Abstract
In vivo, the primary molecular mechanotransductive events mechanically initiating cell differentiation remain unknown. Here we find the molecular stretching of the highly conserved Y654-β-catenin-D665-E-cadherin binding site as mechanically induced by tissue strain. It triggers the increase of accessibility of the Y654 site, target of the Src42A kinase phosphorylation leading to irreversible unbinding. Molecular dynamics simulations of the β-catenin/E-cadherin complex under a force mimicking a 6 pN physiological mechanical strain predict a local 45% stretching between the two α-helices linked by the site and a 15% increase in accessibility of the phosphorylation site. Both are quantitatively observed using FRET lifetime imaging and non-phospho Y654 specific antibody labelling, in response to the mechanical strains developed by endogenous and magnetically mimicked early mesoderm invagination of gastrulating Drosophila embryos. This is followed by the predicted release of 16% of β-catenin from junctions, observed in FRAP, which initiates the mechanical activation of the β-catenin pathway process.
Highlights
Mechanical stresses are inherent to multi-cellular tissues in organisms, and can regulate signalling pathways involved both in their biochemical patterning and biomechanical morphogenesis (Brouzes and Farge, 2004; Chan et al, 2017; Mammoto and Ingber, 2010; Wozniak and Chen, 2009)
We found no significant increase in the fluorescence resonance energy transfer (FRET) lifetime between stage 6 and stage 5 in sna twi RNAi embryos defective in mesoderm invagination (Figure 3d, Figure 3—source data 1, without magnet injected with ultramagnetic liposomes (UMLs) and siRNAi against twi and sna), in contrast to the increase observed in the wild type (WT) (Figure 2c)
To test whether the molecular strain that is observed in mesoderm invagination induces a decrease in b-cat affinity to E-cad, we characterised the dynamics of b-cat in Drosophila embryo mesodermal adherens junctions (AJ) under strain during gastrulation
Summary
Mechanical stresses are inherent to multi-cellular tissues in organisms, and can regulate signalling pathways involved both in their biochemical patterning and biomechanical morphogenesis (Brouzes and Farge, 2004; Chan et al, 2017; Mammoto and Ingber, 2010; Wozniak and Chen, 2009). In the src42A RNAi knockdown background, we found a specific increase in the signal for the un-phosphorylated site of b-cat at AJ under strain, as observed by the increase of Y654-bcat green labelling that adds to, and dominates the b-cat red labelling, leading to a bright green/yellow composite colour (Figure 4b) This signal was measured relative to the junctional b-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). To test whether the molecular strain that is observed in mesoderm invagination induces a decrease in b-cat affinity to E-cad, we characterised the dynamics of b-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). It statistically weakens the interaction of b-cat with E-cad in AJ of the mesoderm cells and thereby favours cytosolic b-cat enrichment
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
