Tissue polarization of junctions

To examine the spatial relationship between AJs and TJs on the tissue level and within individual cell layers (Fig. 1a), we overcame the limited resolution of traditional skin tissue sagittal sections by adapting an epidermal whole-mount technique11 to newborn mouse epidermis and performed high-resolution microscopy. As cadherin-mediated mechanotransduction through vinculin has been implicated in TJ barrier function of simple epithelia12, we first asked how indicators of cadherin-mediated mechanotransduction are organized across epidermal layers. 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). 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). Here it co-localized with E-cadherin (Fig. 1b). Staining with α18-antibody (AB) that detects a mechanosensitive epitope in α-catenin13, further indicated that these junctions were under high mechanical tension (Fig. 1c, d; Supplementary Fig. 1b).

We next examined the architecture of TJs that are exclusively formed in the SG2 layer10, 14, 15. 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). Closer examination revealed that SG2 cells formed an E-cadherin/vinculin-rich basolateral network of AJs, which at its most apical border partially overlapped with the apically localized ZO-1-positive TJ network. Thus, despite their flattened shape, SG2 cells still exhibit junctional cell polarity (Fig. 1g; Supplementary Movie 1). Together the data show that, despite homogenous E-cadherin distribution, AJs display differential molecular compositions along the basal-to-apical tissue axis. Within the SG2 vinculin-positive AJs form a basolateral network that support the apically localized TJs exclusively formed in this layer.

Differential F-actin organization across layers

As mechanical reinforcement of AJs and of functional TJs appears to be restricted to the SG2, we next addressed how these junctions relate to the epidermal organization of the F-actin cytoskeleton. Previous findings using K14-actin-GFP transgenic mice indicated a relatively evenly distributed cortical actin network18. However, actin-GFP marks both G-actin and F-actin and may also interfere with actin dynamics19. 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). Within the SG2 layer, F-actin associated with both the apical positioned linear TJ junctions and the basolateral vinculin-positive AJ networks (Fig. 2d). Interestingly, the strongest F-actin staining was found in scattered cells in the SG1 granular layer above the TJs (Fig. 2a, asterisks, b, e; Supplementary Movie 2). 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. Staining for phosphorylated myosin (pMLC) showed strongest intensity also within the granular layer (Supplementary Fig. 2c), indicating increased actomyosin contractility in this layer. 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.

Fig. 2 Highest F-actin organization in TJ containing granular layer. a Newborn epidermal whole-mount immunofluorescence analysis for Phalloidin revealing strong F-actin organization in granular layer 1 (SG1) and 2 (SG2). Asterisks mark F-actinhigh SG1 cells. Scale bar, 30 µm. b Quantification of relative F-actin distribution in spinous and granular layers of epidermal whole mounts confocal stacks of three biological replicates. Graph shows mean values ± SD as well as single measurements (dots, n = 5 per replicate). c Immunohistochemical analysis for F-actin (phalloidin) on newborn mouse epidermis sections. Scale bar, 20 µm. d F-actin organization in SG2 showing F-actin at the apical junction ring (arrows) and at the lateral, vinculin-positive AJ network (bracket). e Whole-mount analysis using Phalloidin and ZO-1 showing that F-actinhigh cells are in SG1 above the TJ barrier (ZO-1) (arrowheads). Scale bar, 30 µm. f Human adult epidermis whole-mount immunofluorescence analysis for F-actin (phalloidin) and vinculin showing tissue polarity of F-actin and vinculin and presence of F-actinhigh cells in SG1 layer. Scale bar, 20 µm. a, d–f Partial confocal stack projections and virtual sections (v. section) from newborn mouse epidermal whole mounts a, d, e and human adult epidermis (f). a, c–e Representative image of N ≥ 3 biological replicates each. f Representative image of two individuals. Nuclei were stained with DAPI (blue) Full size image

E-cadherin controls junctional tissue polarization

We next addressed how polarized organization of junctional tension and F-actin along the basal-to-apical tissue axis is achieved. As E-cadherin controls apico–basolateral polarity of simple epithelia1 and controls TJ function in the epidermis10, we hypothesized that E-cadherin could mediate tissue-level polarity. 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). 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). Thus, E-cadherin-dependent AJs are the predominant vinculin-positive, tension-bearing AJs in suprabasal epidermal layers.

Fig. 3 E-cadherin regulates tissue polarization of mechanosensitive junctions and cytoskeleton. a Newborn epidermal whole-mount immunofluorescence analysis for tension-sensitive epitope (α18) and total α-catenin and b Vinculin showing loss of α18 and vinculin and strong reduction in total α-catenin in granular layer 2 (SG2) of Ecadepi−/−. Scale bar, 20 µm. c F-actin staining (Phalloidin) showing that loss of E-cadherin results in less F-actin polarization across layers. Scale bar, 30 µm. d ZO-1 and occludin staining showing interrupted TJs in SG2 and increased punctate ZO-1 in lower layers upon loss of E-cadherin. Scale bar, 20 µm. a, b–d Partial confocal stack projections and virtual sections (v. sections) from newborn mouse epidermal whole mounts. Stacks shown in a were deconvolved. a, b–d Representative images of n ≥ 3 biological replicates each. Nuclei were stained with DAPI (blue). e Quantification of F-actin intensities from c normalized to Ctr spinous layer (SS). 20 measurements (dots) per biological replicate and the mean (lines) of each biological replicate are shown, *P = 0.0308; n = 4 biological replicates with Kruskal–Wallis, Dunn’s post hoc test. f Quantification of layer specific cortical ZO-1 enrichment normalized to Ctr SS. 20 measurements (dots) and the respective means (lines) of four biological replicates are shown, ns = not significant, *P = 0.0286; n = 4 biological replicates with Mann–Whitney test Full size image

We next asked whether E-cadherin loss would affect the asymmetric tissue distribution of F-actin across epithelial layers. Surprisingly, despite absence of AJs in the SG2 layer, phalloidin staining of whole mounts revealed no obvious changes in F-actin organization neither in the SG2 nor SG1 layer (Fig. 3c, e). In contrast, the spinous layers showed an increase in cortical F-actin organization (Fig. 3c, e; Supplementary Fig. 3c, d). Moreover, the TJ markers ZO-1 and occludin lost their continuous and linear apical distribution in Ecadepi−/− SG2. Instead, these proteins showed a more punctate pattern at cellular interfaces, revealing many breaks (Fig. 3d), thus explaining why in vivo TJ barrier function is disturbed upon loss of E-cadherin10. Interestingly, this punctate intercellular contact pattern of ZO-1 and occludin was also observed in Ecadepi−/− spinous layers, indicating premature formation of non-functional TJ-like structures (Fig. 3d, f; Supplementary Fig. 3e; ref. 10). 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.

Spatiotemporal coordination of F-actin and barrier formation

We next asked when TJs are formed during morphogenesis. 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. 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). 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). At E16.5, the onset of a functional SC, ZO-1 became strongly enriched at junctions of the uppermost viable dorsal epidermal layer, together with vinculin (Fig. 4a, b), even though total vinculin remained similar (Supplementary Fig. 4b, c). 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. E16.5 ventral regions showed only partially initiated suprabasal F-actin polarization, which was not yet confined to the granular layer (Fig. 4c).

Fig. 4 E-cadherin spatiotemporally coordinates the formation of tension-high adherens junctions, tight junctions, F-actin, and barrier function. a–e Immunohistochemistry analysis on mouse embryonic skin before (E15.5, E16.5 ventral) and after (E16.5, dorsal) epidermal barrier formation for a TJ marker ZO-1 showing that TJ formation in uppermost layer (yellow arrowheads) coincides with barrier formation. Peridermal TJs are marked by magenta arrowheads. b Staining for vinculin showing that suprabasal, junctional recruitment (yellow arrowheads) coincides with barrier formation. Keratin-6 was used to identify periderm (a, b). c Phalloidin staining showing F-actin organization and polarization. d, e. Staining for ZO-1 d and Phalloidin e showing increased depolarization of ZO-1 junctional recruitment and F-actin organization upon loss of E-cadherin during barrier formation. a–c Representative image of n ≥ 3 biological replicates each. d, e Representative image of n = 2 biological replicates each. Nuclei were stained with DAPI (blue). Scale bars, 20 µm Full size image

Importantly, loss of E-cadherin interfered with the tissue polarization of ZO-1 and F-actin in dorsal E16.5 epidermis, resulting in a more uniform distribution across all layers (Fig. 4d, e). 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.

E-cadherin coordinates intercellular forces

To assess whether E-cadherin directly controls junctional tension and actomyosin organization we isolated primary keratinocytes of control and Ecadepi−/− mice and switched these cells from low to high Ca2+-concentration to initiate intercellular junction formation. 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). 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). Importantly, TFM on doublets of cells22 revealed a 3-fold reduction in tension across intercellular contacts in E-cadherin−/− keratinocytes compared to control after 24 h in high Ca2+ (Fig. 5d). This was surprising as desmosomes, considered an important mechanical unit, are formed in these cells10, 23. Moreover, loss of E-cadherin interfered with coordinated transfer of force across multiple cells to cell–matrix adhesion sites at edges of multicellular colonies (Fig. 5e, f)24. These alterations in mechanical behavior were not accompanied by changes in ppMLC2 levels or localization (Supplementary Fig. 5b–d). This inability to mechanically couple cells through junctional zippers was neither due to reduced vinculin expression (Supplementary Fig. 5e, f) nor to a failure of P-cadherin, the other epidermal classical cadherin, to sense and respond to mechanical signals, as vinculin and F-actin were still recruited to individual AJs in E-cadherin−/− keratinocytes (Fig. 5c; Supplementary Fig. 5a). Overexpression of either P-cadherin or E-cadherin also rescued the formation of regularly spaced vinculin-positive AJ zippers (Supplementary Fig. 5g, h). Together, these results indicate that a threshold expression level of classical cadherins is necessary to coordinate the organization of early tension-bearing junctions into a regularly spaced zipper to coordinate force transduction across cells.

Fig. 5 E-cadherin controls AJ zipper formation and intercellular tension. a Immunofluorescence analysis for vinculin and F-actin and b ZO-1 in keratinocytes 2 h in high Ca2+. White arrows indicate cell–matrix contacts a and irregular zipper formation b. c Line plot profile of cell contacts, pink arrows: cell–matrix contacts, black arrows: cell–cell contacts. a–c Representative images/profiles of n ≥ 3 biological replicates. d Traction force microscopy. (Left) Distributions of in-plane traction stresses (blue and red arrows), σ iz , for pairs of control (Ctr) or E-cadherin (Ecad)−/− keratinocytes after 24 h in Ca2+, overlaid on DIC images. White arrows: resultant intercellular tension, T 12 , of cell 2 on cell 1, and vice versa, given by vector sum of in-plane tractions within dashed yellow boundary of opposite cell of the pair. (Right) Quantification of intercellular tensions per cell area for Ctr (n = 14) or Ecad−/− (n = 15) keratinocyte pairs. Error bars: standard deviations. **P = 0.002, Student’s t-test. e In-plane traction stresses (blue and red arrows) overlaid on DIC images and strain energy densities (blue and red heat maps) of Ctr and Ecad−/− keratinocyte colonies 24 h in high Ca2+. f Line plots of normalized strain energy distributions from edge (Δ = 0) to colony interior for Ctr (blue, n = 14) and Ecad−/− (red, n = 14) colonies. f Graph represents fraction of inward displacement from colony edge needed to capture 3/4 of total strain energy. Error bars: standard deviations. *P = 0.0102, Student’s t-test. Scale bars, 20 μm. g AFM force indentation plots, mean plots of n = 8 representative measurements including 2 primary Ctr and E-cad−/− cell lines after 48 h in high Ca2+ are shown. h Distribution of Young’s moduli from a representative indentation experiment on 2 primary Ctr and E-cad−/− cell lines 48 h in high Ca2+ (Ctr: n = 853, E-cad−/−: n = 1099). i Relative Young’s moduli in Ctr and E-cad−/− cells. Median values of biological replicates were used for statistical analysis. ***P = 0.0001; Ctr/Ecad−/− n = 5 biological replicates with Student’s t-test (>300 measurements/replicate). j Transepithelial resistance (TER) measurements in Ctr and Ecad−/− keratinocytes. Representative example of n > 10 independent isolates. k TER measurements in primary keratinocytes upon Blebbistatin treatment 24 h after switching to high Ca2+. Scale bars, 20 µm Full size image

E-cadherin coordinates TJ barrier through actomyosin

As both tension-bearing AJs and TJ formation were restricted to the suprabasal SG2 layer in vivo (Fig. 1), which does not contain tension-bearing cell–matrix contacts, we next asked whether E-cadherin would specifically regulate mechanical properties of suprabasal cells. 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. As the three-dimensional nature of this multi-layered structure where forces are dissipated in multiple directions precludes laser ablation or TFM, we quantified cell mechanics in this layer using force indentation spectroscopy with an AFM. Using blebbistatin, we first confirmed that the cortical actomyosin network controls the measured elastic properties of these cells (Supplementary Fig. 5i). 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). These alterations in cell mechanics were accompanied by the inability of E-cadherin−/− keratinocytes to establish a functional barrier, as measured by transepithelial resistance (TER) measurements (Fig. 5j). 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. Importantly, inhibiting myosin activity interfered with barrier formation in control keratinocytes (Fig. 5k), showing that actomyosin tension directly controls the TJ barrier. Together, these results indicate that proper coupling of E-cadherin-containing AJs to the actomyosin cytoskeleton is crucial to couple and coordinate tension across a stratifying epithelial sheet essential to establish and reinforce the barrier.

Vinculin not required for junctional tissue polarization

We next addressed the role of vinculin recruitment to AJs in early AJ zipper formation and in vivo epidermal polarization of junctions and the cytoskeleton. To this end, we generated vinculin-floxed mice (Supplementary Fig. 6a, b) and crossed them with K14-Cre mice25 to delete vinculin in the epidermis (Supplementary Fig. 6c). In vivo, loss of epidermal vinculin did not result in perinatal lethality unlike loss of E-cadherin10. 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). 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).

Fig. 6 Vinculin is not essential for junctional tissue polarization. a Newborn epidermal whole-mount fluorescence analysis for ZO-1 showing its distribution in granular layer 2 (SG2), spinous layer (SS), and cross section (v. section). b Newborn epidermal whole-mount fluorescence analysis for phalloidin showing F-actin organization in SG2 and in all epidermal layers (v. section). a, b Partial confocal stack (deconvolved) projections and virtual section (v. section). Nuclei stained with DAPI (blue). Scale bars, 20 µm. c Quantification of layer specific cortical actin enrichment normalized to Ctr SS, showing no change upon loss of vinculin. d Immunofluorescence analysis for E-cadherin, F-actin (phalloidin) and active myosin (ppMLC2) in primary control and vinculin−/− keratinocytes after 2 h in high Ca2+. Scale bar, 10 µm. e Quantification of AJ length. **P < 0.05; n = 6 Ctr, n = 3 vinculin−/−, n = 4 E-cadherin−/− biological replicates (n = 100 AJs per replicate) with Kruskal–Wallis, Dunn’s post hoc test. f Low magnification images of F-actin organization corresponding to d. Scale bar, 20 µm. d, f Representative image of n ≥ 3 independent keratinocyte isolations. g Transepithelial resistance measurements in primary Ctr and vinculin−/− keratinocytes. Representative example of n = 4 biological replicates. h Histogram showing the distribution of Young’s moduli obtained from a representative indentation experiment on 3 primary Ctr and 3 vinculin−/− cell lines after 48 h in high Ca2+ (Ctr: n = 1194, vinculin−/−: n = 1207 measurements) Full size image

We then assessed whether vinculin was essential for initial adhesion zipper formation. 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), further confirming that these are cell–cell and not cell–matrix junctions. However, these junctions were strongly elongated compared to those in control or E-cadherin−/− cells (Fig. 6d, e). Moreover, unlike in controls, F-actin was still predominantly organized in stress fibers, indicating that vinculin is necessary for early transition of F-actin from stress fibers into a cortical organization (Fig. 6f). In agreement, staining for ppMLC2 showed a more diffuse pattern compared to control (Fig. 6d). Thus, vinculin controls the length of the individual primordial AJs as well as overall cortical organization of the F-actin cytoskeleton. However, unlike E-cadherin deficiency, vinculin loss does not obviously impair the regularly spaced organization of early AJs into zippers (Fig. 5b; Supplementary Fig. 6e). Although TER measurements showed a reduced TJ barrier function upon loss of vinculin (Fig. 6g), this reduction was much less severe than that induced by loss of E-cadherin (Fig. 5j). In agreement, loss of vinculin did not obviously affect cortical stiffness of the suprabasal keratinocyte sheet as measured by AFM (Fig. 6h; Supplementary Fig. 6g, h). Thus, vinculin is important for initial junctional organization, suggesting it promotes mechanical reinforcement of newly formed junctions. 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. Our results suggest that other mechanically relevant AJ-associated proteins either control barrier formation or compensate for the in vivo loss of vinculin.

E-cadherin regulates EGFR localization and activity

We proceeded to identify the molecular mechanism by which E-cadherin controls tissue-level organization of cell mechanics and TJ positioning. As EGFR activity status has been linked to intercellular junctional and actomyosin organization and function26,27,28,29, we examined this receptor in more detail. Staining of control tissue sections showed localization of EGFR not only in the basal layer, as expected30, but also in suprabasal layers (Supplementary Fig. 7a, b). Whole-mount analysis revealed that EGFR was enriched at or near the apical ZO-1-containing TJs in SG2 (Fig. 7a, b). EGFR was also cortically enriched in the SG2 of human epidermis (Supplementary Fig. 7c). 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). In vitro, EGFR localized to early AJs showing a regular zipper-like distribution, which was strongly reduced in E-cadherin−/− keratinocytes (Supplementary Fig. 7d). Loss of E-cadherin also resulted in increased EGFR activity both in vivo (Fig. 7e, f; Supplementary Fig. 9) and in vitro (Supplementary Fig. 7e, f). Interestingly, this increased activity was not associated with enhanced proliferation in vitro or in vivo (ref. 10; Supplementary Fig. 7g). Importantly, reintroduction of full length E-cadherin reversed this increased EGFR activity in E-cadherin−/− keratinocytes to control levels (Supplementary Fig. 7h, i), showing that E-cadherin directly regulates EGFR. To address whether E-cadherin inhibits EGFR by promoting tension we treated cells with blebbistatin, resulting in increased EGFR activity, indicating that tension is important (Fig. 7g; Supplementary Fig. 7j). Interestingly, however, an E-cadherin mutant that cannot interact with β-catenin and thus α-catenin also rescued increased EGFR activity in E-cadherin−/− keratinocytes, suggesting that a direct connection of junctions to actin is not required for E-cadherin-dependent regulation of EGFR (Supplementary Fig. 7h, i).

Fig. 7 E-cadherin regulates EGFR tissue polarization and activity. a Newborn epidermal whole-mount fluorescence analysis for EGFR and ZO-1 showing enrichment of EGFR at TJs in control SG2 layer. b Quantification of junctional EGFR fluorescence intensity. n = 6 biological replicates with 10 junctions each. Graph shows mean values ± SEM and single means of biological replicates (dots/squares). Granular layer (SG) intensity was normalized to spinous layer (SS) intensity. *P = 0.0214 with D’Agostino & Pearson omnibus normality test followed by one sample t-test. c Newborn epidermal whole-mount fluorescence analysis for EGFR and ZO-1 showing SG2, SS, and cross section (v. section), showing increased junctional ZO-1 and EGFR (arrowheads) in the SS of E-cadherin (Ecad)−/− epidermis compared to control (Ctr). a, c Partial projections and v. sections of confocal stacks from newborn epidermal whole mounts. For better visualization fluorescence intensities of SS projections have been increased relative to SG. Nuclei were stained with DAPI (blue). d Quantification of cortical EGFR enrichment in SG normalized to Ctr SS. Shown are respective means (lines) of three biological replicates with 20 measurements (dots) per replicate, *P < 0.05; n = 3 biological replicates with Kruskal–Wallis, Dunn’s post hoc test. e Western blot for total and phosphorylated EGFR (pEGFR) from Ctr and Ecadepi−/− newborn epidermis. f Western blot quantification of pEGFR levels in newborn Ctr and Ecadepi−/− epidermis. **P = 0.0042; Ctr n = 6, Ecadepi−/− n = 5 biological replicates with Student’s t-test. g Quantification of western blot for phospho-EGFR (72 h in high Ca2+, blebbistatin added after 24 h). *P < 0.05; n = 4 biological replicates with one sample t-test, hypothetical value = 1 (normalized to control treated DMSO). h Staining of embryonic skin for EGFR before (E15.5) and after epidermal barrier formation (E16.5, dorsal skin). i Immunofluoresence analysis of E15.5 control and Ecadepi−/− skin for EGFR shows premature suprabasal localization of EGFR. Nuclei were stained with DAPI (blue). j Quantification of cell membrane EGFR intensities (mean gray value) in suprabasal vs. basal layers. The ratio of suprabasal vs. basal layer membrane intensity is plotted. Dots represent means of biological replicates. *P < 0.05; n = 3 biological replicates each group with Kruskal–Wallis, Dunn’s post hoc test. Scale bars, 20 µm Full size image

We then examined the in vivo spatiotemporal relationship of EGFR localization, junctional tension asymmetry, and acquisition of barrier properties. Interestingly, during early stages of epidermal morphogenesis, EGFR was restricted to basal intercellular contacts and was recruited to suprabasal junctions of dorsal epidermis only at E16.5 (Fig. 7h, j). This coincided with ZO-1 and vinculin recruitment to the most viable suprabasal layer (Fig. 4a, b) and with initiation of barrier function20. Most importantly, loss of E-cadherin resulted in premature recruitment of EGFR to suprabasal intercellular contacts at E15.5 (Fig. 7i, j). 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). In conclusion, E-cadherin controls the polarized localization and activity status of suprabasal EGFR, which is uncoupled from growth10.

EGFR activity controls apical stiffness and tight junctions

As loss of epidermal EGFR is associated with skin barrier dysfunction30, we next asked whether spatiotemporal regulation of EGFR activity would affect formation of TJ barrier function. EGF stimulation substantially lowered TER in control keratinocytes (Fig. 8a), but did not further reduce TER in E-cadherin−/− keratinocytes (Fig. 8a), consistent with the observation that EGFR activity is already increased in these cells. Vice versa, sustained inhibition of EGFR was sufficient to abrogate TJ barrier function (Fig. 8b). These findings show that either too little or too much EGFR activity interferes with formation of a functional TJ barrier.

Fig. 8 Spatiotemporal control of EGFR localization and activity controls TJ barrier function. a, b Transepithelial resistance (TER) measurements in primary keratinocytes. a Control (Ctr) keratinocytes were stimulated with EGF (100 ng ml−1 final concentration) 24 h after switching to high Ca2+ (dashed line). b Ctr keratinocytes were treated with EGFR inhibitor gefitinib starting with switching to high Ca2+ (0 h). c Immunofluorescence analysis of occludin organization in Ctr and E-cadherin (Ecad)−/− primary keratinocytes after 48 h in high Ca2+. Scale bar, 20 µm. Nuclei were stained with DAPI (blue). d Cell surface biotinylation/internalization assay. Graphs represent the relative amount of internalized protein per surface labeled protein 30 min after internalization. Indiviudal experiments are connected by dashed lines. e TER measurement, E-cadherin−/− keratinocytes were treated with different doses of EGFR inhibitor gefitinib and EGFR 24 h after switching to high Ca2+. Note dose dependent rescue of TER in E-cadherin−/− keratinocytes. a, b, f Representative example of n = 3 biological replicates. f Histogram showing the distribution of Young’s moduli obtained from a representative indentation experiment from primary either DMSO or gefitinib treated E-cadherin−/− keratinocytes after 48 h in high Ca2+ (treatment after 24 h) (n > 500 measurements each treatment). g Model showing how E-cadherin integrates mechanical and chemical signals to restrict the formation of tight junctions to the stratum granulosum 2 Full size image

We then asked whether enhanced EGFR activity induced by loss of E-cadherin directly controlled TJs. Immunofluoresence analysis revealed occludin-positive vesicle-like punctae in E-cadherin−/− keratinocytes but not in controls when cultured in conditions that allow for functional TJs (Fig. 8c). 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). 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. Importantly, EGFR inhibition also reversed the reduced cortical stiffness of E-cadherin−/− keratinocytes (Fig. 8f; Supplementary Fig. 8b), thus providing a direct link between chemical and mechanical signaling in the regulation of the barrier. 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. Thus, E-cadherin-mediated spatiotemporal control of EGFR localization and activity is crucial for proper force transduction to correctly position the formation of a stable TJ barrier in multi-layered epithelia such as the epidermis (Fig. 8g).