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Formation of adherens junctions leads to the emergence of a tissue-level tension in epithelial monolayers.

Harris AR, Daeden A, Charras GT - J. Cell. Sci. (2014)

Bottom Line: Adherens junctions and desmosomes integrate the cytoskeletons of adjacent cells into a mechanical syncitium.Though much is known about the biological mechanisms underlying junction formation, little is known about how tissue-scale mechanical properties are established.As a consequence, inhibition of any of the molecular mechanisms participating in adherens junction initiation, remodelling and maturation significantly impeded the emergence of tissue-level tension in monolayers.

View Article: PubMed Central - PubMed

Affiliation: London Centre for Nanotechnology, University College London, London WC1H 0AH, UK Department of Physics, University College London, London WC1E 6BT, UK Engineering Doctorate Program, Department of Chemistry, University College London, London WC1H 0AJ, UK.

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Inhibition of actin polymerisation and lamellipodial crawling impede the emergence of monolayer tension. (A) Localisation of LifeAct–Ruby in control monolayers and monolayers treated with latrunculin B 150 min after replating. (B) Temporal evolution of the apparent stiffness of control monolayers (black) and monolayers treated with latrunculin B (blue) to depolymerise the actin cytoskeleton. (C) Differential interference contrast images of cells during monolayer reformation (see supplementary material Movie 3). Cells formed lamellipodia to close existing gaps. Arrowhead, the position of a lamellipodium; yellow dashed line, initial position of the lamellipodium; white dashed line, lamellipodial position at subsequent time-points. (D) Upper image, differential interference contrast imaging of a lamellipodial protrusion during monolayer formation; lower image, phalloidin staining of the same location. Arrowhead, the lamellipodium. (E) Localisation of LifeAct–Ruby in control monolayers and monolayers treated with CK666 to inhibit Arp2/3-mediated lamellipodial protrusion. In A and E, the upper images show a single xy confocal plane and the lower images show a single zx profile. The location of zx profiles is shown by dashed yellow lines on the xy images. Scale bars: 10 µm. (F) Temporal evolution of the apparent stiffness in control monolayers (black) and monolayers treated with CK666 (green). In B and F, the number of measurements for each condition is indicated below each box; boxes, median, 1st quartile and 3rd quartile; whiskers, maximum and minimum; dotted line, temporal evolution of monolayer apparent stiffness for each condition; *P<0.01 between control monolayers and treated monolayers at a given time-point (Student's t-test).
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f05: Inhibition of actin polymerisation and lamellipodial crawling impede the emergence of monolayer tension. (A) Localisation of LifeAct–Ruby in control monolayers and monolayers treated with latrunculin B 150 min after replating. (B) Temporal evolution of the apparent stiffness of control monolayers (black) and monolayers treated with latrunculin B (blue) to depolymerise the actin cytoskeleton. (C) Differential interference contrast images of cells during monolayer reformation (see supplementary material Movie 3). Cells formed lamellipodia to close existing gaps. Arrowhead, the position of a lamellipodium; yellow dashed line, initial position of the lamellipodium; white dashed line, lamellipodial position at subsequent time-points. (D) Upper image, differential interference contrast imaging of a lamellipodial protrusion during monolayer formation; lower image, phalloidin staining of the same location. Arrowhead, the lamellipodium. (E) Localisation of LifeAct–Ruby in control monolayers and monolayers treated with CK666 to inhibit Arp2/3-mediated lamellipodial protrusion. In A and E, the upper images show a single xy confocal plane and the lower images show a single zx profile. The location of zx profiles is shown by dashed yellow lines on the xy images. Scale bars: 10 µm. (F) Temporal evolution of the apparent stiffness in control monolayers (black) and monolayers treated with CK666 (green). In B and F, the number of measurements for each condition is indicated below each box; boxes, median, 1st quartile and 3rd quartile; whiskers, maximum and minimum; dotted line, temporal evolution of monolayer apparent stiffness for each condition; *P<0.01 between control monolayers and treated monolayers at a given time-point (Student's t-test).

Mentions: Next, we examined the role of the biological mechanisms involved in adherens junction formation in the establishment of tissue-level tension. In the current consensus view of adherens junction formation (Harris and Tepass, 2010), lamellipodial crawling, powered by Arp2/3-complex-mediated F-actin polymerisation, allows cells to make initial contacts with their neighbours that can be subsequently broadened by further lamellipodial extension. Consistent with a central role for actin polymerisation, incubating the cells with latrunculin B to depolymerise actin filaments prevented the formation of intercellular junctions and the generation of traction stresses (Fig. 5A). Temporal increases in apparent stiffness following replating were severely diminished (Fig. 5B). Differential interference contrast imaging during monolayer formation (Fig. 5C; supplementary material Movie 3) and phalloidin staining of F-actin (Fig. 5D) confirmed the presence of lamellipodial crawling under our experimental conditions. To examine the role of Arp2/3 in the establishment of tissue tension, we treated reforming monolayers with the Arp2/3-complex inhibitor CK666 (Nolen et al., 2009), which has been shown to inhibit lamellipodial protrusion in neutrophils (Wilson et al., 2013) and aplysia growth cones (Yang et al., 2012). Treatment of mature monolayers with CK666 led to loss of junctional localisation of the Arp2/3 complex (supplementary material Fig. S4D), confirming the effectiveness of the inhibitor in MDCK cells, in agreement with previous work (Tang and Brieher, 2012). When we treated reforming monolayers with CK666, cells no longer formed lamellipodia and this resulted in poorly interconnected monolayers (Fig. 5E). Lack of junction formation and decreased traction stresses severely impeded the establishment of monolayer stiffness (Fig. 5F).


Formation of adherens junctions leads to the emergence of a tissue-level tension in epithelial monolayers.

Harris AR, Daeden A, Charras GT - J. Cell. Sci. (2014)

Inhibition of actin polymerisation and lamellipodial crawling impede the emergence of monolayer tension. (A) Localisation of LifeAct–Ruby in control monolayers and monolayers treated with latrunculin B 150 min after replating. (B) Temporal evolution of the apparent stiffness of control monolayers (black) and monolayers treated with latrunculin B (blue) to depolymerise the actin cytoskeleton. (C) Differential interference contrast images of cells during monolayer reformation (see supplementary material Movie 3). Cells formed lamellipodia to close existing gaps. Arrowhead, the position of a lamellipodium; yellow dashed line, initial position of the lamellipodium; white dashed line, lamellipodial position at subsequent time-points. (D) Upper image, differential interference contrast imaging of a lamellipodial protrusion during monolayer formation; lower image, phalloidin staining of the same location. Arrowhead, the lamellipodium. (E) Localisation of LifeAct–Ruby in control monolayers and monolayers treated with CK666 to inhibit Arp2/3-mediated lamellipodial protrusion. In A and E, the upper images show a single xy confocal plane and the lower images show a single zx profile. The location of zx profiles is shown by dashed yellow lines on the xy images. Scale bars: 10 µm. (F) Temporal evolution of the apparent stiffness in control monolayers (black) and monolayers treated with CK666 (green). In B and F, the number of measurements for each condition is indicated below each box; boxes, median, 1st quartile and 3rd quartile; whiskers, maximum and minimum; dotted line, temporal evolution of monolayer apparent stiffness for each condition; *P<0.01 between control monolayers and treated monolayers at a given time-point (Student's t-test).
© Copyright Policy - open-access
Related In: Results  -  Collection

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f05: Inhibition of actin polymerisation and lamellipodial crawling impede the emergence of monolayer tension. (A) Localisation of LifeAct–Ruby in control monolayers and monolayers treated with latrunculin B 150 min after replating. (B) Temporal evolution of the apparent stiffness of control monolayers (black) and monolayers treated with latrunculin B (blue) to depolymerise the actin cytoskeleton. (C) Differential interference contrast images of cells during monolayer reformation (see supplementary material Movie 3). Cells formed lamellipodia to close existing gaps. Arrowhead, the position of a lamellipodium; yellow dashed line, initial position of the lamellipodium; white dashed line, lamellipodial position at subsequent time-points. (D) Upper image, differential interference contrast imaging of a lamellipodial protrusion during monolayer formation; lower image, phalloidin staining of the same location. Arrowhead, the lamellipodium. (E) Localisation of LifeAct–Ruby in control monolayers and monolayers treated with CK666 to inhibit Arp2/3-mediated lamellipodial protrusion. In A and E, the upper images show a single xy confocal plane and the lower images show a single zx profile. The location of zx profiles is shown by dashed yellow lines on the xy images. Scale bars: 10 µm. (F) Temporal evolution of the apparent stiffness in control monolayers (black) and monolayers treated with CK666 (green). In B and F, the number of measurements for each condition is indicated below each box; boxes, median, 1st quartile and 3rd quartile; whiskers, maximum and minimum; dotted line, temporal evolution of monolayer apparent stiffness for each condition; *P<0.01 between control monolayers and treated monolayers at a given time-point (Student's t-test).
Mentions: Next, we examined the role of the biological mechanisms involved in adherens junction formation in the establishment of tissue-level tension. In the current consensus view of adherens junction formation (Harris and Tepass, 2010), lamellipodial crawling, powered by Arp2/3-complex-mediated F-actin polymerisation, allows cells to make initial contacts with their neighbours that can be subsequently broadened by further lamellipodial extension. Consistent with a central role for actin polymerisation, incubating the cells with latrunculin B to depolymerise actin filaments prevented the formation of intercellular junctions and the generation of traction stresses (Fig. 5A). Temporal increases in apparent stiffness following replating were severely diminished (Fig. 5B). Differential interference contrast imaging during monolayer formation (Fig. 5C; supplementary material Movie 3) and phalloidin staining of F-actin (Fig. 5D) confirmed the presence of lamellipodial crawling under our experimental conditions. To examine the role of Arp2/3 in the establishment of tissue tension, we treated reforming monolayers with the Arp2/3-complex inhibitor CK666 (Nolen et al., 2009), which has been shown to inhibit lamellipodial protrusion in neutrophils (Wilson et al., 2013) and aplysia growth cones (Yang et al., 2012). Treatment of mature monolayers with CK666 led to loss of junctional localisation of the Arp2/3 complex (supplementary material Fig. S4D), confirming the effectiveness of the inhibitor in MDCK cells, in agreement with previous work (Tang and Brieher, 2012). When we treated reforming monolayers with CK666, cells no longer formed lamellipodia and this resulted in poorly interconnected monolayers (Fig. 5E). Lack of junction formation and decreased traction stresses severely impeded the establishment of monolayer stiffness (Fig. 5F).

Bottom Line: Adherens junctions and desmosomes integrate the cytoskeletons of adjacent cells into a mechanical syncitium.Though much is known about the biological mechanisms underlying junction formation, little is known about how tissue-scale mechanical properties are established.As a consequence, inhibition of any of the molecular mechanisms participating in adherens junction initiation, remodelling and maturation significantly impeded the emergence of tissue-level tension in monolayers.

View Article: PubMed Central - PubMed

Affiliation: London Centre for Nanotechnology, University College London, London WC1H 0AH, UK Department of Physics, University College London, London WC1E 6BT, UK Engineering Doctorate Program, Department of Chemistry, University College London, London WC1H 0AJ, UK.

Show MeSH
Related in: MedlinePlus