Identification of Mechanically Sensitive Proteins in Early Development

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DESCRIPTION (provided by applicant): Embryos undergo dramatic cell and tissue rearrangements that are required for morphogenesis and the sculpting of the various body plans encoded within the genomes of all animals. These underlying movements result in the generation of forces that are sensed both locally and globally by other cells and tissues in the embryo. Mechanisms of mechanotransduction are responsible for sensing these forces and converting them to chemical signals. Thus, physical force may serve to instruct and guide key aspects of early development. This hypothesis is supported by mounting evidence that mechanical stimuli influence gene expression, differentiation, cell adhesion and morphogenesis. Cell and tissue responses to mechanical forces are also underlying factors in varied pathologies that include hypertension and atherosclerosis, tumorigenesis and metastasis, bone degeneration, and deafness. Despite the importance of mechanotransduction to development, normal physiology and disease, the molecular mechanisms involved remain poorly understood. One of the key questions surrounding mechanotransduction concerns the mechanism(s) by which cells sense local stresses, which typically are transduced through adhesive specializations involving cadherins, integrins and the cytoskeleton. The primary hypothesis to be tested by these studies is that mechanosensation involves force-dependent changes in the conformations of proteins involved in cell adhesion and related signaling events. Approaches are needed that will not only test rigorously the overall hypothesis, but that will simultaneously make a significant impact on this important new field by identifying classes of proteins subject to conformational changes occurring under physiologically relevant conditions. We will apply cysteine shotgun mass spectrometry (CSMS) to identify conformational changes in proteins obtained from an embryonic tissue known to be mechanosensitive. The mesendoderm of Xenopus laevis gastrula stage embryos undergoes a collective form of cell migration that requires both cell-cell cohesion and engagement of a fibronectin (FN) substrate in order to move directionally. Recently, we discovered a novel mechanosensitive cadherin complex with links to the intermediate filament cytoskeleton that is required for directed motility of this tissue. There are two specific aims. In Aim 1, cells will be treated with fluorescent cys-reactive dyes to label thiol groups exposed following application of stress to cadherins or integrins. Labeled proteins will be separated, identified by changes in labeling intensity, subjected to tandem mass spectrometry and the sequence data analyzed using bioinformatics approaches. The second aim will focus on detecting protein conformational changes in intact mesendoderm. Patterned elastic substrates will also be developed that will enable simultaneous application of force to both cadherin and integrin adhesions in order to mimic tissue-level stresses in single cells. These studies will contribute a catalog of candidate, mechanosensitive proteins that will comprise an important resource for investigators interested in the role of force in morphogenesis, normal tissue biology and disease. PUBLIC HEALTH RELEVANCE: In recent years it has become increasingly apparent that cells and tissues generate and respond to physical forces in a number of ways that are important for embryo development, tissue regeneration, normal physiology and disease progression. The pulling and tugging forces that cells exert on one another and their extracellular environments can influence cell behavior and even profiles of gene expression, however, little is known about the mechanisms cells use to sense force and convert these stimuli to chemical signals within the cell. This project will use state-of-the-art approaches to discover structural changes in proteins following mechanical stimulation of cells and thus, will allow us to begin elucidating the cellular mechanisms involved in force sensation.