Dynamic molecular interactions among cell adhesion proteins, the extracellular matrix (ECM) and the cytoskeleton give rise to tissue-level mechanics, but delineating the mechanism of emergence of spatiotemporally regulated tissue mechanics remains a challenge. This proposal addresses this problem utilizing an integrated cross-scale approach that quantifies molecular scale protein movement and protein-protein association as well as tissue mechanics using cell tracking and quantitative metrics from fluid mechanics. Specifically, the assembly of the paraxial mesoderm from motile mesodermal progenitors is examined. The proposal seeks to elucidate a regulatory mechanism in which Fibronectin fibrillogenesis is confined to the surface of the paraxial mesoderm despite the presence of Fibronectin and its primary Integrin receptors throughout the tissue. Fluorescence cross correlation spectroscopy (FCCS) in live embryos revealed a protein complex containing Integrin ?5 and Cadherin 2 and genetic experiments indicate that this complex represses Fibronectin matrix assembly within the mesenchyme of the paraxial mesoderm. On the surface of the paraxial mesoderm, Integrin ?5 is de-repressed and Fibronectin matrix coats the tissue surface.
Aim 1 is to define the role of Integrin ?V, the other main Fibronectin receptor, in this process using FCCS and genetic mosaics. In addition, fluorescent timer fusion proteins are used to reveal the patterns of adhesion protein turnover in vivo, and a FRET/FLIM assay is used to examine Integrin conformation in live embryos.
Aim 2 is to identify other components of this protein complex using co-IP and Mass Spectroscopy. These proteins will be further characterized by in vivo FCCS, gene knockout and systems analysis of cell motion in the mutants.
This project seeks to understand the in vivo biophysics of cell-cell and cell-ECM adhesion using FCS and FCCS. These molecular level analyses are complemented by systems analysis of tissue mechanics to provide a cross-scale study of the biomechanics of the assembly of the paraxial mesoderm.
