Mechanical stresses are inherent to multi-cellular tissues in organisms, and can regulate signalling pathways involved both in their biochemical patterning and biomechanical morphogenesis (Brouzés and Farge, 2004; Chan et al., 2017; Mammoto and Ingber, 2010; Wozniak and Chen, 2009). Mechanical regulation is involved not only in physiological developmental processes, but also in pathological processes such as in tumorigenesis (Mitrossilis et al., 2017; Dupont et al., 2011; Baeyens et al., 2015; Yang et al., 2016; Wu et al., 2015; Engler et al., 2006; Vogel and Sheetz, 2009; McBeath et al., 2004; Paszek et al., 2014; Uyttewaal et al., 2012; Zhang et al., 2011; Farge, 2003). Such regulation is based on molecular mechanotransductional processes, which consist in the translation of mechanical strains submitted to living cells into biochemical signals (Gospodarowicz et al., 1978; Nakache and Gaub, 1988; Kanno et al., 2007; Bissell et al., 2005; Rubashkin et al., 2014; Delarue et al., 2014; Liu et al., 2015; Mammoto et al., 2009; Yan et al., 2012). The molecular basis of mechanotransduction is the activation or modulation of biochemical reactions in response to cell or tissue mechanical strain-deformations. These are transmitted by the stress to subcellular mesoscopic structures or individual biomolecules. Mechanical induction of biochemical reactions is thus enabled by the coupling of the two major properties of molecular living matter: its high biochemical reactivity, and its high physical deformability. Since free-energy barriers between protein conformations are usually smaller than a few tens of kT (Lannon et al., 2012) (i.e several ten times the molecular thermal energy), mechanical stresses applied to biomolecular structures can a priori easily trigger conformational changes. Such deformations can subsequently lead to changes in the molecular biochemical reactivity and states, thereby initiating the activation or modulation of biochemical signal-transduction pathways (Brujic et al., 2007; Fernandez-Sanchez et al., 2015a; Grashoff et al., 2010; Vargas et al., 2001). Mechanical stresses have been shown to initiate a variety of molecular protein-conformation, or membrane shape changes, in cell culture. One example is the rendering of accessibility of the phosphorylation target site for kinases, like for Src-kinase, in the p130Cas protein involved in tumorigenic cell migration, in response to mechanical stress (Sawada et al., 2006). Another example is the mechanically induced inhibition of the formation of endocytic vesicles that lead to the inhibition or the degradation of interactions of secreted ligand-receptor complexes and thus activate or enhance downstream signalling pathways. This was shown in cell culture for BMP2 inducing myoblast/osteoblast trans-differentiation (Rauch et al., 2002).

However, none of the underlying molecular mechanism of mechanotransduction characterized so far has been found to be functionally involved in cell specification and differentiation, in vivo.

Among the many mechanosensitive pathways, the β-catenin (β-cat) pathway is mechanotransductively involved in major biochemical patterning processes in both developmental and tumorous contexts (Fernandez-Sanchez et al., 2015a; Farge, 2003; Desprat et al., 2008; Brunet et al., 2013; Benham-Pyle et al., 2015; Fernández-Sánchez et al., 2015b; Samuel et al., 2011; Whitehead et al., 2008; Mouw et al., 2014). Initially, it was found that mechanical activation of β-cat and downstream transcriptional signaling is vitally involved in embryonic anterior endoderm specification in response to the first morphogenetic movements of gastrulation (Farge, 2003; Desprat et al., 2008) In addition, it is involved in ex-vivo reconstructed epithelial cells in response to mechanical stretching (Benham-Pyle et al., 2015; Benham-Pyle et al., 2016), in tumor progression in healthy tissues in response to neighboring tumor growth pressure and stiffness (Fernández-Sánchez et al., 2015b; Samuel et al., 2011; Whitehead et al., 2008), or in follicle development initiation (Shyer et al., 2017). In both zebrafish and Drosophila embryos, early mesoderm differentiation was also found to be mechanically induced by the very first morphogenetic movements of embryogenesis. This induction occurs through the mechanical activation of the β-cat pathway, where the mechanical strain generated by the onset of embryonic morphogenesis mechanically induces the Src-family kinase (SFK) dependent phosphorylation of the highly conserved β-cat Y654 site (Brunet et al., 2013; Fernández-Sánchez et al., 2015b), a major site of interaction with E-cadherin (E-cad) in adherens junctions (AJ) (Roura et al., 1999; van Veelen et al., 2011). This chemical modification leads to β-cat release from AJ and its translocation to the nucleus, where it activates the transcription of early mesodermal key specification genes (Brunet et al., 2013). The molecular mechanism of mechanotransduction that mechanically initiates such SFK-dependent β-cat phosphorylation in vivo, as well as its primary mechano-sensor, are still unknown.

Here we identified the primary mechanotransductive molecular process and the mechano-sensor. We tested in vivo the hypothesis that the major Y654-β-cat/E-cad-D665 interaction site of the AJ complex (Fernandez-Sanchez et al., 2015a) could be, by itself, the primary mechanosensor that activates the β-cat pathway through its mechanical opening. This hypothesis implied to characterize the mechanical opening of the molecular interaction between the Y654 β-cat tyrosine with the D665-E-cad aspartic acid. This mechanical event would favour phosphorylation by Src42A, that is known to lead to a 86% decrease in the Y654 β-cat-D665-E-cadherin affinity (Roura et al., 1999; van Veelen et al., 2011). It further releases β-cat into the cytosol and nucleus, where it targets functional patterning gene transcription during mesoderm invagination at gastrulation (Brunet et al., 2013).

β-cat is highly conserved across metazoan, with a central armadillo repeat domain consisting of twelve repeats (Figure 1a). These repeats form a spring like superhelix featuring a long, positively charged groove that interacts with several key residues of the cytoplasmic tail of E-cad (Figure 1—figure supplement 1a) (Huber and Weis, 2001; Orsulic and Peifer, 1996). The strength of interaction is dynamically modulated, mostly by the phosphorylation of β-cat on tyrosine Y654 (Figure 1—figure supplement 1b,c in red), reducing the affinity of E-cad to β-cat by approximately 86% (Roura et al., 1999; van Veelen et al., 2011). In mammalian cells, phosphorylation of β-cat Y654 has been shown to involve SFK (Roura et al., 1999). In Drosophila, the Y667 site of the β-cat homologue Armadillo shows sequence similarity to the conserved Src-phosphorylation site at Y654 in mouse (Brunet et al., 2013; Roura et al., 1999; van Veelen et al., 2011) (Figure 1—figure supplement 1d). Its Drosophila E-cad D1170 interacting site similarly shows sequence conservation with the D665 mice site (Figure 1—figure supplement 1e) (Huber and Weis, 2001).

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Figure 1—source data 1

Y654-ß-cat-D665-E-cad interacting site distances under local H3-ß-cat-HII-E-cad stretching due to the global stretching of the ß-cat-E-cad complex, molecular dynamics simulation.

https://doi.org/10.7554/eLife.33381.006

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To test the hypothesis of a mechanical opening of the Y654-β-cat/E-cad-D665 site as the primary mechantransductive molecular event initiating the mechanical activation of the β-cat pathway, and gain insight at the molecular level, we performed molecular dynamics (MD) simulations of the β-cat-E-cad complex submitted to a stretching uniaxial force (Huang and MacKerell, 2013; Phillips et al., 2005).

The force is applied on E-Cad by both the cell’s and the neighbouring cell’s apical constrictions that drive Drosophila embryos mesoderm invagination (Sweeton et al., 1991). It thus acts perpendicular to the plasma membrane directed to the outside, with β-cat fixed at its Y142 interacting site with α-catenin which is connected to the Actin cytoskeleton (Xu and Kimelman, 2007) (Figure 1a). Note that the timescales of conventional MD simulations are currently limited to the hundreds of nanoseconds to the microsecond. To observe protein unfolding, or conformational changes in response to mechanical force on such a reduced timescale, it is therefore common to use forces that are higher than experimental ones. Due to this high force, the same events that biologically occur on a timescale that is not accessible by the MD simulations are sped up and hence become amenable to molecular simulations.

Here, we generated 20 independent simulations for a total simulation time of 30 ns each (600 ns total) on a protein complex of significant size (more than 500 residues). Our simulations under strain employed a constant force of 150 pN in order to observe relevant conformational changes on the 30-ns timescale of the simulation (Figure 1—figure supplement 2a). We estimated a posteriori the biological force that is mimicked by our procedure as follows. The mean elongation of the simulated complex showing the opening of the Y654-D665 site on the timescale of 12 ns is of 6 nm (Figure 1—figure supplement 2a). The proteins including β-cat are formed by α-helices only, with a modulus of elasticity of K_h of an α-helix on the order of 1 pN/nm (Kim et al., 2010). Therefore we find that this elongation corresponds to a force of 6 pN, consistent with the 2-3 pN characteristic force of E-cad in adherent cells without external force (Borghi et al., 2012).

After a global extension of 8 nm of the overall complex, a local and fine stretching deformation is observed between the A648 site of the α-helix HII-E-cad and the C-terminus of the α-helix H3-β-cat, which are linked by the Y654-β-cat-D665-E-cad (Y654-D665) interacting site, of 1.25 ± 0.6 nm (45% strain). This change led, at 6 nm of extension mimicking an actual strain of 6 pN, to a discrete opening of the Y654-D665 interacting site of 0.42 ± 0.08 nm in 3 simulations out of 20, namely in 15% of cases (Figure 1b,c,d, Videos 1 and 2 and Figure 1—figure supplement 2a,b,c,d, respectively; See Figure 1c,d and Figure 1—source data 1).

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We then tested in vivo the prediction of the existence of a strain deformation in the β-cat-E-cad complex of 1.25 nm between the A648-E-cad and the β-cat-C-terminus, due to mesoderm invagination in Drosophila embryos. In this case, mechanical induction of the Y654-β-cat phosphorylation by mesoderm invagination is specifically required for mesoderm specification (Brunet et al., 2013). To do so, we carried out fluorescence lifetime imaging experiments (FLIM) to measure fluorescence resonance energy transfer (FRET) efficiency between the loop domain of E-cad region II directly linked to A648 and β-cat-C-terminus. For the purpose of introducing fluorescent groups, we used embryos that express a version of β-cat tagged with GFP as a donor at the C-terminus. The acceptor is an injected antibody (JCAb20) fluorescently labelled with Alexa555 and specifically designed against the linear domain of region II in the E-cadherin protein, which is directly linked to A648-E-cad (acceptor) (Huber and Weis, 2001) (Figure 1—figure supplement 1, Figure 2a and Figure 2—figure supplement 1. Schematized in Figure 2a, the two fluorochromes are linked to the two green spheres of Figure 1 simulation, see Materials and methods).

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Fluorescence lifetime measurements of the β-cat-C-terminus-GFP donor in the presence of the E-cad linear region II Alexa555 acceptor during mesoderm invagination.

https://doi.org/10.7554/eLife.33381.012

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FLIM signal of the β-cat-GFP donor in the absence of acceptor during mesoderm invagination.

https://doi.org/10.7554/eLife.33381.013

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The choice of the E-cad site was based on the crystalline structure of the β-cat/E-cad complex of mice (Huber and Weis, 2001), and on the high conservation of the sequence of E-cad around D665 between Drosophila and mice (Figure 1—figure supplement 1e). The JCAb20 antibody targets an E-cad loop at 1143–1158 (in Drosophila) neighbouring directly the alpha helix of region II. The latter contains the D665 (D1170 in Drosophila), which interacts with the Y654 site of β-cat (Y667 in Drosophila), and is directly labelled with Alexa 555 (in orange in Figure 1—figure supplement 1c and Figure 1—figure supplement 1a, from Protein Data Bank and Huber and Weis, 2001, with accession code 1I7X, schematized in red Figure 2a) (Huber and Weis, 2001). The free E-cad 1143–1158 loop of the crystalline structure is shown in purple and green in Figure 1—figure supplement 1a. The antibody was tested for its specificity by immuno-staining, western-blot analysis and immunoprecipitation (see Figure 2—figure supplement 1a, b). The peptide sequence of the immunogen was checked for possible similarities with other proteins by a standard protein-protein BLAST search against the Drosophila proteome (taxid: 7227) (see Table 1). The GFP, about 4 nm long, is located at the C-terminus of the β-cat (schematized in green in Figure 2a) (Peifer, 2004).

At the time of antibody injection, the cells in the embryo still form a syncytium, that is they are open to the same internal yolk volume, which allows the antibody to diffuse into any cell. Afterwards, cells are closed in a process called cellularization followed by the first embryonic morphogenetic movement, the invagination of the mesodermal tissue during gastrulation (Wieschaus and Nusslein-Volhard, 1998). FRET experiments were performed by measuring the lifetime of the β-cat-GFP donor (FLIM), as the E-cad Alexa555 labelled antibody acceptor acts as a quencher that reduces the lifetime of the donor. A relatively low lifetime indicates higher FRET efficiency and closer distances, while a lifetime close to the normal lifetime of the fluorophore indicates low FRET efficiency and higher distances (Figure 2a).

FLIM measurements were performed during the two following different developmental stages during gastrulation: just before mesoderm invagination (last 10 min of cellularization, late embryonic stage 5), and during invagination (stage 6) (Figure 2b) when the acto-myosin contractile mechanical forces acting along the AJ increase in the mesoderm (Martin et al., 2010). At stage 6, measurements showed an increase of the GFP lifetime in the mesoderm, observed by an increase of bright green and of yellow spots in junctions of the invaginating mesoderm (e.g. yellow arrows in Figure 2b), of 0.073 ± 0.029 ns compared to the ectoderm, and of 0.094 ± 0.059 ns compared to stage 5 mesoderm (2.473 ns) (Figure 2c, Figure 2—source data 1).

As a control, no change in the GFP donor lifetime was observed during gastrulation in the absence of injected antibody acceptor (E-cad Alexa 555 labelled AB), as well as in embryos injected with PBS alone in the absence of the antibody acceptor (Figure 2—figure supplement 2a,b and Figure 2c-right). Such a fine increase in lifetime is quantitatively in line with a mean increase in distance on the order of ~1 nm (see ‘Calculation of the strain from FRET efficiency variations’ in the Materials and methods section) between the GFP tagged β-cat and the fluorescently tagged E-cad antibody. Indeed, such ~1 nm stretching is quantitatively predicted by the MD simulations (Figure 1c). The lifetime increase observed specifically in the mesoderm at gastrulation thus shows the existence of a mechanical stretching strain between the fluorescently labelled linear domain II of E-cad, and the C-terminus of β-cat, respectively, in the invaginating mesoderm.

Note that the lifetime of the controls with GFP alone in the embryo junctions and with PBS injection without antibody, of 2.60 ± 0.04 ns, (Figure 2—figure supplement 2a,b, Figure 2—source data 2 and Figure 2—source data 1), was consistently higher than in the presence of the acceptor before stretching, of 2.473 ± 0.074 ns (Figure 2—source data 1). These values are similar to the lifetime of GFP tagged proteins measured in cellula, namely of 2.6 ns coupled to Histones in the nucleus in the absence of acceptor and of 2.4 ns in the presence of an acceptor (Llères et al., 2017). Furthermore, despite the fact that all junctions are labelled with the E-cad Alexa 555 acceptor (see Figure 2—figure supplement 1b), modulations in the labelling efficiency may be at the origin of variations observed in the lifetime. In addition, it is not possible to interpret the observed mean increase of lifetime, which is in the order of ~0.1 ns in all FLIM experiments, in terms of β-cat release. The reason is that the released fraction of free β-cat in the cytoplasm should escape detection and not lead to a higher lifetime value due to an increased population of GFP in a no-FRET configuration (see Figure 2—figure supplement 2). Indeed, the free fraction released should necessarily be at much of 100% with a strong dilution from a 2πr junctional line into the πr²l cell volume, where r = 2.5 μm is the cell apex radius and l = 15 μm is the cell length. Hence, the contribution of the free fraction of 100%*2πr*/πr²l*d² = 5.10⁻⁶ is negligible, with d² = (10 nm)² being the cross-section surface of the molecular complex.

These experiments thus demonstrate the existence of a mechanical deformation involving a specific site connecting two interacting proteins. The molecular detail of this deformation involves the stretching of the β-cat and E-cad helices which interact through their respective Y654 and D665 residues, during mesoderm invagination.

It is important to confirm that the mechanical strain at the molecular level around the Y654-D665 interacting sites during mesoderm invagination is caused by the mechanical strain developed at the cell tissue level by the invaginating cells of the mesoderm tissue (Costa et al., 1993). For this purpose, we first evaluated the FLIM signal in Snail (Sna) and Twist (Twi) deficient embryos that are defective in mesoderm invagination. Snail and Twist were knocked-down by injecting sna and twi siRNAs for RNAi (Mitrossilis et al., 2017). We then rescued the mechanical strains that are lacking in the sna twi RNAi mesoderm invagination defective embryos. This was achieved by injecting ultramagnetic liposomes (UMLs) into mesoderm cells (Brunet et al., 2013; Mitrossilis et al., 2017) and applying a magnetic field gradient tangential to the mesoderm, and perpendicular to its antero-posterior axis (Figure 3a and Method section). This gradient resulted in a force that constricts cells laterally, with a deformation of the apexes which is similar to the uniaxial apical constriction generating mesoderm invagination (Figure 3b,c) (Sweeton et al., 1991).

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FLIM measurements in the mesoderm in response to magnetically induced cell apex constriction mimicking the onset of mesoderm invagination.

https://doi.org/10.7554/eLife.33381.016

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We found no significant increase in the FRET lifetime between stage 6 (stage of invagination in the WT) and stage 5 in sna twi RNAi embryos defective in mesoderm invagination (Figure 3d, Figure 3—source data 1, without magnet injected with UMLs and siRNAi against twi and sna), in contrast to the increase observed in the WT (Figure 2c). This observation shows a defect of mechanical strain deformation between the linear domain II of E-cad and the C-terminus of β-cat induced in sna twi embryos defective in mesoderm invagination. In response to the magnetically induced constriction, junctional FRET live imaging showed an increase of the lifetime, observed by an increase of bright green and of yellow spots in junctions of the deformed tissue (e.g. yellow arrows in Figure 3b), of 0.098 ± 0.057 ns, comparable to the increase of 0.094 ± 0.059 ns observed in the mesoderm during its invagination. This lifetime increase shows an increase of distance between the two fluorophores on the order of ~1 nm (Figure 3d,e and Materials and methods). As controls, no increase in the lifetime between stage 5 and stage 6 was observed after injection of the UMLs and RNAi without magnet (Figure 3d), or with magnets after UML and PBS injection without the E-cad Alexa 555 acceptor (Figure 3d).

Thus, the defect of molecular mechanical strain deformation between the fluorescently labelled linear domain II of E-cad and the C-terminus of β-cat in sna twi RNAi embryos is rescued mechanically. Consequently, it is associated to the lack of mechanical strains in mesoderm defective embryo. These observations demonstrate that the mechanical strains developed by mesoderm invagination induce a mechanical stretching deformation in the order of ~1 nm in the molecular hetero-complex between the HII-E-cad and H3-β-cat helices, which are linked by the Y654-D665 site. These results confirm quantitatively the 1.25 ± 0.6 nm deformation predicted in our MD simulations.

We then tested if the increase of endogenous mechanical strain stretching the HII-E-cad and H3-β-cat helices causing the Y654-D665 binding site to open, leads to the increase of accessibility of Y654, as predicted by the opening of the site, with a probability of 15% by MD. To do so, we made use of an antibody directed against the non-phosphorylated Y654 site in the β-cat protein (Y654-Ab). To prevent disturbing competition between the Y654-β-cat antibody and E-cad on the Y654 site by injection of the E-cad anti-body in vivo before gastrulation, immuno-fluorescent staining was performed on fixed embryos using the ultra-rapid MeOH heat procedure that maintains junctional labelling only (Müller and Wieschaus, 1996), at stage 5, early stage 6 and late-stage 6 embryos. The apical punctual nature of the labelling can be observed at the onset of gastrulation, as curvature is still low enough to prevent compacting apical junctions together.

We found an increase in the signal for the un-phosphorylated site of β-cat at AJ relative to the junctional β-cat signal during gastrulation in the invaginating tissue. The increase is specific of gastrulation early stage 6 and late-stage 6 compared to pre-gastrulation stage 5, of 19.6 ± 6.2% and 23.0 ± 5.7% respectively (Figure 4a,c, Figure 4—source data 1). As a control, no increase in the signal for the un-phosphorylated site of β-cat at AJ relative to the junctional β-cat signal observed in the ectoderm, which is less strained (Figure 4d, Figure 4—source data 1). Consistently, an increase of 16.5 ± 7.5% and 18.0 ± 5.5% respectively, is observed in the signal for the un-phosphorylated site of β-cat at AJ in the mesoderm normalized to the un-phosphorylated site of site of β-cat in the ectoderm (Figure 4e, Figure 4—source data 1). This result thus shows that under the molecular strain induced by the morphogenetic movement of invagination, the Y654 site increases its accessibility by nearly 20 ± 5% to specific molecular interactions, before being phosphorylated. This is in line with our quantitative predictions by the model, and based on the simulation of the Y654-D665 binding site opening of 15% under strain.

Figure 4

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Increase of accessibility of Y654-β-cat in the mesoderm during invagination.

https://doi.org/10.7554/eLife.33381.018

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The mechanically induced opening of the Y654-D665 binding site should thus favour the accessibility of the Y654 tyrosine to Src42A and thereby its phosphorylation by Src42A which leads to the release of the β-cat from AJ (Brunet et al., 2013). However, such Src42A interaction with the Y654 site might be difficult to detect, as it should be highly transient, and thus would yield most likely poor FRET signals by probing any increase of the accessibility effect. On the other hand, Src42A would compete with Y654-Ab in its interaction with Y654. The increase of accessibility of the Y654 site to Y654-Ab under strain should thus be enhanced in the absence Src42A. An alternative prediction of our model, leading to the same conclusion, concerns the knock down of the responsible kinase Src42A that reduces Y654-β-cat phosphorylation (Takahashi et al., 2005). The knock down should thus lead firstly to stable and consequently enhanced molecular stretching effects during gastrulation, and thus to an increased number of stretched Y654 sites opened to non-phospho Y654 labelling. Both effects should lead to an enhancement of the accessibility of the Y654 sites.

To test such predictions, we thus measured the Y654 accessibility to the non-phospho antibody in src42A RNAi embryos (Desprat et al., 2008). In the src42A RNAi knockdown background, we found a specific increase in the signal for the un-phosphorylated site of β-cat at AJ under strain, as observed by the increase of Y654-βcat green labelling that adds to, and dominates the β-cat red labelling, leading to a bright green/yellow composite colour (Figure 4b).

This signal was measured relative to the junctional β-cat signal during gastrulation (44.3 ± 7.3% and 45.1 ± 7.9%, respectively), and of about 25 ± 5% compared to wild type, both at early stage 6 and late-stage 6 (Figure 4b,c). As a control, no significant increase in the signal for the un-phosphorylated site of β-cat at AJ relative to the junctional β-cat signal is observed in the ectoderm, that is less strained, in the src42A RNAi embryos compared to the WT (Figure 4d). Consistently, an increase of 12.4 ± 5.8% and 9.3 ± 4.3% respectively, is observed in the signal for the un-phosphorylated site of site of β-cat at AJ in the mesoderm normalized to the un-phosphorylated site of β-cat ectoderm, in the src42A RNAi embryos compared to the WT (Figure 4e). These results showed the increase of accessibility of Y654 to Y654-Ab of nearly 20% in the absence of Src42A under strain.

Therefore, we conclude that during mesoderm invagination, the accessibility of the site of Y654 β-cat to Src42A increases due to its mechanical opening. At the same time, the Y654 β-cat residue becomes specifically more available for Src42A phosphorylation at AJ, as the strain builds up at the Y654 β-cat-D665-E-cad molecular complex interaction site. The mechanical induction of the increase in accessibility of the Y654 site under strain for Src42A phosphorylation should thus lead to the release of an amount of junctional β-cat into the cytosol proportional to the subset of β-cat/E-cad complex that opens at Y654-D665 under strain (Brunet et al., 2013; Roura et al., 1999; van Veelen et al., 2011).

To test whether the molecular strain that is observed in mesoderm invagination induces a decrease in β-cat affinity to E-cad, we characterised the dynamics of β-cat in Drosophila embryo mesodermal AJs under strain during gastrulation. This was achieved by fluorescent recovery after photobleaching (FRAP) experiments at three different developmental stages: just before invagination (end of cellularization, late embryonic stage 5), at the onset of invagination (early 6) and during invagination (mid-stage 6) (Figure 5a,b).

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Increase of the mobile fraction of β-cat-GFP in the mesoderm during invagination.

https://doi.org/10.7554/eLife.33381.021

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We found upon photobleaching that the fluorescent recovery amplitude, the so-called mobile fraction, of β-cat upon photobleaching is increased by 16 ± 6% when mesoderm invagination has built up at mid-stage 6. This observation indicated that about 16% more molecules at the AJ are able to be recycled and exchanged at stage 6 compared to stage 5 (Figure 5c,d, Figure 5—source data 1). This result is consistent with studies examining β-cat turnover at cellular junctions under strain during germ band extension (Tamada et al., 2012). This effect is quantitatively predicted by the 15% probability of opening the β-cat/E-cad complex observed in our MD simulations under strain. As the opening of the Y654-D665 interacting site will lead to an increased Src42A phosphorylation, it will therefore lead to a decreased affinity between β-cat and E-cad.

This relationship shows that the morphogenetic movements of invagination locally cause a mechanical stretch at the Y654-D665 interaction site in the β-cat/E-cad molecular complex. It indeed statistically weakens the interaction of β-cat with E-cad in AJ of the mesoderm cells and thereby favours cytosolic β-cat enrichment.

In the gastrulating Drosophila embryo, the Src42A kinase required for the mechanical induction of Y654-β−cat phosphorylation is already activated (phosphorylated on Y400) before tissue deformation. Therefore, Src42A is surprisingly not mechanically ‘over-activated’ by tissue deformation during the morphogenetic movements of gastrulation (Desprat et al., 2008), and is thus not upstream of the mechanotransduction pathway leading to Y654-β-cat phosphorylation. In contrast, the already activated form of Src42A is permissively required in the mechanotransductive process leading to the activation of β-cat release from the junctions into the cytoplasm and then to the nucleus. This observation opened the question of the underlying molecular mechanism of the mechanotransductive process leading to the β-cat mechanical activation, which is conserved and generically involved in many diverse physiological and pathological biological conditions (Fernandez-Sanchez et al., 2015a; Farge, 2003; Desprat et al., 2008; Brunet et al., 2013; Benham-Pyle et al., 2015; Fernández-Sánchez et al., 2015b; Samuel et al., 2011; Whitehead et al., 2008; Benham-Pyle et al., 2016; Sen et al., 2008; Shyer et al., 2017) and suggests the Y654 β-cat interaction site with E-cad as the underlying molecular mechanosensor.

Here we showed that the highly evolutionary conserved Y654 β-cat-D665-E-cad major interaction site of the AJ complex acts as primary mechanosensor. The site activates the β-cat pathway through its mechanical opening in vivo that enables phosphorylation by Src42A kinase. Phosphorylation in turn leads to the decrease of the Y654 β-cat-D665-E-cad affinity (Roura et al., 1999; van Veelen et al., 2011), and to the release of β-cat into the cytosol. This release finally leads to target developmental patterning gene transcription in the nucleus (Figure 5—figure supplement 1) (Brunet et al., 2013).

We thereby demonstrate in vivo the existence of a primary molecular mechano-chemical translator. It is functionally involved in the trans-scale activation of a molecular physiological differentiation pathway, in response to an endogenously produced macroscopic mechanical strain. Here, the mechanosensor is the Y654-β-cat/D665-E-cad interaction site of the hetero β-cat/E-cad complex at AJ. It opens under strain and favours the phosphorylation of β-cat by Src42A. This process initiates early mesoderm differentiation and specification in response to the strain associated to mesoderm invagination at the onset of Drosophila’s embryonic gastrulation. The mechanical activation of the β-cat pathway shows a fine regulation under physiological strains. The soft stretching of 1 nm between the two α--helices surrounding the Y654-β-cat/D665 E-cad major interaction site leads to a probability of 15% opening and release of signalling β-cat from the junctions after phosphorylation by Src42A. Such finely tuned process could trigger the mechanotransductive signalling translocation from AJ to the nucleus of low levels of β-cat, known to be sufficient to trigger transcription into the nucleus (Peifer and Wieschaus, 1990), and ensure the maintenance of tissue coherence by the maintenance of 85% of the β-cat in the junctions. This scenario would explain the high sensitivity of β-cat target gene expression to low levels of β-cat expression (Peifer and Wieschaus, 1990). A possible interpretation for the observed 15% efficiency of site opening is that, once under tension, the deformation of the β-cat/E-cad complex can a priori statistically occur at different locations all along the complex structure. In 15% of cases, the deformation would occur on the effective Y654-β-cat/D665-E-cad site and open it. Future molecular dynamics investigations will be required to quantitatively test this hypothesis.

Finally, both the Y654-β-cat and D665 E-cad major sites between the two molecules are strongly conserved across all metazoa (Figure 1—figure supplement 3). This mechanotransductive molecular sensor may thus be generic in the overall animal kingdom, and be involved in physiological mechanical induction processes, including mesoderm differentiation across all bilaterians (Brunet et al., 2013), and pathological processes, including mechanical induction of tumour differentiation in response to tumour growth pressure in most epithelia of most animals (Fernández-Sánchez et al., 2015b).

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for: Figure 1c,d; Figure 1-supplementary; Figure 2b; Figure 2c; Figure 3d; Figure 4c,d,e; Figure 5d; Figure 2-supplementary figure 2b.

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Author details

1. Jens-Christian Röper

   Mechanics and Genetics of Embryonic and Tumoral Development, Institut Curie, INSERM, CNRS UMR 168, PSL University, Paris, France

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Visualization, Methodology, Writing—original draft, Designed experiments (with EF), Realized experiments on FLIM controls and in the presence of magnetic forces (with DM) and immunoprecipitation (with MEFS)

   Competing interests

   No competing interests declared

2. Démosthène Mitrossilis

   Mechanics and Genetics of Embryonic and Tumoral Development, Institut Curie, INSERM, CNRS UMR 168, PSL University, Paris, France

   Contribution

   Data curation, Investigation, Methodology, Set up magnetically induced mechanical deformation experiments

   Contributed equally with

   Guillaume Stirnemann

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-3382-296X

3. Guillaume Stirnemann

   CNRS Laboratoire de Biochimie Théorique, Institut de Biologie Physico-Chimique, PSL University, Paris, France

   Contribution

   Data curation, Software, Formal analysis, Investigation, Visualization, Methodology, Designed and generated numerical molecular simulations

   Contributed equally with

   Démosthène Mitrossilis

   Competing interests

   No competing interests declared

4. François Waharte

   Space-Time Imaging of Endomembranes Dynamics, Cell and Tissue Imaging Facility, Institut Curie, CNRS UMR 144, PSL University, Inria, France

   Contribution

   Resources, Data curation, Formal analysis, Methodology, Designed FRET experiments (with JCR and JS)

   Competing interests

   No competing interests declared

5. Isabel Brito

   CBIO-Centre for Computational Biology, MINES ParisTech, Institut Curie, INSERM, PSL University, Paris, France

   Contribution

   Software, Wrote the R script to compute the Fisher (1932) theory for p-values of combined p-values allowing the calculation between two curves

   Competing interests

   No competing interests declared

6. Maria-Elena Fernandez-Sanchez

   Mechanics and Genetics of Embryonic and Tumoral Development, Institut Curie, INSERM, CNRS UMR 168, PSL University, Paris, France

   Contribution

   Investigation, Performed the immunoprecipitation experiment and produced FLIM controls (with JCR)

   Competing interests

   No competing interests declared

7. Marc Baaden

   CNRS Laboratoire de Biochimie Théorique, Institut de Biologie Physico-Chimique, PSL University, Paris, France

   Contribution

   Resources, Validation, Supervised coordination between numerical and wetlab experiments (with EF)

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-6472-0486

8. Jean Salamero

   Space-Time Imaging of Endomembranes Dynamics, Cell and Tissue Imaging Facility, Institut Curie, CNRS UMR 144, PSL University, Inria, France

   Contribution

   Conceptualization, Resources, Validation, Designed FRET experiments (with JCR and FW)

   Competing interests

   No competing interests declared

9. Emmanuel Farge

   Mechanics and Genetics of Embryonic and Tumoral Development, Institut Curie, INSERM, CNRS UMR 168, PSL University, Paris, France

   Contribution

   Conceptualization, Supervision, Funding acquisition, Validation, Methodology, Writing—original draft, Project administration, Writing—review and editing, Supervised coordination between numerical and wetlab experiments (with MB), Designed experiments (with JCR), Coordinated the overall work

   For correspondence

   efarge@curie.fr

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-5063-7179

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