Final Report Summary - BCATENIN_MECHANICS (Mechanical activation of beta-catenin signalling) While mechanical forces and physical properties of cells have been extensively studied in in vitro culture systems only recently, we begin to understand the mechanical forces that shape the embryo. On the other hand, scientific studies of gene activation have focused so far on chemical signals and little is known about the involvement of mechanical stimuli in gene activation during development. Especially, the link between the molecular mechanisms sensing mechanical force and the underlying signalling cascade in an in vivo system is an open and burning question. This project specifically addresses the question of how the mechanical force stimulus is primarily sensed and leads to the activation of a gene expression cascade. The project presents therefore an innovative and modern approach to the understanding of mechanical activation of gene transcription during animal development. During embryonic morphogenesis, cells do not only generate forces necessary for shape changes and tissue movement but also sense and respond to the forces exerted on them by neighbouring cells and tissues. Recent studies demonstrate that these forces can act as mechanical cues during development regulating cytoskeletal remodelling, cell proliferation and gene expression. However, in most cases the molecular mechanism by which cells sense and transduce a mechanical signal into a biochemical signal remains to be discovered. This is specifically the case in vivo, for which no primary molecular mechanism of mechano-biochemical transduction was found to our knowledge so far. Therefore, this project consists in finding the underlying molecular mechanism of mechanotransduction in vivo, in the initiation of the mechanical activation of the beta-catenin (β-cat) pathway, which is involved in the mechanical induction of bilaterian mesoderm differentiation at gastrulation.An important innovativeness of this project lies in its combination of state-of-the-art live imaging techniques, powerful genetic tools of Drosophila and innovative molecular biochemical assays. Using state-of-the-art live imaging techniques we found that upon strain, which builds up in the mesodermal tissue during drosophila gastrulation, the release of β-cat from adherens junctions is facilitated indicating that the interaction of β-cat with its binding partner is weakened. Indeed, we found also that upon strain increase a particular phosphorylation site in the β-cat molecule becomes more available for phosphorylation. Phosphorylation at this site had been shown before to lead to the un-reversible inhibition of the intermolecular interaction. Therefore, we conclude that the site of β-cat opens and becomes more available for phosphorylation at the adherens junctions as the strain builds up during development indicating that β-cat is the primary mechanosensor. Experiments to assess directly the opening of the β-cat phosphorylation site upon strain in vivo are currently performed and preliminary data point in the same direction. This mechanotransductive process is probably generically involved in many mechanotransduction processes, as we found the mechanosensitive site of β-cat conserved all across the multi-cellular metazoan kingdom by comparing the molecular sequence of the β-cat protein of different species. Indeed, the activation of the β-cat pathway in response to mechanical strain has been also observed in other processes such as bone mass formation and homeostasis in mice in response to shocks due to muscle oscillations as well as in the inhibition of adipogenesis. They are also important initiators of a now well-characterized functional process of mechanically induced differentiation, that has been suggested to be at the evolutionary origin of bilaterians mesoderm emergence as well as of a process of mechanical induction of tumourogenesis in response to tumour growth pressure exerted on the weeks time scales in mice, in vivo. Therefore, the final results will not only lead to a better understanding of the mechano-activation of β-cat signalling during Drosophila gastrulation and the interplay of biochemical signals and mechanical forces during embryonic development but could have implications in therapeutic approaches. Ultimately, the information gained about the mechanosensitive element will be used to design a fluorescent probe to directly monitor the mechano-activation in response to the morphogenetic movements during Drosophila gastrulation. To our knowledge, this would be the first time in vivo to visualise directly the activation of a primary mechanosensor with a fully identified downstream physiological function of transcription and major differentiation events. These findings will also have potential impact in cancer progression, as the mechanical activation of β-cat by tumor growth pressure in healthy tissues has been very recently demonstrated to be tumorigenic.
