E-cadherin integrates mechanotransduction and EGFR signaling to control junctional tissue polarization and tight junction positioning

Classified as probably not a substantial claim.

Traction scales directly with keratinocyte spread area.

“The magnitudes of these two tensions, T21 and T12, were divided by the spread area, K2 or K1, of cell 2 or cell 1, respectively, because traction scales directly with keratinocyte spread area64.”

• cites [64] Mertz, A. F. et al. Scaling of traction forces with the size of cohesive cell colonies. Phys. Rev. Lett. 108, 1–5 (2012). sourceDisputed

  The paper defines colony radius as the radius of a disk with the same area, then reports that total traction force approaches linear scaling with radius for large keratinocyte colonies (Fig. 3 discussion and model; page unavailable). That is scaling with the square root of area, not directly with spread area. The paper does not establish the claimed area-proportional relationship for individual keratinocytes. (LLM, conf=0.87)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured

May combine two claims.

A functional SC barrier is first formed in dorsal epidermis at embryonic day (E)16.5, spreading only to the ventral side one day later.

“A functional SC barrier is first formed in dorsal epidermis at embryonic day (E)16.5, spreading only to the ventral side one day later10, 20.”

• cites [10] Tunggal, J. A. et al. E-cadherin is essential for in vivo epidermal barrier function by regulating tight junctions. EMBO J. 24, 1146–1156 (2005). sourcePartly supported

  The section “The outside-in barrier is functional in K14-Cre/Ecad Fl/− mice” (page not supplied) reports dye penetration on the ventral side at E16.5, consistent with a barrier forming first elsewhere. It reports impermeability at E18.5 but does not establish that the barrier reached the ventral side exactly one day after E16.5. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [20] Hardman, M. J., Sisi, P., Banbury, D. N. & Byrne, C. Patterned acquisition of skin barrier function during development. Development 1552, 1541–1552 (1998). sourceNot checked — no full text of the source

May combine two claims.

In the epidermis, loss of E-cadherin expressed in all viable layers interferes with the TJ barrier, resulting in perinatal death.

“In the epidermis loss of E-cadherin expressed in all viable layers interferes with the TJ barrier, resulting in perinatal death10.”

• cites [10] Tunggal, J. A. et al. E-cadherin is essential for in vivo epidermal barrier function by regulating tight junctions. EMBO J. 24, 1146–1156 (2005). sourceSupported

May combine two claims.

When primary keratinocytes are exposed to high Ca2+ for 24-72 h, cells stratify and TJs become localized to the suprabasal layer of a multi-layered epithelial sheet.

“When primary keratinocytes are exposed to high Ca2+ for 24–72 h, cells stratify and TJs become localized to the suprabasal layer of a multi-layered epithelial sheet18.”

• cites [18] Vaezi, A., Bauer, C., Vasioukhin, V. & Fuchs, E. Actin cable dynamics and Rho/Rock orchestrate a polarized cytoskeletal architercture in the early steps of assembling a stratified epithelium. Dev. Cell. 3, 367–381 (2002). sourcePartly supported

  The Experimental Procedures describe raising calcium in primary keratinocyte cultures, and the Results report that some colonies had stratified by 24 hours. The Results identify desmosomes and adherens junctions in those cultures, not tight junctions localized to a suprabasal layer. The Discussion says tight junctions form later, but our copy does not establish their suprabasal localization after 24–72 hours. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

Classified as probably not a substantial claim. May combine two claims.

Increased tension across junctions results in the unfolding of α-catenin, exposing a binding site for vinculin, which then reinforces interactions with the actomyosin cytoskeleton.

“On a molecular level, increased tension across junctions results in the unfolding of α-catenin, exposing a binding site for vinculin, which then reinforces interactions with the actomyosin cytoskeleton7, 8.”

• cites [7] Yonemura, S. A mechanism of mechanotransduction at the cell-cell interface: Emergence of α-catenin as the center of a force-balancing mechanism for morphogenesis in multicellular organisms. BioEssays 33, 732–736 (2011). sourceNot checked — no full text of the source
• cites [8] Leckband, D. E. & de Rooij, J. Cadherin adhesion and mechanotransduction. Annu. Rev. Cell Dev. Biol. 30, 291–315 (2014). sourcePartly supported

  The sections “α-Catenin Is a Central Molecule in Cadherin Mechanotransduction” and “Vinculin Is a Key Effector of α-Catenin Mechanosensing” describe tension-dependent α-catenin opening, exposure of a vinculin-binding site, and vinculin recruitment to stressed junctions. The Figure 4 discussion says vinculin stabilizes junctions but explicitly states that how it does so is unclear; reinforcement through its interaction with F-actin is presented as a possibility, not an established mechanism. Page numbers are unavailable in our copy. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

Many of the molecular players required for barrier formation are shared between simple and multi-layered epithelia.

“even though many of the molecular players required for barrier formation are shared between simple and multi-layered epithelia2”

• cites [2] Tellkamp, F., Vorhagen, S. & Niessen, C. M. Epidermal polarity genes in health and disease. Cold Spring Harb. Perspect. Med. 4, a015255 (2014). sourceSupported

May combine two claims.

Control keratinocytes form regularly spaced cadherin-catenin-complex-positive early intercellular junctions (zippers) that recruit vinculin, F-actin and ZO-1.

“Control cells formed regularly spaced cadherin-catenin-complex-positive early intercellular junctions, also known as zippers21, which recruit both vinculin, F-actin and ZO-1 (Fig. 5a–c; Supplementary Fig. 5a).”

• cites [21] Vasioukhin, V., Bauer, C., Yin, M. & Fuchs, E. Directed actin polymerization is the driving force for epithelial cell-cell adhesion. Cell 100, 209–219 (2000). sourceSupported

May combine two claims.

In vivo, loss of epidermal vinculin does not result in perinatal lethality, unlike loss of E-cadherin.

“In vivo, loss of epidermal vinculin did not result in perinatal lethality unlike loss of E-cadherin10.”

• cites [10] Tunggal, J. A. et al. E-cadherin is essential for in vivo epidermal barrier function by regulating tight junctions. EMBO J. 24, 1146–1156 (2005). sourceSupported

Classified as probably not a substantial claim.

Classical cadherins such as E-cadherin and P-cadherin are key adhesive components of AJs that couple intercellular adhesion to the cytoskeleton via β-catenin and α-catenin.

“Classical cadherins such as E-cadherin and P-cadherin are key adhesive components of AJs that couple intercellular adhesion to the cytoskeleton via β-catenin and α-catenin5.”

• cites [5] Niessen, C. M., Leckband, D. & Yap, A. S. Tissue organization by cadherin adhesion molecules: dynamic molecular and cellular mechanisms of morphogenetic regulation. Physiol. Rev. 91, 691–731 (2011). sourceSupported

Classified as probably not a substantial claim.

Studies in lower organisms and mammalian cell cultures have identified a central role for E-cadherin in coordinating the establishment of apico-basolateral polarity with the formation of AJs, desmosomes, and TJs.

“Studies in lower organisms and mammalian cell cultures have identified a central role for E-cadherin in coordinating the establishment of apico–basolateral polarity with the formation of AJs, desmosomes, and TJs1, 9.”

• cites [1] Nelson, W. J. Adaptation of core mechanisms to generate cell polarity. Nature 422, 766–74 (2003). sourceNot checked — no full text of the source
• cites [9] Watabe, M., Nagafuchi, A., Tsukita, S. & Takeichi, M. Induction of polarized cell-cell association and retardation of growth by activation of the E-cadherin-catenin adhesion system in a dispersed carcinoma line. J. Cell Biol. 127, 247–256 (1994). sourceSupported

Classified as probably not a substantial claim.

EGFR activity status has been linked to intercellular junctional and actomyosin organization and function.

“As EGFR activity status has been linked to intercellular junctional and actomyosin organization and function26–29”

• cites [26] Itallie, C. M., Van Balda, M. S. & Anderson, J. M. Epidermal growth factor induces tyrosine phosphorylation and reorganization of the tight junction protein ZO-1 in A431 cells. J. Cell Sci. 1742, 1735–1742 (1995). sourceNot checked — no full text of the source
• cites [27] Duan, L. et al. Negative regulation of EGFR-Vav2 signaling axis by Cbl ubiquitin ligase controls EGF receptor-mediated epithelial cell adherens junction dynamics and cell migration. J. Biol. Chem. 286, 620–633 (2011). sourcePartly supported

  The Results report that EGFR signaling reorganized junctional actin and disrupted adherens junctions in human mammary epithelial cells. The cited passages do not establish an effect on myosin or actomyosin function, so that part of the claim goes beyond what this paper shows. (LLM, conf=0.83)

  Experimental — accuracy not yet measured
• cites [28] Muhamed, I. et al. E-Cadherin-mediated force transduction signals regulate global cell mechanics. J. Cell Sci. 129, 1843–1854 (2016). sourceSupported
• cites [29] Chiasson-MacKenzie, C. et al. NF2/Merlin mediates contact-dependent inhibition of EGFR mobility and internalization via cortical actomyosin. J. Cell Biol. 211, 1–15 (2015). sourceSupported

May combine two claims.

Epidermal inactivation of EGFR in mice showed that skin rashes were a direct consequence of epidermal EGFR, which was attributed to chemokine-driven inflammation.

“Epidermal inactivation of EGFR in mice showed that these rashes were a direct consequence of epidermal EGFR, which was attributed to chemokine-driven inflammation51–53.”

• cites [51] Franzke, C.-W. et al. Epidermal ADAM17 maintains the skin barrier by regulating EGFR ligand-dependent terminal keratinocyte differentiation. J. Exp. Med. 209, 1105–1119 (2012). sourcePartly supported

  The Results section reports that mice with epidermal EGFR inactivation developed inflammatory skin changes, with elevated Ccl2 and Cxcl1 and increased inflammatory-cell infiltrates (page not provided). But the Discussion attributes the inflammation primarily to an earlier skin-barrier defect; it does not establish that chemokines drive the rash. (LLM, conf=0.87)

  Experimental — accuracy not yet measured
• cites [52] Mascia, F. et al. Genetic ablation of epidermal EGFR reveals the dynamic origin of adverse effects of anti-EGFR therapy. Sci. Transl. Med. 5, 199ra110 (2013). sourceSupported
• cites [53] Lichtenberger, B. M. et al. Epidermal EGFR controls cutaneous host defense and prevents inflammation. Sci. Transl. Med. 5, 199ra111 (2013). sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

Myosin-II-mediated junctional tension in the epidermis is required for TJ barrier function.

“The observation that myosin-II-mediated junctional tension in the epidermis was shown to be required for TJ barrier function44”

• cites [44] Sumigray, K. D., Foote, H. P. & Lechler, T. Noncentrosomal microtubules and type II myosins potentiate epidermal cell adhesion and barrier formation. J. Cell. Biol. 199, 513–525 (2012). sourcePartly supported

  The Results section, “Loss of myosin IIA/B in the epidermis leads to tight junction defects” (page unavailable), reports a profound tight-junction functional defect in myosin IIA/B–null epidermis. The Discussion links myosin-dependent junctional strengthening to barrier function, but says the data *suggest* that adherens-junction tension controls tight-junction activity; it does not establish that junctional tension itself is required. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

E-cadherin itself was shown to interact with and regulate EGFR in vitro.

“Along the same lines, E-cadherin itself was shown to interact with and regulate EGFR in vitro46–49.”

• cites [46] Curto, M., Cole, B. K., Lallemand, D., Liu, C.-H. & McClatchey, A. I. Contact-dependent inhibition of EGFR signaling by Nf2/Merlin. J. Cell Biol. 177, 893–903 (2007). sourcePartly supported

  The Results (page unavailable) show that E-cadherin associates with EGFR in cultured cells and that disrupting cadherin-based adhesion increases EGFR signaling. But the reported association depends on NHE-RF1, and the paper attributes contact-dependent EGFR inhibition to Merlin; it does not show that E-cadherin itself directly interacts with or independently regulates EGFR. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [47] Perrais, M., Chen, X., Perez-Moreno, M. & Gumbiner, B. M. E-cadherin homophilic ligation inhibits cell growth and epidermal growth factor receptor signaling independently of other cell interactions. Mol. Biol. Cell 18, 2013–25 (2007). sourceNot checked — no full text of the source
• cites [48] Qian, X., Karpova, T., Sheppard, A. M., McNally, J. & Lowy, D. R. E-cadherin-mediated adhesion inhibits ligand-dependent activation of diverse receptor tyrosine kinases. EMBO J. 23, 1739–1748 (2004). sourceSupported
• cites [49] Erasmus, J. C., Welsh, N. J. & Braga, V. M. M. Cooperation of distinct Rac-dependent pathways to stabilise E-cadherin adhesion. Cell Signal. 27, 1905–1913 (2015). sourcePartly supported

  The Results section on EGFR and junction-dependent Rac activation reports that EGFR co-precipitates with E-cadherin in keratinocytes, supporting an association in vitro. The Introduction and Discussion describe EGFR-dependent signalling following E-cadherin contact formation, but the cited passages do not show that E-cadherin regulates EGFR itself. (LLM, conf=0.83)

  Experimental — accuracy not yet measured

May combine two claims.

Loss of vinculin in the epidermis does not affect either apical ZO-1 positioning or F-actin distribution in SG2 and does not result in major barrier defects in vivo, as vinculinepi−/− mice are viable, unlike Ecadepi−/− mice generated using the same K14-Cre line.

“In contrast, loss of vinculin in the epidermis did not affect either apical ZO-1 positioning or F-actin distribution in SG2 and did not result in major barrier defects in vivo, as vinculinepi−/− mice are viable, unlike Ecadepi−/− mice generated using the same K14-Cre line10.”

• cites [10] Tunggal, J. A. et al. E-cadherin is essential for in vivo epidermal barrier function by regulating tight junctions. EMBO J. 24, 1146–1156 (2005). sourcePartly supported

  Split into 2 atoms — 0 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 1 unverifiable. Expand the claim to see per-atom verdicts.

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

Epidermal keratinocytes require continuous upward extrusion in order to turn over and renew the tissue.

“In contrast, epidermal keratinocytes require continuous upward extrusion in order to turn over and renew the tissue37.”

• cites [37] Fuchs, E. & Horsley, V. More than one way to skin. Genes Dev. 22, 976–985 (2008). sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

Earlier in vitro work indicated vinculin as a crucial effector of cadherin-mediated mechanotransduction.

“Earlier in vitro work indicated vinculin as a crucial effector of cadherin-mediated mechanotransduction38–42.”

• cites [38] le Duc, Q. et al. 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, 1107–15 (2010). sourceSupported
• cites [39] Huveneers, S. et al. Vinculin associates with endothelial VE-cadherin junctions to control force-dependent remodeling. J. Cell Biol. 196, 641–652 (2012). sourceSupported
• cites [40] Alenghat, F. J., Fabry, B., Tsai, K. Y., Goldmann, W. H. & Ingber, D. E. Analysis of cell mechanics in single vinculin-deficient cells using a magnetic tweezer. Biochem. Biophys. Res. Commun. 277, 93–99 (2000). sourceNot checked — no full text of the source
• cites [41] Galbraith, C. G., Yamada, K. M. & Sheetz, M. P. The relationship between force and focal complex development. J. Cell Biol. 159, 695–705 (2002). sourcePartly supported

  The Discussion (page unavailable) suggests vinculin has an essential role in strengthening force-dependent adhesions, but specifies connections between integrin receptors and the cytoskeleton. It does not establish vinculin as a crucial effector of cadherin-mediated mechanotransduction. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [42] Barry, A. K. et al. α-Catenin cytomechanics: role in cadherin-dependent adhesion and mechanotransduction. J. Cell Sci. 127, 1779–1791 (2014). sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

Actomyosin-dependent forces also constrain EGFR activity in simple epithelia.

“In line with our data, actomyosin-dependent forces also constrain EGFR activity in simple epithelia29.”

• cites [29] Chiasson-MacKenzie, C. et al. NF2/Merlin mediates contact-dependent inhibition of EGFR mobility and internalization via cortical actomyosin. J. Cell Biol. 211, 1–15 (2015). sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

VE-cadherin and VEGFR2/3 can form a mechanosensitive complex in endothelial cells that controls VEGFR signaling in a shear-force-dependent manner.

“VE-cadherin and VEGFR2/3 can form a mechanosensitive complex in endothelial cells that controls VEGFR signaling in a shear-force-dependent manner45.”

• cites [45] Coon, B. G. et al. Intramembrane binding of VE-cadherin to VEGFR2 and VEGFR3 assembles the endothelial mechanosensory complex. J. Cell Biol. 208, 975–986 (2015). sourceSupported

May combine two claims.

E-cadherin controls the polarized localization and activity status of suprabasal EGFR, which is uncoupled from growth.

“In conclusion, E-cadherin controls the polarized localization and activity status of suprabasal EGFR, which is uncoupled from growth10.”

• cites [10] Tunggal, J. A. et al. E-cadherin is essential for in vivo epidermal barrier function by regulating tight junctions. EMBO J. 24, 1146–1156 (2005). sourceCould not verify

  The ‘Increased permeability of tight junctions’ section reports E-cadherin-dependent localization of activated aPKC and Rac in suprabasal epidermis, not EGFR. A search of our copy for EGFR and epidermal growth factor receptor found no passage addressing EGFR localization, activity, or whether its activity is uncoupled from growth. No page numbers were available for these passages. (LLM, conf=0.76)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

In simple epithelia, vinculin was shown to control apical positioning of the ZA.

“In simple epithelia, vinculin was shown to control apical positioning of the ZA43.”

• cites [43] Watabe-Uchida, M. et al. α-Catenin-vinculin interaction functions to organize the apical junctional complex in epithelial cells. J. Cell. Biol. 142, 847–857 (1998). sourcePartly supported

  The Results section, “Apical Junctional Organization in Vinculin-deficient Cells” (page unavailable), shows that loss of vinculin disrupted apical ZO-1 networks in F9 cells and that restoring vinculin rescued them. The Discussion proposes a role for vinculin in extending the ZA along apicolateral surfaces, but does not show that vinculin controls the ZA’s apical position in simple epithelia; it also cautions that F9 cells may not represent mature epithelial cells. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

May combine two claims.

Either too much or too little EGFR activity at TJs will impair barrier function and alter cytokine expression, both of which contribute to skin inflammatory skin diseases as well as skin cancer.

“Either too much or too little EGFR activity at TJs will impair barrier function and alter cytokine expression, both of which contribute to skin inflammatory skin diseases3 as well as skin cancer54.”

• cites [3] Kubo, A., Nagao, K. & Amagai, M. Review series Epidermal barrier dysfunction and cutaneous sensitization in atopic diseases. J. Clin. Invest. 122, 440–447 (2012). sourcePartly supported

  The discussion of TJ barriers (p. 5) links impaired tight-junction function to epidermal barrier defects and possible atopic dermatitis, and Figure 2 (p. 4) describes cytokine production after barrier damage. Neither passage supports the specific claim that too much or too little EGFR activity at TJs causes those effects. Skin cancer is attached to a different citation in the citing sentence and is not judged here. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [54] Cipolat, S., Hoste, E., Natsuga, K., Quist, S. R. & Watt, F. M. Epidermal barrier defects link atopic dermatitis with altered skin cancer susceptibility. Elife 3, e01888 (2014). sourcePartly supported

  The Results and Discussion report that mice lacking three epidermal barrier proteins had altered cytokine production and were protected against chemically induced skin tumours. That supports a link between barrier defects, immune signalling and skin cancer, but the paper does not establish that either excessive or insufficient EGFR activity at tight junctions causes those changes. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

May combine two claims.

In vitro, EGFR localizes to early AJs with a regular zipper-like distribution, which is strongly reduced in E-cadherin−/− keratinocytes.

“In vitro, EGFR localized to early AJs showing a regular zipper-like distribution, which was strongly reduced in E-cadherin−/− keratinocytes (Supplementary Fig. 7d).”

May combine two claims.

Phalloidin staining of whole mounts and tissue sections reveals a highly organized cortical F-actin network in the SG2 layer, whereas cortical staining is much less pronounced in the spinous layer.

“Phalloidin staining of whole mounts and tissue sections revealed a highly organized cortical F-actin network in the SG2 layer, whereas cortical staining was much less pronounced in the spinous layer (Fig. 2a–c).”

May combine two claims.

Junctional actin in the granular layer and actin polarization along the basal-to-apical tissue axis are not obviously affected by loss of vinculin.

“More surprisingly, junctional actin in the granular layer or actin polarization along the basal-to-apical tissue axis were both not obviously affected by the loss of vinculin (Fig. 6b, c).”

May combine two claims.

Little junctional vinculin is observed in E15.5 epidermis or in E16.5 ventral epidermis prior to SC formation, despite uniform distribution of E-cadherin at all intercellular contacts in both E16.5 ventral and dorsal epidermis.

“Similarly, little junctional vinculin was observed in E15.5 epidermis or in E16.5 ventral epidermis, prior to SC formation, despite uniform distribution of E-cadherin at all intercellular contacts in both E16.5 ventral and dorsal epidermis (Fig. 4b).”

May combine two claims.

Primary vinculin−/− keratinocytes organize E-cadherin-ZO-1 positive junctions into regular zipper-like structures that recruit F-actin but not paxillin.

“Primary vinculin−/− keratinocytes were able to organize E-cadherin-ZO-1 positive junctions into regular zipper-like structures that recruited F-actin but not paxillin (Fig. 6d, f; Supplementary Fig. 6d–f)”

May combine two claims.

ZO-1 is found at intercellular membranes in all epidermal layers but shows enrichment at intercellular contacts of the SG2 layer.

“The TJ marker ZO-1 is found at intercellular membranes in all layers, but, as previously reported16, 17, ZO-1 showed an enrichment at intercellular contacts of the SG2 layer (Fig. 1e, f).”

• cites [16] Yoshida, K. et al. Functional tight junction barrier localizes in the second layer of the stratum granulosum of human epidermis. J. Dermatol. Sci. 71, 89–99 (2013). sourceNot checked — no full text of the source
• cites [17] Brandner, J. M. et al. Organization and formation of the tight junction system in human epidermis and cultured keratinocytes. Eur. J. Cell. Biol. 81, 253–263 (2002). sourceNot checked — no full text of the source

May combine two claims.

AFM measurements show a reduction in cortical stiffness upon loss of E-cadherin but not upon knockdown of the TJ protein ZO-1.

“Interestingly, AFM measurements showed a reduction in cortical stiffness upon loss of E-cadherin (Fig. 5g–i) but not upon knockdown of the TJ protein ZO-1 (Supplementary Fig. 5j).”

May combine two claims.

E-cadherin-containing AJs are present at sites of cell-cell contacts in all epidermal layers, with no apparent enrichment in the granular layer where TJs are formed.

“Staining for E-cadherin confirmed the presence of E-cadherin-containing AJs at sites of cell–cell contacts in all layers, with no apparent enrichment in the granular layer, where TJs are formed (Fig. 1b, e, f).”

May combine two claims.

In E14.5 and E15.5, ZO-1 is absent from intercellular epidermal contacts and only stains TJs of the periderm.

“Interestingly, in E14.5 and E15.5, ZO-1 was absent from intercellular epidermal contacts, and only stained TJs of the periderm, a simple provisional barrier-forming epithelium (Fig. 4a; Supplementary Fig. 4a).”

May combine two claims.

Epidermal whole mounts from vinculinepi−/− mice show no obvious defects in polarized ZO-1 tissue distribution or in its linear apical TJ localization within SG2.

“Epidermal whole mounts from vinculinepi−/− mice revealed no obvious defects in polarized ZO-1 tissue distribution or in its linear apical TJ localization within SG2 (Fig. 6a).”

May combine two claims.

A basal-to-apical polarization of F-actin becomes obvious only in E16.5 dorsal epidermis, with cortical F-actin enriched in the granular layer, in contrast to uniform F-actin in E14.5/15.5 embryos.

“Similarly, a basal-to-apical polarization of F-actin became only obvious in E16.5 dorsal epidermis, with cortical F-actin now being enriched in the granular layer (Fig. 4c) in contrast to uniform F-actin in E14.5/15.5 embryos.”

May combine two claims.

In E-cadherin-deficient epidermis, total α-catenin in suprabasal layers and the mechanosensitive α18 epitope in the SG2 layer are strongly reduced.

“Whole-mount analysis revealed a strong reduction in both total α-catenin in suprabasal layers and the mechanosensitive α18 epitope in the SG2 layer in E-cadherin-deficient epidermis (from here on Ecadepi−/−)(Fig. 3a).”

May combine two claims.

Junctional vinculin is lost in the granular layer of E-cadherin-deficient epidermis, with no apparent change in total protein level.

“Furthermore, junctional vinculin was lost in the granular layer (Fig. 3b; Supplementary Fig. 3a), albeit with no apparent change in total protein level (Supplementary Fig. 3b).”

May combine two claims.

Detection of F-actin using Lifeact-GFP transgenic mice reveals a similarly polarized distribution for F-actin, including occurrence of scattered F-actinhigh cells in SG1, excluding that reduced phalloidin staining in lower layers resulted from epitope masking or insufficient permeabilization.

“Importantly, detection of F-actin using Lifeact-GFP transgenic mice revealed a similarly polarized distribution for F-actin including occurrence of scattered F-actinhigh cells in SG1 (Supplementary Fig. 2a, asterisks), excluding that reduced phalloidin staining in lower layers resulted from epitope masking or insufficient permeabilization.”

May combine two claims.

Upon loss of E-cadherin, EGFR staining in SG2 becomes more punctate and more prominent in spinous layers, where it partially co-localizes with ZO-1.

“Upon loss of E-cadherin, EGFR staining in the SG2 layer became more punctate in addition to a more prominent staining in the spinous layers, where it partially co-localized with ZO-1 (Fig. 7c, d).”

May combine two claims.

Vinculin exhibits a polarized tissue distribution and is highly enriched (4-fold) at intercellular contact sites in the second granular layer (SG2).

“Surprisingly, vinculin, which is recruited to AJs upon mechanosensitive unfolding of α-catenin8, 13, exhibited a polarized tissue distribution and was highly enriched (4-fold) at intercellular contact sites in the second granular layer (SG2, Fig. 1a, b, f; Supplementary Fig. 1a).”

May combine two claims.

E-cadherin−/− keratinocytes show strong impairment in early zipper formation, with most vinculin localized to focal contacts at the cell-matrix interphase.

“E-cadherin−/− keratinocytes showed strong impairment in early zipper formation, with most of the vinculin localized to focal contacts at the cell–matrix interphase (Fig. 5a, c; Supplementary Fig. 5a, white arrows).”

May combine two claims.

Inhibition of EGFR or its downstream mediator PKCα rescues TJ barrier function, but only when inhibitors are applied after junctions are allowed to form for 24 h in high calcium.

“Furthermore, inhibition of EGFR or its downstream mediator PKCα rescued TJ barrier function (Fig. 8e; Supplementary Fig. 8a), but, interestingly, only when inhibitors were applied after junctions were allowed to form for 24 h in high calcium, a time point that coincides with stratification and initial barrier formation31.”

• cites [31] Kirschner, N. et al. Contribution of tight junction proteins to ion, macromolecule, and water barrier in keratinocytes. J. Invest. Dermatol. 133, 1161–9 (2013). sourceNot checked — no full text of the source

May combine two claims.

EGF stimulation substantially lowers TER in control keratinocytes but does not further reduce TER in E-cadherin−/− keratinocytes.

“EGF stimulation substantially lowered TER in control keratinocytes (Fig. 8a), but did not further reduce TER in E-cadherin−/− keratinocytes (Fig. 8a)”

May combine two claims.

EGFR recruitment to suprabasal junctions at E16.5 coincides with ZO-1 and vinculin recruitment to the most viable suprabasal layer and with initiation of barrier function.

“This coincided with ZO-1 and vinculin recruitment to the most viable suprabasal layer (Fig. 4a, b) and with initiation of barrier function20.”

• cites [20] Hardman, M. J., Sisi, P., Banbury, D. N. & Byrne, C. Patterned acquisition of skin barrier function during development. Development 1552, 1541–1552 (1998). sourceNot checked — no full text of the source

May combine two claims.

Cell surface biotinylation analysis reveals increased internalization of occludin and EGFR, but not claudin-1, which is reversed upon inhibiting EGFR activity.

“Quantitative cell surface biotinylation analysis revealed increased internalization of occludin and EGFR, but not of claudin-1, which was reversed upon inhibiting EGFR activity (Fig. 8d).”

May combine two claims.

In vitro multi-layered epithelial sheets do not form an SC but still restrict TJs to the most apical layer.

“As the in vitro multi-layered epithelial sheets do not form an SC but still restrict TJs to the most apical layer (Fig. 5j)”

May combine two claims.

Vinculin is dispensable for polarized tissue organization of junctions, cytoskeleton, and tension across epithelial layers and contributes only to a small extent to proper epidermal barrier formation.

“It is, however, dispensable for polarized tissue organization of junctions, the cytoskeleton, and tension across epithelial layers and contributes only to a small extent to proper epidermal barrier formation.”

May combine two claims.

E-cadherin controls basal-to-apical tissue organization of vinculin-positive AJs and F-actin across epidermal layers and regulates the positioning of functional TJs to the SG2 layer.

“Thus, E-cadherin controls basal-to-apical tissue organization of vinculin-positive AJs and F-actin across epidermal layers and regulates the positioning of functional TJs to the SG2 layer.”

May combine two claims.

Control keratinocytes efficiently develop a functional barrier over time, showing that E-cadherin-mediated barrier formation is intrinsic and does not require a SC.

“In contrast, control keratinocytes efficiently developed a functional barrier over time, showing that E-cadherin-mediated barrier formation is intrinsic and does not require a SC.”

May combine two claims.

Vinculin is important for initial junctional organization, suggesting it promotes mechanical reinforcement of newly formed junctions.

“Thus, vinculin is important for initial junctional organization, suggesting it promotes mechanical reinforcement of newly formed junctions.”

May combine two claims.

Other mechanically relevant AJ-associated proteins either control barrier formation or compensate for the in vivo loss of vinculin.

“Our results suggest that other mechanically relevant AJ-associated proteins either control barrier formation or compensate for the in vivo loss of vinculin.”

May combine two claims.

Polarized tissue distribution of vinculin-positive junctions and F-actin is also observed in human adult whole-mount epidermis, demonstrating that tissue asymmetry for junctions and cytoskeleton is a general phenomenon of stratifying epithelia.

“Polarized tissue distribution of vinculin-positive junctions and F-actin was also observed in human adult whole-mount epidermis (Fig. 2f; Supplementary Fig. 2b), demonstrating that tissue asymmetry for junctions and cytoskeleton is a general phenomenon of stratifying epithelia.”

May combine two claims.

Vinculin controls the length of individual primordial AJs and overall cortical organization of the F-actin cytoskeleton.

“Thus, vinculin controls the length of the individual primordial AJs as well as overall cortical organization of the F-actin cytoskeleton.”

Classified as probably not a substantial claim.

Apico-basolateral polarity promotes the asymmetric positioning of intercellular junctions and the cytoskeleton within the cell to drive functional barrier formation in simple epithelia.

“For example, apico–basolateral polarity promotes the asymmetric positioning of intercellular junctions and the cytoskeleton within the cell to drive functional barrier formation in simple epithelia1”

• cites [1] Nelson, W. J. Adaptation of core mechanisms to generate cell polarity. Nature 422, 766–74 (2003). sourceNot checked — no full text of the source

May combine two claims.

E-cadherin directs the polarized tissue distribution of junction tension, F-actin, and TJs at E16.5, which coincides with initiation of functional SC barrier activity.

“Thus, E-cadherin directs the polarized tissue distribution of junction tension, F-actin, and TJs at E16.5, which coincides with initiation of functional SC barrier activity.”

Classified as probably not a substantial claim. May combine two claims.

Classical cadherins not only withstand tension but also sense mechanical forces and transduce this tension across cells in an actomyosin-dependent manner to expand junctional size and complexity.

“Recently, it has become clear that classical cadherins not only withstand tension but also sense mechanical forces and transduce this tension across cells in an actomyosin-dependent manner to expand junctional size and complexity6.”

• cites [6] Hoffman, B. D. & Yap, A. S. Towards a dynamic understanding of cadherin-based mechanobiology. Trends Cell. Biol. 25, 803–814 (2015). sourceNot checked — no full text of the source

May combine two claims.

Inhibiting myosin activity interferes with barrier formation in control keratinocytes, showing that actomyosin tension directly controls the TJ barrier.

“Importantly, inhibiting myosin activity interfered with barrier formation in control keratinocytes (Fig. 5k), showing that actomyosin tension directly controls the TJ barrier.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The numbers of independent experiments and biological replicates performed for all experiments, P values and the statistical tests used are indicated in the figure legends.

“The numbers of independent experiments and biological replicates performed for all experiments, P values and the statistical tests that were used are indicated in the figure legends.”

May combine two claims.

Cadherin engagement, force transduction, and the observed tight, spatiotemporal regulation of EGFR activity may be fundamentally different in the two systems.

“Cadherin engagement, force transduction, and the observed tight, spatiotemporal regulation of EGFR activity may thus be fundamentally different in the two systems.”

May combine two claims.

The data show that actomyosin cytoskeleton controls cortical stiffness, EGFR activation and TJ formation, whereas tight regulation of EGFR itself determines cortical stiffness as well as TJ stability and function.

“Our data furthermore show that actomyosin cytoskeleton controls cortical stiffness, EGFR activation and TJ formation, whereas tight regulation of EGFR itself determines cortical stiffness as well as TJ stability and function.”

May combine two claims.

In vitro data provide indirect evidence that E-cadherin regulates EGFR in a tension-dependent manner that appears independent of stable association with the actin cytoskeleton.

“Our in vitro data provide indirect evidence that E-cadherin regulates EGFR in a tension-dependent manner that, however, appears independent of stable association with the actin cytoskeleton (Fig. 7g and Supplementary Fig. 7h, i).”

May combine two claims.

Junctional tension generated by E-cadherin is required to localize and constrain EGFR activity, which in turn is required for cortical reinforcement and subsequent formation of TJs.

“Collectively, these data suggest that junctional tension generated by E-cadherin is required to localize and constrain EGFR activity, which in turn is required for cortical reinforcement and subsequent formation of TJs.”

May combine two claims.

The results identify a key role for E-cadherin in regulation of actomyosin-dependent tension, junctional and cytoskeletal tissue polarization, EGFR localization and activation, and TJ positioning and barrier formation.

“Our results identify a key role for E-cadherin in: (1) regulation of actomyosin-dependent tension, (2) junctional and cytoskeletal tissue polarization (3) EGFR localization and activation, and (4) TJ positioning and barrier formation.”

May combine two claims.

In lower layers, all intercellular interfaces contain E-cadherin-AJs that are under low tension and permit moderate EGFR activity, thus further propagating low cortical tension and unstable TJs by promoting internalization.

“In lower layers, all intercellular interfaces contain E-cadherin-AJs that are under low tension and permit moderate EGFR activity, thus further propagating low cortical tension and unstable TJs by promoting internalization.”

May combine two claims.

Mislocalization of EGFR is the earliest change induced by loss of E-cadherin and precedes misguided localization of ZO-1 and actin in lower layers at E16.5.

“Thus, mislocalization of EGFR is the earliest change induced by loss of E-cadherin and precedes misguided localization of ZO-1 and actin in lower layers at E16.5 (Fig. 4d, e).”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

Images to be used for deconvolution were obtained at optimal resolution according to Nyquist.

“Images to be used for deconvolution were obtained at optimal resolution according to Nyquist.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

In all AFM indentation experiments, forces of up to 3 nN were applied and the velocities of cantilever approach and retraction were kept constant at 2 μm s−1, ensuring detection of elastic properties only.

“For all indentation experiments, forces of up to 3 nN were applied, and the velocities of cantilever approach and retraction were kept constant at 2 μm s−1 ensuring detection of elastic properties only.”

May combine two claims.

Together, in vitro and in vivo data suggest a two-step model in which E-cadherin actively inhibits premature TJ formation in lower epidermal suprabasal layers while promoting TJ formation in the SG2 layer.

“Together, the in vitro and in vivo data suggest a two-step model in which E-cadherin actively inhibits premature TJ formation in lower epidermal suprabasal layers while promoting TJ formation in the SG2 layer (Fig. 8g).”

May combine two claims.

SG2 junctions show structural resemblance to tensile lateral networks in cultured simple epithelia but function more like the ZA in providing a continuous tensile network that mechanically couples contractile forces across epidermal sheets to promote barrier-forming TJs.

“Although SG2 junctions show structural resemblance to the tensile lateral networks recently described in cultured simple epithelia36, they function more like the ZA in providing a continuous tensile network that mechanically couples contractile forces across epidermal sheets to promote barrier-forming TJs.”

• cites [36] Wu, S. K. et al. Cortical F-actin stabilization generates apical-lateral patterns of junctional contractility that integrate cells into epithelia. Nat. Cell. Biol. 16, 167–78 (2014). sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

To control for efficient stripping of surface-bound biotin after internalization, additional control samples were stripped immediately after biotin labeling without medium incubation.

“To control for efficient stripping of surface bound biotin after internalization, additional control samples were stripped immediately after biotin labeling without medium incubation.”

Classified as probably not a substantial claim.

To normalize for overall different fluorescence intensities between different experiments, the ratio of fluorescence intensities between layers of each experiment was compared.

“To normalize for overall different fluorescence intensities between different experiments, the ratio of fluorescence intensities between layers of each experiment were compared.”

May combine two claims.

Either too little or too much EGFR activity interferes with formation of a functional TJ barrier.

“These findings show that either too little or too much EGFR activity interferes with formation of a functional TJ barrier.”

May combine two claims.

E-cadherin controls the number and regular spacing of initial junctions assembling into zipper-like structures, whereas vinculin is only required for local F-actin reorganization at these early junctions but does not control zipper formation itself.

“In addition, whereas E-cadherin controls the number and regular spacing of initial junctions assembling into zipper-like structures, vinculin is only required for local F-actin reorganization at these early junctions but does not control zipper formation itself.”

Probably not a substantial claim: may not pass S3 (load-bearing).

EGFR might interact with TJ proteins at least after the barrier has formed, providing an alternative mechanism for recruitment.

“suggesting that EGFR might interact with TJ proteins at least after the barrier has formed, providing an alternative mechanism for recruitment.”

May combine two claims.

Cell-level apical-basolateral polarity is observed within the epidermal SG2 layer, with TJs apically positioned from AJs, resembling apical positioning of simple epithelial TJs above the ZA.

“Furthermore, cell-level apical–basolateral polarity is also observed within the epidermal SG2 layer, with the TJs being apically positioned from the AJs, thus resembling the apical positioning of simple epithelial TJs above the zonula adherens (ZA)”

May combine two claims.

Junctional tension generated by E-cadherin is required to localize and constrain EGFR activity, which in turn controls cortical stiffness and subsequent formation of TJs.

“Collectively, these data suggest that junctional tension generated by E-cadherin is required to localize and constrain EGFR activity, which in turn controls cortical stiffness and subsequent formation of TJs.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Cultured cells were regularly monitored for mycoplasma contamination and discarded when the result was positive.

“Cultured cells were regularly monitored for mycoplasma contamination and discarded in case of positive results.”

Classified as probably not a substantial claim.

Loss of epidermal EGFR is associated with skin barrier dysfunction.

“As loss of epidermal EGFR is associated with skin barrier dysfunction30”

• cites [30] Sibilia, M. & Wagner, E. F. Strain-dependent epithelial defects in mice lacking the EGF receptor. Science 269, 234–238 (1995). sourceNot checked — no full text of the source

May combine two claims.

This increased tension reduces EGFR activity, thus further increasing cortical stiffness while stabilizing TJs at the cell surface.

“This in turn reduces EGFR activity, thus further increasing cortical stiffness while at the same time stabilizing TJs at the cell surface.”

May combine two claims.

In the SG2 layer, AJs are absent from the apical intercellular interface, resulting in an anisotropy of actomyosin force distribution that may be sufficient to increase tension on E-cadherin junctions allowing for increased unfolding of α-catenin and recruitment of vinculin.

“In contrast in the SG2 layer, AJs are absent from the apical intercellular interface, resulting in an anisotropy of actomyosin force distribution that may be sufficient to increase tension on E-cadherin junctions allowing for increased unfolding of α-catenin and recruitment of vinculin.”