, traction forces remain increased. Along similar lines, Trepat and co-workers (17) report that contractile forces are transmitted over supracellular length scales via focal adhesions. Therefore, we asked the question to what extent cells that are not in direct contact with the defect also display altered cortical mechanics due to cell-cell mechanotransduction. ~ 0 , is still We found that the apparent pretension, T increased in fourth-row neighbors (named ‘‘neighbor 4’’), which are separated by three cells and hence are several tens of microns away from the defect (Fig. 4 A). Also, for the incomplete cell layer, a far-reaching mechanical effect
FIGURE 4 Cellular mechanics as a function of distance from the wound or defect. (A) Elevated apparent pretension, T~0 , is also measurable in cells belonging to outer shells of the defect (with ‘‘neighbor 1’’ being the direct neighbor and ‘‘neighbor 4’’ being separated from the wound by three cells) and (B) at larger distances from the migration front of a subconfluent layer displaying clear borders. Error bars indicate the mean 5 SE. (C) The apparent pretension, T~0 , stays constant (p > 0.05) when the same cells are measured before (dashed blue line) and after (solid orange line) inducing a defect at larger distances (>100 mm away from the measuring area). Shown is the cumulative probability, with the corresponding histogram (inset). To see this figure in color, go online.
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Please cite this article in press as: Karsch et al., Single-Cell Defects Cause a Long-Range Mechanical Response in a Confluent Epithelial Cell Layer, Biophysical Journal (2017), https://doi.org/10.1016/j.bpj.2017.10.025
Karsch et al.
was measurable. Compared to cells in a fully confluent cell layer, with its cobblestone morphology, the cell shape is less well defined under these conditions. Hence, not the degree of neighborhood but the distance from the wound margin was considered. Here, even up to 90 mm away from the ~0, migration front, an increased apparent pretension, T was found (Fig. 4 B). In the case of the single-cell wound, where the increased pretension is less pronounced, the effect had already vanished when cells >100 mm away from the defect were measured before and after inducing the defect (Fig. 4 C). This means that the information is spread over greater distances than a single cell, which is probably due to the strong connectivity of MDCKII cells acting as a mechanical syncytium rather than individual cells (Fig. 5). Still, this far-reaching mechanical response is in line with correlation lengths of several cell widths found for migrating MDCKII cells (32,33). As the disturbance by a single-cell defect is smaller, smaller spatial extension of the effect is also expected. All in all, cortical mechanics also seem to be highly correlated between cells in close proximity, most likely due to cell-cell contacts. Another finding is that mechanical properties of cells as measured by force indentation are also affected by cellular morphology, such as size and geometry (34). Therefore, we also investigated size and shape parameters of the cells during wound closure and examined whether simple morphological changes could also account for the observed increase in pretension. The analysis shows that the circularity of cells neighboring a defect stays constant over time (Fig. S5 A) and also that the aspect ratio reveals no strong tendency (Fig. S5 B). This shows that the cells do not change their overall shape during single-cell wound
FIGURE 5 Spatial extension of mechanical changes. A manually skeletonized image shows the affected cells of the singlecell defect, with their assigned shell numbers, before (gray) and 60 min after (red) wounding of the center cell (X). The scale bar represents 20 mm. To see this figure in color, go online.
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closure in a way that would explain the elevation in pretension. However, 40 min after wounding, variations in the aspect ratio of adjacent cells increase. At this stage, the individual cells slightly elongate toward the wounded spot, a process that is necessary for the typical rosette formation. As these changes are still marginal compared to the overall cell size and very individual for every single cell, a switch in morphology cannot explain the obvious increase in pretension. Additionally, the projected area stays constant over time, with only small variations (Fig. S5 C). This further implies that the area loss during wounding is not compensated by the direct neighbors alone but rather by a cooperative and collective process. Therefore, after the wound is finally closed, the area loss has been collectively compensated for by a large number of marginal size changes in more cells than the five to seven direct neighbors. DISCUSSION It is consensus that contractility at the wound margin is a key feature to realize proper wound closure (19–21). Here, we show that in addition, the apical actomyosin cortex is reinforced during this process, leading to a larger resistance against deformation during indentation experiments. This might be due to the coherence of the actomyosin cortex supporting the cell plasma membrane at the cell periphery (35). As the effect of increased cortical tension is also seen in larger wounds, where the purse string is less dominant, it implies that contractility of the purse string might not be the only factor underlying a higher prestress stored in the apical cell membrane. Notably, a reduction of cortical tension at cell-cell contacts is required to maintain connectivity and reach homeostasis in a cell layer (36). This is due to the fact that the energy gain due to cell-cell adhesion (e.g., by forming molecular contacts such as those established by E-cadherins) is not sufficient to enlarge the interfacial area and generate appreciable adhesion (37). As cortical tension comprises cell-cell adhesion (38), it has to be reduced for proper layer integrity (36). This can also nicely explain the large pretension found for front cells at the boundary of a subconfluent cell layer. Here, the tension is largely increased, as no layer homeostasis can be established at this point. Along the same line, disturbing layer integrity by inducing a single-cell wound seems to disturb homeostasis and leads to a shift in the mechanical phenotype toward increased cortical tension. This is also in line with earlier studies showing the same effect, e.g., for the epithelial-tomesenchymal transition (31). There, a mesenchymal cell displayed much larger cortical tension than an epithelial phenotype. Also, our findings suggest that a confluent epithelial cell layer mechanically responds in a collective manner to wounding, even though the perturbation might be small, namely, the size of a single cell. This mechanical behavior
Please cite this article in press as: Karsch et al., Single-Cell Defects Cause a Long-Range Mechanical Response in a Confluent Epithelial Cell Layer, Biophysical Journal (2017), https://doi.org/10.1016/j.bpj.2017.10.025
Biophysical Letter
of the unperturbed neighbors points toward a mechanical syncytium rather than an assembly of individual cells (Fig. 5). How exactly tension is transmitted to cells far away from the defect is still unclear. Both the action of an actomyosin purse string and crawling cells dragging their neighbors toward the wound are conceivable factors in the increase of tension in the apical sheet. In any case, cell-cell contacts such as the adherens junction are prime candidates to explain the long-distance transmission of stress: it was shown that long-scale interactions depend on the functionality of cell-cell junctions (39) and apical-lateral junction contractility is fundamental for proper cell layer homeostasis (40). Force-activated enhancement of cell-cell junctions and cadherin-cytoskeleton interactions might be a key factor in signal transduction via cell-cell junctions (41–43). Also, traveling stress patterns inside the layer, mediated by cellsubstrate adhesion complexes, seem to be involved in epithelial wound healing on elastic substrates (17,44). However, as both of the adhesion complexes seem to be tightly coupled in a cell layer (45,46), both adhesion mechanisms might be involved at the same time during wound closure. In addition, chemical signaling through GTPase activity at cell-cell junctions during wound closure points to a junction-mediated coupling between neighbors (47). Another factor is traveling calcium waves, which were observed after wounding (48,49) and can induce F-actin reorganization (50) after wounding.
ACKNOWLEDGMENTS Financial support from the Deutsche Forschungsgemeinschaft through CRC 937 (A10, A14) and priority program SPP 1782 is acknowledged.
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CONCLUSIONS The repair of epithelial MDCKII cells subjected to external generation of wounds has been investigated by means of AFM in conjunction with optical microscopy. We found that cortical mechanics is significantly altered adjacent to a wound and extends over supracellular length scales. This suggests that the mechanotransduction most likely accomplished via cell-cell contacts is relayed over large distances within an intact cell monolayer. Cortical tension might therefore be used by the cell layer as a source of information to prompt a large number of cells to help with closing the wound. This far-reaching mechanical effect distributes the burden of wound closure over many cells.
SUPPORTING MATERIAL Supporting Materials and Methods and five figures are available at http:// www.biophysj.org/biophysj/supplemental/S0006-3495(17)31145-1.
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AUTHOR CONTRIBUTIONS
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S.K. and D.K. performed the ablation experiments. S.K. performed AFM and imaging experiments and analyzed data. S.K. and A.J. wrote the manuscript with contributions from all authors (J.G. and D.K).
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39. Vedula, S. R. K., M. C. Leong, ., B. Ladoux. 2012. Emerging modes of collective cell migration induced by geometrical constraints. Proc. Natl. Acad. Sci. USA. 109:12974–12979. 40. Wu, S. K., G. A. Gomez, ., A. S. Yap. 2014. Cortical F-actin stabilization generates apical-lateral patterns of junctional contractility that integrate cells into epithelia. Nat. Cell Biol. 16:167–178. 41. Liu, Z., J. L. Tan, ., C. S. Chen. 2010. Mechanical tugging force regulates the size of cell-cell junctions. Proc. Natl. Acad. Sci. USA. 107:9944–9949. 42. le Duc, Q., Q. Shi, ., J. de Rooij. 2010. Vinculin potentiates E-cadherin mechanosensing and is recruited to actin-anchored sites within adherens junctions in a myosin II-dependent manner. J. Cell Biol. 189:1107–1115. 43. Barry, A. K., H. Tabdili, ., D. E. Leckband. 2014. a-catenin cytomechanics—role in cadherin-dependent adhesion and mechanotransduction. J. Cell Sci. 127:1779–1791. 44. Serra-Picamal, X., V. Conte, ., X. Trepat. 2012. Mechanical waves during tissue expansion. Nat. Phys. 8:628–634. 45. Mertz, A. F., Y. Che, ., V. Horsley. 2013. Cadherin-based intercellular adhesions organize epithelial cell-matrix traction forces. Proc. Natl. Acad. Sci. USA. 110:842–847. 46. Nelson, C. M., D. M. Pirone, ., C. S. Chen. 2004. Vascular endothelial-cadherin regulates cytoskeletal tension, cell spreading, and focal adhesions by stimulating RhoA. Mol. Biol. Cell. 15:2943–2953. 47. Clark, A. G., A. L. Miller, ., W. M. Bement. 2009. Integration of single and multicellular wound responses. Curr. Biol. 19:1389–1395. 48. Klepeis, V. E., A. Cornell-Bell, and V. Trinkaus-Randall. 2001. Growth factors but not gap junctions play a role in injury-induced Ca2þ waves in epithelial cells. J. Cell Sci. 114:4185–4195.
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Biophysical Letter
Single-Cell Defects Cause a Long-Range Mechanical Response in a Confluent Epithelial Cell Layer Susanne Karsch,1 Deqing Kong,2 Jo¨rg Großhans,2 and Andreas Janshoff1,* 1 Institute for Physical Chemistry, University of Go¨ttingen, Go¨ttingen, Germany and 2Institute for Developmental Biochemistry, Medical School, University of Go¨ttingen, Go¨ttingen, Germany
ABSTRACT Epithelial cells are responsible for tissue homeostasis and form a barrier to maintain chemical gradients and mechanical integrity. Therefore, rapid wound closure is crucial for proper tissue function and restoring homeostasis. In this study, the mechanical properties of cells surrounding a single-cell wound are investigated during closure of the defect. The single-cell wound is induced in an intact layer using micropipette action and responses in neighboring cells are monitored with atomic force microscopy. Direct neighbors reveal a rise in the apparent pretension, which is dominated by cortical tension. The same effect was observed for a single-cell wound induced by laser ablation and during closure of a not fully confluent layer. Moreover, changes in the apparent pretension are far reaching and persist even in cells separated by three cell widths from the defect. This shows that epithelial cells respond to minimal wounds in a collective fashion by increased contractility with substantial reach.
INTRODUCTION Wound repair and closure is a very important physiological process in living organisms (1), which is why a lot of tissue types share similarities (2). Especially for epithelial cells, which form a tight barrier that prevents molecules from crossing the epithelium and provide mechanical stability (3), the integrity and tight regulation of the cell layer are indispensable (4). Therefore, proper wound closure must be ensured to restore tissue homeostasis, but parameters like wound size (5,6) and wound geometry (7) can influence the closing. For closing larger wounds, migration, and hence formation of lamellipodia and leader cells, is important (6,8–10). Contrastingly, for small wounds of the size of one or only a few cells, the wound is mostly closed by a multistep mechanism relying on the action of an actomyosin purse string (11–15). Depending also on the method of wounding, both mechanisms can be present to different extents (16). In this context, Trepat and co-workers (17) used traction force microscopy for wounds the size of about 20 cells and thereby found a force pattern that can be explained by a two-stage process including both mechanisms. At an early stage, leading actin protrusions from cells adjacent to the
Submitted June 16, 2017, and accepted for publication October 16, 2017. *Correspondence: [email protected] Editor: Jeffrey Fredberg. https://doi.org/10.1016/j.bpj.2017.10.025
wound generate traction forces pointing away from the defect, indicating that wound closure is driven by cell migration. At a later stage, traction forces also point inward due to the action of the actomyosin ring lining the wound and generating tension via focal adhesions to the underlying substrate, which deforms and drags the cell sheet inward. However, for wounding of a single cell in a confluent layer, as performed in this study, it was found that an explicit multistep mechanism takes place: initially, the dying cell provides a signal for the adjacent cells by exerting tension on the neighboring cells through a contractile apical F-actin ring (18,19). Then, Rho and Rho-kinase localize at the wound margin and a multicellular actomyosin purse string is established at the wound margin (20,21). In a next step, myosin light chain kinase is activated and the actomyosin ring starts to contract while moving in an apical to basal direction (19,21). Lastly, F-actin protrusions from the neighbors become visible at the basal plane, which leads to the final cell extrusion (19,21). Caspase activity and the sphingosine 1-phosphate pathway are essential for the final extrusion process (22,23). Here, the aim is to enlarge the known parameter space by mapping the apical mechanical properties of cells neighboring a wound to address the impact of wounding on cellular elasticity and cortex tension. The above-mentioned multistep purse-string mechanism was investigated and
Ó 2017 Biophysical Society.
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compared to a large cell-free wound devoid of a multicellular purse string. With site-specific force-indentation experiments, we can show that in both cases the cell cortex stiffens over supracellular length scales, pointing to a collective mechanical behavior when layer integrity is impaired. MATERIALS AND METHODS Cell culture Madin-Darby canine kidney cells (strain II) (MDCKII cells) were obtained from the Health Protection Agency (Salisbury, United Kingdom) and cultivated in Earl’s minimum essential medium (without L-glutamine; Lonza, Basel, Switzerland) supplemented with 4 mM glutamine (Biochrom, Berlin, Germany) and 10% (v/v) fetal calf serum (BioWest, Nuaille, France) at 37 C in a 5% CO2 humidified incubator. Cells were subcultured every 2–3 days after reaching confluency via trypsinization (trypsin/EDTA 0.5%/0.2%; Biochrom). For experiments, penicillin-streptomycin (0.2 mg/mL; PAA, Pasching, Germany) and HEPES (10 mM; Biochrom) were added to the culture medium.
(UPLFLN100xO2PH, NA 1.3; Olympus). Phase-contrast images were taken with an inverted microscope (IX 81; Olympus) and a 40 air objective. Geometrical parameters were measured in ImageJ by manually selecting the cell boundaries.
Atomic force microscopy Force indentation experiments were performed with a NanoWizard II/IV atomic force microscope (AFM) (JPK Instruments). The thermal-noise method (24) was applied to calculate the sensitivity and measure the spring constant of the pyramidal-tip cantilever. C levers (nominal spring constant of 10 pN/nm) from MLCT-Cantilevers (Bruker AFM Probes, Camarillo, CA) were used. Before each experiment, the cantilevers were cleaned with isopropanol and demineralized water. To facilitate tether pulling, the tips were incubated for 90 min in a 2.5 mg/mL concanavalin A-FITC conjugate (Sigma-Aldrich, Steinheim, Germany) in PBS, as described earlier (25). Force curves were taken in force-mapping mode with a resolution of 2 or 4 mm/pixel. A tip velocity of 3 mm/s and a set point of 1 nN was chosen. After reaching the set point, a dwell time of 0.5 s at constant height was employed to increase the likelihood of tether pulling upon retraction. Before analysis, the force curves were baseline-corrected and the contact point was assigned by visual inspection. Then, a polynomial (Eq. 1)),
Cell manipulation For single-cell manipulation cells were grown for 2 days to confluency using a gridded petri dish (m-Dish, 35 mm, grid-500; ibidi, Martinsried, Germany) and mounted on an inverted microscope (Olympus IX 81; Olympus, Tokyo, Japan) equipped with a heater (set to 37 C; JPK Instruments, Berlin, Germany). The cells were mechanically manipulated via a microinjection system (Femtojet and InjectMan NI2, both from Eppendorf, Hamburg, Germany). Glass capillaries (Femtotip; Eppendorf) were mounted on the micromanipulator and moved carefully across a cell cortex for a few seconds until a change in the phase-contrast image was visible, indicative of successful wounding. As a control, single cells were destroyed using a laser ablation setup (DSPL-355/14, 355 nm, 70 mJ/pulse; Rapp Optoelectronic, Wedel, Germany) with 40 pulses within 200 ms and a laser power of 2–3%. For the ablation, a 40 oil objective (NA 1.3) was used. For the incomplete cell monolayer, cells were seeded in a slightly lower density and also grown for 2 days using the same culture conditions.
Cell labeling At the desired time lag after manipulation, cells were washed with phosphate-buffered saline (PBS) without Ca2þ and Mg2þ (Biochrom) and fixed with 4% paraformaldehyde in PBS for 20 min. For blocking and permeabilization, the cells were treated for 30 min with blocking buffer (5% (w/v) bovine serum albumin and 0.3% (w/v) Triton-X 100 in PBS). To stain actin, 165 nM Alexa Fluor 546-phalloidin (Invitrogen, Darmstadt, Germany) was added to the cells for 1 h. Nuclei were labeled via a 15 min incubation with 50 ng/mL 40 ,6-diamino-2-phenylindole (stock 500 ng/mL in methanol). All dyes were diluted in dilution buffer (1% (w/v) bovine serum albumin and 0.3% (w/v) Triton-X 100 in PBS) to the desired concentration. Between labeling steps, cells were rinsed three times with PBS for 5 min and placed on a shaker.
Optical microscopy Fluorescence images were taken with a confocal laser scanning microscope (CLSM FluoView1200; Olympus) using an oil immersion objective
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~0d þ K ~ A d3 ; FðdÞ ¼ T
(1)
was fitted to the contact regime of the force (F)-indentation (d) curve with ~ A as the two fit parameters, which represent the mechanical propT~ 0 and K erties of the cell (vide infra). All these steps were performed with homewritten MATLAB (The MathWorks, Natick, MA) scripts. Force curves for which the contact point could not be unequivocally assigned or a drop in the force signal was found in the indentation part (‘‘breakthrough event’’) were excluded from analysis. Tether forces were obtained from measuring the final rupture force at the end of a force plateau during the retraction of the cantilever. For analysis of force curves according to the Sneddon model (26), baseline correction and definition of contact region were the same as described above. The resulting force distance curve was fitted with Eq. 2:
FðdÞ ¼
2 tan q E 2 d : p ð1 y2 Þ
(2)
y is the Poisson ratio ðy ¼ 0:5Þ , q the half-opening angle of the conical indenter, and E the Young’s modulus. Statistical analysis was done with the Wilcoxon rank sum test.
RESULTS Wounding of a single cell Single cells as part of a confluent epithelial cell layer are mechanically compromised using either micropipette action or laser ablation (Fig. 1 A). The wounded cell is extruded from the cell layer (Fig. 1 B) by contraction of the supracellular actomyosin ring acting essentially as a purse string (Fig. 1 C). Finally, the wound is closed after 100 min (Fig. 1 D). During the first 10 min after wounding, the wound size stays roughly constant, which is in accordance with findings from others showing that during this time
Please cite this article in press as: Karsch et al., Single-Cell Defects Cause a Long-Range Mechanical Response in a Confluent Epithelial Cell Layer, Biophysical Journal (2017), https://doi.org/10.1016/j.bpj.2017.10.025
Biophysical Letter FIGURE 1 (A) Schematics (top) and phase-contrast image (bottom) of singlecell wounding with a micropipette. (B) Confocal image including xz and yz projections of a wounded cell (arrows). The cell shows an increase in cell height before extrusion (red, actin; blue, nucleus). (C) Fluorescence micrographs 30 and 105 min after wounding show an accumulation of actin filaments (red) surrounding the wounded cell. The filaments remain visible even after the wound is closed. (blue, nucleus). (D) Typical time trace of the normalized wound size (normalized to the initial area of the cell) during closure. Inset: example of cell shapes before (thin black line) and 50 min after wounding (thick red line) of the center cell, showing the typical rosette formation. (E) Example of an AFM indentation experiment where the apical cell membrane is deformed (dark gray curve). The force (F)-distance (d) curve is subject to fitting with Eq. 1. During cantilever retraction (light gray curve), tethers were extracted from the cell, and the associated rupture forces provide a means to calculate the membrane tension. Polynomial fit and tether-force measurement are indicated in red. All scale bars, 20 mm.
frame Rho-mediated actomyosin ring assembly takes place before wound shrinkage (20). Mechanical parameters obtained from AFM force cycles AFM force indentation cycles (Fig. 1 E) were carried out to monitor changes in mechanical properties during wound closure. Force-indentation curves were analyzed by means of a tension-based model that was previously introduced to describe the elasticity of epithelial cells (25,27,28). The tension model treats the cell as a pre-stressed composite shell with lateral tension being the prime source of restoring force to deformation. The constant isotropic tension, T, resisting apical indentation is composed of a pretension, T0, and a term representing linear area dilatation of the bilayer, becoming important especially at larger indentations (i.e., the smaller the area compressibility modulus, KA, the easier stretching is accomplished) (see Eq. 3): DA T ¼ T0 þ KA : A0
(3)
Here, DA denotes the area change compared to the initial area, A0. As a lipid bilayer is only laterally extendable up to 3% before it ruptures (29), large expansion of the membrane cortex shell can only be accomplished by recruiting membrane from reservoirs like membrane protrusions or caveolae.
Assuming conformal contact with the indenter and a locally planar geometry of the apical cell membrane, indentation data were fitted with a simple polynomial (see Eq. 1). Derivation of the model and exact calculation of the ~ 0 , and the apparent area compressapparent pretension, T ~ ibility modulus, K A , can be found in the Supporting Material (Indentation theory according to the tension model; Fig. S1). The apical dominant pretension, T0 is a sum of the membrane tension and tension due to actomyosin contraction. To separate these contributions, we determine membrane tension, Tm, independently from tether pulling during retraction of the cantilever. The rupture force of individual tethers, FTether , directly provides a measure of the local membrane tension with Tm f F2Tether if the contribution from membrane viscosity is neglected. This tension originates in the membrane in-plane tension and is predominantly governed by the adhesion of the membrane to the cytoskeleton (30). Mechanical properties of cells neighboring a single-cell defect In the following, we recorded force cycles on cells adjacent to the wound (Fig. 2 A) and farther away to find out whether cells adjacent to a wound display altered mechanical properties. For this purpose, we focused on the three above ~0, K ~ A , and Tm . mentioned parameters, T ~ A , of cells The apparent area compressibility modulus, K did not show a dependency on cell position (namely,
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Karsch et al.
FIGURE 2 (A) AFM indentation maps allow measurement of cell mechanics during wound closure. (Left) Phase-contrast image. (Right) AFM force map overlaid with height reconstruction. The scale bar represents 20 mm. (B) Apparent pretension, T~0 , of cells next to a micropipette-induced single-cell wound (dashed red line) compared to control cells in an undisturbed cell layer (solid black line) (p < 0.001). Shown is the cumulative probability of T~0 and the corresponding histogram (inset). (C) Comparing the same cells before any disturbance (dashed blue line) and during closure of a neighboring defect (solid orange line) led to a significant increase (p ¼ 0.034). (D) Measuring undisturbed cells twice with the same time delay as needed for wounding did not change the apparent pretension (p ¼ 0.28). (E) Defect induction with a laser ablation setup led to the same effect as observed for mechanical micropipette action: cells neighboring the defect (dashed red line) display a higher apparent pretension than control cells in an undisturbed layer (solid black line) (p < 0.001). To see this figure in color, go online.
neighboring the defect or in an intact cell layer) during seven independently performed experiments (Fig. S2). Hence, the regulation of recruiting membrane reservoirs is not inherently altered by the closing process. Here, one also has to consider that the values vary over four orders ~ A , is dominantly of magnitude. As the measured value, K measurable at high strain, i.e., in large indentation depths, the forces recorded there might also be influenced by cell organelles, such as the nucleus, and hence depend on the position of the indentation on the cell. ~ 0 , is significantly In contrast, the apparent pretension, T different in cells neighboring a wound compared to cells of an intact cell monolayer (Fig. S3). Combining all independent experiments (N ¼ 7), a significant increase in the apparent pretension is visible for cells directly adjacent to a defect compared to control cells in a homogeneous layer (Fig. 2 B). This result was also reproducible when the same cells were measured two times, first undisturbed and then after inducing a single-cell wound in the center of the measurement area (Fig. 2 C). To rule out that the in~ 0 at the border of the wound is due to perturbacrease in T tions by the cantilever indentation, exactly the same spot was measured two times with the same time delay needed for a wounding process but without inducing any defect in the cell layer. No significant shift was measurable leading to the conclusion that wound induction is responsible for
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an increase in apparent pretension in cells neighboring and hence closing the single-cell wound (Fig. 2 D). To verify that the effect is independent of the way the wound was generated, we also used laser ablation to generate single-cell wounds. Energies of 80 mJ per cell led to the same closure process observed for the mechanical ablation. Also, after induction of the single-cell defect, an increase in the apparent pretension of neighbors is measurable (Fig. 2 E). This showed that the stiffening is an intrinsic process generally occurring in single-cell wound closure. The question now was whether the increase in pretension is limited to single-cell wounds where the purse-string mechanism prevails or whether closing of a larger cellfree wound shows the same general trend. Therefore, the front cells of an incomplete cell layer exhibiting a clear border were mapped (Fig. 3 A) and an even larger increase in the apparent pretension was detected in cells to the border (Fig. 3 B). This result shows that even in larger cell-free wounds the pretension increases in adjacent cells, most likely due to cells close to the wound crawling and dragging neighbors toward the defect in the cell layer. This is in accordance with findings that cells undergoing the epithelial-to-mesenchymal transition display higher cortical tension of the mesenchymal phenotype (31). As stated above, the cells’ pretension, T0 compiles contributions from membrane tension and actomyosin
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Biophysical Letter
most of the force curves were not accurately described by the model, which already raises questions as to the fulfillment of the underlying assumptions of this model. However, with this method, a stiffening of cells neighboring a defect is also detectable (Fig. 3 C), indicating that cell mechanics are affected by wounding independent of the exact model assumptions. Spatial extension of altered mechanics in the cell monolayer
FIGURE 3 (A) Mechanical investigation of the migration front of an incomplete cell layer with a clear border. (Left) Phasecontrast image. (Right) AFM force map (height reconstruction). The scale bar represents 20 mm. (B) Apparent pretension in cells at the migration front (dashed red line) compared to bulk cells far away from a border (solid black line) (p < 0.001). Shown is the cumulative probability of T~0 and the corresponding histogram (inset). (C) Generic Sneddon model analysis reveals an increase in the Young’s modulus in cells neighboring a defect (dashed red line) compared to cells in an intact cell layer (solid black line) (p < 0.001). Shown is the cumulative probability of the Young’s modulus and the corresponding histogram (inset). To see this figure in color, go online.
contractility of the cortex attached to the plasma membrane. To disentangle the two contributions and to see which one is responsible for the increase in pretension, tether forces were measured upon retraction of the cantilever giving access to the membrane tension. Tether rupture forces showed no difference in resilience between cells neighboring a wound and control cells surrounded by an intact cell layer (Fig. S4). As a consequence, ~ 0 , can the measured increase of the apparent pretension, T only originate from changes in cortical tension. We suggest that the increase of the apical pretension must be evoked by increased actomyosin contractility in the cell cortex of cells responding to the existence of a neighboring wound. Thus, generation of contractility happens not only in the purse string but also in the apical cell cortex during closure. As this increase is also seen in front cells of a not fully confluent layer, where the purse string is not the dominant closure mechanism, we do not state that the purse string directly induces the apical cortex stiffening. However, for single-cell wounding, both effects seem to coincide. At this point, the advantages of the tension model come to light, as it allows for distinguishing different contributions to the overall cell mechanics. Nonetheless, a generic set of force curves (experiment G) was additionally fitted with the traditional Sneddon model. As only one fit parameter exists,
Spatz and co-workers (32) investigated the force correlation of cells as a function of distance from a larger wound and found that up to 10 cells away, traction forces remain increased. Along similar lines, Trepat and co-workers (17) report that contractile forces are transmitted over supracellular length scales via focal adhesions. Therefore, we asked the question to what extent cells that are not in direct contact with the defect also display altered cortical mechanics due to cell-cell mechanotransduction. ~ 0 , is still We found that the apparent pretension, T increased in fourth-row neighbors (named ‘‘neighbor 4’’), which are separated by three cells and hence are several tens of microns away from the defect (Fig. 4 A). Also, for the incomplete cell layer, a far-reaching mechanical effect
FIGURE 4 Cellular mechanics as a function of distance from the wound or defect. (A) Elevated apparent pretension, T~0 , is also measurable in cells belonging to outer shells of the defect (with ‘‘neighbor 1’’ being the direct neighbor and ‘‘neighbor 4’’ being separated from the wound by three cells) and (B) at larger distances from the migration front of a subconfluent layer displaying clear borders. Error bars indicate the mean 5 SE. (C) The apparent pretension, T~0 , stays constant (p > 0.05) when the same cells are measured before (dashed blue line) and after (solid orange line) inducing a defect at larger distances (>100 mm away from the measuring area). Shown is the cumulative probability, with the corresponding histogram (inset). To see this figure in color, go online.
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was measurable. Compared to cells in a fully confluent cell layer, with its cobblestone morphology, the cell shape is less well defined under these conditions. Hence, not the degree of neighborhood but the distance from the wound margin was considered. Here, even up to 90 mm away from the ~0, migration front, an increased apparent pretension, T was found (Fig. 4 B). In the case of the single-cell wound, where the increased pretension is less pronounced, the effect had already vanished when cells >100 mm away from the defect were measured before and after inducing the defect (Fig. 4 C). This means that the information is spread over greater distances than a single cell, which is probably due to the strong connectivity of MDCKII cells acting as a mechanical syncytium rather than individual cells (Fig. 5). Still, this far-reaching mechanical response is in line with correlation lengths of several cell widths found for migrating MDCKII cells (32,33). As the disturbance by a single-cell defect is smaller, smaller spatial extension of the effect is also expected. All in all, cortical mechanics also seem to be highly correlated between cells in close proximity, most likely due to cell-cell contacts. Another finding is that mechanical properties of cells as measured by force indentation are also affected by cellular morphology, such as size and geometry (34). Therefore, we also investigated size and shape parameters of the cells during wound closure and examined whether simple morphological changes could also account for the observed increase in pretension. The analysis shows that the circularity of cells neighboring a defect stays constant over time (Fig. S5 A) and also that the aspect ratio reveals no strong tendency (Fig. S5 B). This shows that the cells do not change their overall shape during single-cell wound
FIGURE 5 Spatial extension of mechanical changes. A manually skeletonized image shows the affected cells of the singlecell defect, with their assigned shell numbers, before (gray) and 60 min after (red) wounding of the center cell (X). The scale bar represents 20 mm. To see this figure in color, go online.
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closure in a way that would explain the elevation in pretension. However, 40 min after wounding, variations in the aspect ratio of adjacent cells increase. At this stage, the individual cells slightly elongate toward the wounded spot, a process that is necessary for the typical rosette formation. As these changes are still marginal compared to the overall cell size and very individual for every single cell, a switch in morphology cannot explain the obvious increase in pretension. Additionally, the projected area stays constant over time, with only small variations (Fig. S5 C). This further implies that the area loss during wounding is not compensated by the direct neighbors alone but rather by a cooperative and collective process. Therefore, after the wound is finally closed, the area loss has been collectively compensated for by a large number of marginal size changes in more cells than the five to seven direct neighbors. DISCUSSION It is consensus that contractility at the wound margin is a key feature to realize proper wound closure (19–21). Here, we show that in addition, the apical actomyosin cortex is reinforced during this process, leading to a larger resistance against deformation during indentation experiments. This might be due to the coherence of the actomyosin cortex supporting the cell plasma membrane at the cell periphery (35). As the effect of increased cortical tension is also seen in larger wounds, where the purse string is less dominant, it implies that contractility of the purse string might not be the only factor underlying a higher prestress stored in the apical cell membrane. Notably, a reduction of cortical tension at cell-cell contacts is required to maintain connectivity and reach homeostasis in a cell layer (36). This is due to the fact that the energy gain due to cell-cell adhesion (e.g., by forming molecular contacts such as those established by E-cadherins) is not sufficient to enlarge the interfacial area and generate appreciable adhesion (37). As cortical tension comprises cell-cell adhesion (38), it has to be reduced for proper layer integrity (36). This can also nicely explain the large pretension found for front cells at the boundary of a subconfluent cell layer. Here, the tension is largely increased, as no layer homeostasis can be established at this point. Along the same line, disturbing layer integrity by inducing a single-cell wound seems to disturb homeostasis and leads to a shift in the mechanical phenotype toward increased cortical tension. This is also in line with earlier studies showing the same effect, e.g., for the epithelial-tomesenchymal transition (31). There, a mesenchymal cell displayed much larger cortical tension than an epithelial phenotype. Also, our findings suggest that a confluent epithelial cell layer mechanically responds in a collective manner to wounding, even though the perturbation might be small, namely, the size of a single cell. This mechanical behavior
Please cite this article in press as: Karsch et al., Single-Cell Defects Cause a Long-Range Mechanical Response in a Confluent Epithelial Cell Layer, Biophysical Journal (2017), https://doi.org/10.1016/j.bpj.2017.10.025
Biophysical Letter
of the unperturbed neighbors points toward a mechanical syncytium rather than an assembly of individual cells (Fig. 5). How exactly tension is transmitted to cells far away from the defect is still unclear. Both the action of an actomyosin purse string and crawling cells dragging their neighbors toward the wound are conceivable factors in the increase of tension in the apical sheet. In any case, cell-cell contacts such as the adherens junction are prime candidates to explain the long-distance transmission of stress: it was shown that long-scale interactions depend on the functionality of cell-cell junctions (39) and apical-lateral junction contractility is fundamental for proper cell layer homeostasis (40). Force-activated enhancement of cell-cell junctions and cadherin-cytoskeleton interactions might be a key factor in signal transduction via cell-cell junctions (41–43). Also, traveling stress patterns inside the layer, mediated by cellsubstrate adhesion complexes, seem to be involved in epithelial wound healing on elastic substrates (17,44). However, as both of the adhesion complexes seem to be tightly coupled in a cell layer (45,46), both adhesion mechanisms might be involved at the same time during wound closure. In addition, chemical signaling through GTPase activity at cell-cell junctions during wound closure points to a junction-mediated coupling between neighbors (47). Another factor is traveling calcium waves, which were observed after wounding (48,49) and can induce F-actin reorganization (50) after wounding.
ACKNOWLEDGMENTS Financial support from the Deutsche Forschungsgemeinschaft through CRC 937 (A10, A14) and priority program SPP 1782 is acknowledged.
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CONCLUSIONS The repair of epithelial MDCKII cells subjected to external generation of wounds has been investigated by means of AFM in conjunction with optical microscopy. We found that cortical mechanics is significantly altered adjacent to a wound and extends over supracellular length scales. This suggests that the mechanotransduction most likely accomplished via cell-cell contacts is relayed over large distances within an intact cell monolayer. Cortical tension might therefore be used by the cell layer as a source of information to prompt a large number of cells to help with closing the wound. This far-reaching mechanical effect distributes the burden of wound closure over many cells.
SUPPORTING MATERIAL Supporting Materials and Methods and five figures are available at http:// www.biophysj.org/biophysj/supplemental/S0006-3495(17)31145-1.
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