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Hydraulic fracture during epithelial stretching.

Casares L, Vincent R, Zalvidea D, Campillo N, Navajas D, Arroyo M, Trepat X - Nat Mater (2015)

Bottom Line: Here, we demonstrate that for a variety of synthetic and physiological hydrogel substrates the formation of epithelial cracks is caused by tissue stretching independently of epithelial tension.We show that the origin of the cracks is hydraulic; they result from a transient pressure build-up in the substrate during stretch and compression manoeuvres.Our findings demonstrate that epithelial integrity is determined in a tension-independent manner by the coupling between tissue stretching and matrix hydraulics.

View Article: PubMed Central - PubMed

Affiliation: Institute for Bioengineering of Catalonia, 08028 Barcelona, Spain.

ABSTRACT
The origin of fracture in epithelial cell sheets subject to stretch is commonly attributed to excess tension in the cells' cytoskeleton, in the plasma membrane, or in cell-cell contacts. Here, we demonstrate that for a variety of synthetic and physiological hydrogel substrates the formation of epithelial cracks is caused by tissue stretching independently of epithelial tension. We show that the origin of the cracks is hydraulic; they result from a transient pressure build-up in the substrate during stretch and compression manoeuvres. After pressure equilibration, cracks heal readily through actomyosin-dependent mechanisms. The observed phenomenology is captured by the theory of poroelasticity, which predicts the size and healing dynamics of epithelial cracks as a function of the stiffness, geometry and composition of the hydrogel substrate. Our findings demonstrate that epithelial integrity is determined in a tension-independent manner by the coupling between tissue stretching and matrix hydraulics.

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Epithelial fracture during stretch/unstretch maneuversa, Scheme of the stretching device (see Methods and Supplementary Fig. 1). b, Zoomed view of the region enclosed by a dashed rectangle in (a). c, LifeAct-GFP MDCK cluster before, during and after a 10 min pulse of 10% biaxial strain. The bottom row is a zoom of the region highlighted in the upper row. Arrowheads point at cracks after stretch cessation. The acquisition time of each snap shot is marked by a black dot on the time axis (top). d, Live fluorescence images of MDCK cells expressing LifeAct-Ruby (left) and a fluorescently-labeled plasma membrane green marker (right). Images were obtained 30 s after stretch cessation. Scale bar, 5μm. e, Live fluorescence images of MDCK cells expressing LifeAct-GFP (left) and E-cad-RFP (right). Images were obtained 30 s after stretch cessation. Scale bar, 5μm. f-g, Confocal x-y, x-z and y-z sections of cracks. Cells were fixed immediately after stretch cessation and stained for F-actin (phalloidin, red) and ZO-1 (green) (Supplementary methods). Sections show that ZO-1 remained intact at the apical surface (white arrows). In (f), a discontinuous actin layer was present at the basal surface of the cluster (blue arrowheads) and the largest crack diameter was located in the medial plane. In (g), no basal actin layer was present and the largest crack diameter was located in the basal plane. See Supplementary Fig. 6 for confocal sections of additional cracks (Scale bar, 5 μm). h, Crack area in epithelial clusters at high density (37±3 cells/pattern, mean±SEM, n=5) and low density (19±1 cells/pattern, mean±SEM, n=5). Representative images of the clusters before and after stretch are shown in Supplementary Fig. 9. i, Dependence of crack area with strain (n=6). In (h) and (i), crack area was expressed as a percentage of the total pattern area. Epithelial clusters are 80 μm in diameter.
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Figure 1: Epithelial fracture during stretch/unstretch maneuversa, Scheme of the stretching device (see Methods and Supplementary Fig. 1). b, Zoomed view of the region enclosed by a dashed rectangle in (a). c, LifeAct-GFP MDCK cluster before, during and after a 10 min pulse of 10% biaxial strain. The bottom row is a zoom of the region highlighted in the upper row. Arrowheads point at cracks after stretch cessation. The acquisition time of each snap shot is marked by a black dot on the time axis (top). d, Live fluorescence images of MDCK cells expressing LifeAct-Ruby (left) and a fluorescently-labeled plasma membrane green marker (right). Images were obtained 30 s after stretch cessation. Scale bar, 5μm. e, Live fluorescence images of MDCK cells expressing LifeAct-GFP (left) and E-cad-RFP (right). Images were obtained 30 s after stretch cessation. Scale bar, 5μm. f-g, Confocal x-y, x-z and y-z sections of cracks. Cells were fixed immediately after stretch cessation and stained for F-actin (phalloidin, red) and ZO-1 (green) (Supplementary methods). Sections show that ZO-1 remained intact at the apical surface (white arrows). In (f), a discontinuous actin layer was present at the basal surface of the cluster (blue arrowheads) and the largest crack diameter was located in the medial plane. In (g), no basal actin layer was present and the largest crack diameter was located in the basal plane. See Supplementary Fig. 6 for confocal sections of additional cracks (Scale bar, 5 μm). h, Crack area in epithelial clusters at high density (37±3 cells/pattern, mean±SEM, n=5) and low density (19±1 cells/pattern, mean±SEM, n=5). Representative images of the clusters before and after stretch are shown in Supplementary Fig. 9. i, Dependence of crack area with strain (n=6). In (h) and (i), crack area was expressed as a percentage of the total pattern area. Epithelial clusters are 80 μm in diameter.

Mentions: The principle of the technique is as follows. A thin layer of soft hydrogel is polymerized and chemically attached on a stretchable polydimethylsiloxane (PDMS) membrane (Fig. 1a,b). The resulting double-layered substrate is mounted on a custom-made stretching device compatible with inverted and upright optical microscopy (Fig. 1a). The substrate is stretched over a lubricated O-ring by applying negative pressure underneath its outer annular area (Supplementary Fig. 1a). The device produces homogeneous and equibiaxial strain with user-controlled amplitude and time-course (Supplementary Fig. 1b,c).


Hydraulic fracture during epithelial stretching.

Casares L, Vincent R, Zalvidea D, Campillo N, Navajas D, Arroyo M, Trepat X - Nat Mater (2015)

Epithelial fracture during stretch/unstretch maneuversa, Scheme of the stretching device (see Methods and Supplementary Fig. 1). b, Zoomed view of the region enclosed by a dashed rectangle in (a). c, LifeAct-GFP MDCK cluster before, during and after a 10 min pulse of 10% biaxial strain. The bottom row is a zoom of the region highlighted in the upper row. Arrowheads point at cracks after stretch cessation. The acquisition time of each snap shot is marked by a black dot on the time axis (top). d, Live fluorescence images of MDCK cells expressing LifeAct-Ruby (left) and a fluorescently-labeled plasma membrane green marker (right). Images were obtained 30 s after stretch cessation. Scale bar, 5μm. e, Live fluorescence images of MDCK cells expressing LifeAct-GFP (left) and E-cad-RFP (right). Images were obtained 30 s after stretch cessation. Scale bar, 5μm. f-g, Confocal x-y, x-z and y-z sections of cracks. Cells were fixed immediately after stretch cessation and stained for F-actin (phalloidin, red) and ZO-1 (green) (Supplementary methods). Sections show that ZO-1 remained intact at the apical surface (white arrows). In (f), a discontinuous actin layer was present at the basal surface of the cluster (blue arrowheads) and the largest crack diameter was located in the medial plane. In (g), no basal actin layer was present and the largest crack diameter was located in the basal plane. See Supplementary Fig. 6 for confocal sections of additional cracks (Scale bar, 5 μm). h, Crack area in epithelial clusters at high density (37±3 cells/pattern, mean±SEM, n=5) and low density (19±1 cells/pattern, mean±SEM, n=5). Representative images of the clusters before and after stretch are shown in Supplementary Fig. 9. i, Dependence of crack area with strain (n=6). In (h) and (i), crack area was expressed as a percentage of the total pattern area. Epithelial clusters are 80 μm in diameter.
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Figure 1: Epithelial fracture during stretch/unstretch maneuversa, Scheme of the stretching device (see Methods and Supplementary Fig. 1). b, Zoomed view of the region enclosed by a dashed rectangle in (a). c, LifeAct-GFP MDCK cluster before, during and after a 10 min pulse of 10% biaxial strain. The bottom row is a zoom of the region highlighted in the upper row. Arrowheads point at cracks after stretch cessation. The acquisition time of each snap shot is marked by a black dot on the time axis (top). d, Live fluorescence images of MDCK cells expressing LifeAct-Ruby (left) and a fluorescently-labeled plasma membrane green marker (right). Images were obtained 30 s after stretch cessation. Scale bar, 5μm. e, Live fluorescence images of MDCK cells expressing LifeAct-GFP (left) and E-cad-RFP (right). Images were obtained 30 s after stretch cessation. Scale bar, 5μm. f-g, Confocal x-y, x-z and y-z sections of cracks. Cells were fixed immediately after stretch cessation and stained for F-actin (phalloidin, red) and ZO-1 (green) (Supplementary methods). Sections show that ZO-1 remained intact at the apical surface (white arrows). In (f), a discontinuous actin layer was present at the basal surface of the cluster (blue arrowheads) and the largest crack diameter was located in the medial plane. In (g), no basal actin layer was present and the largest crack diameter was located in the basal plane. See Supplementary Fig. 6 for confocal sections of additional cracks (Scale bar, 5 μm). h, Crack area in epithelial clusters at high density (37±3 cells/pattern, mean±SEM, n=5) and low density (19±1 cells/pattern, mean±SEM, n=5). Representative images of the clusters before and after stretch are shown in Supplementary Fig. 9. i, Dependence of crack area with strain (n=6). In (h) and (i), crack area was expressed as a percentage of the total pattern area. Epithelial clusters are 80 μm in diameter.
Mentions: The principle of the technique is as follows. A thin layer of soft hydrogel is polymerized and chemically attached on a stretchable polydimethylsiloxane (PDMS) membrane (Fig. 1a,b). The resulting double-layered substrate is mounted on a custom-made stretching device compatible with inverted and upright optical microscopy (Fig. 1a). The substrate is stretched over a lubricated O-ring by applying negative pressure underneath its outer annular area (Supplementary Fig. 1a). The device produces homogeneous and equibiaxial strain with user-controlled amplitude and time-course (Supplementary Fig. 1b,c).

Bottom Line: Here, we demonstrate that for a variety of synthetic and physiological hydrogel substrates the formation of epithelial cracks is caused by tissue stretching independently of epithelial tension.We show that the origin of the cracks is hydraulic; they result from a transient pressure build-up in the substrate during stretch and compression manoeuvres.Our findings demonstrate that epithelial integrity is determined in a tension-independent manner by the coupling between tissue stretching and matrix hydraulics.

View Article: PubMed Central - PubMed

Affiliation: Institute for Bioengineering of Catalonia, 08028 Barcelona, Spain.

ABSTRACT
The origin of fracture in epithelial cell sheets subject to stretch is commonly attributed to excess tension in the cells' cytoskeleton, in the plasma membrane, or in cell-cell contacts. Here, we demonstrate that for a variety of synthetic and physiological hydrogel substrates the formation of epithelial cracks is caused by tissue stretching independently of epithelial tension. We show that the origin of the cracks is hydraulic; they result from a transient pressure build-up in the substrate during stretch and compression manoeuvres. After pressure equilibration, cracks heal readily through actomyosin-dependent mechanisms. The observed phenomenology is captured by the theory of poroelasticity, which predicts the size and healing dynamics of epithelial cracks as a function of the stiffness, geometry and composition of the hydrogel substrate. Our findings demonstrate that epithelial integrity is determined in a tension-independent manner by the coupling between tissue stretching and matrix hydraulics.

Show MeSH
Related in: MedlinePlus