id="title" class="nomar"Assembly, Dynamics and Evolution of Cell-Cell and Cell-Matrix Adhesions
This biological project consists of investigators from the Sanford-Burnham Medical Research Institute, Stanford University, the University of California at San Diego, and the University of North Carolina. It is partnered with the PSI High-Throughtput Structure Determination center NYSGRC.
W. James Nelson
Cell adhesion complexes are a bio-medically important class of multi-protein assemblies. They are involved in sensing interactions between cells and their external environment, and then initiating and regulating intracellular signals that control cell-cell adhesion and cell migration, cell shape and functional organization, proliferation and survival, and gene expression. They are evolutionarily old, critical for normal development and homeostasis, and are defective in genetic and acquired diseases of all organ systems.
Cell adhesion is mediated by specific plasma membrane receptors that link other cells (e.g., cadherins, Igsuperfamily proteins) or the extracellular matrix (integrins) to a hierarchical assembly of cytoplasmic adaptor, signaling and cytoskeleton complexes. A parts-list of mostly binary interactions between proteins in these complexes is accumulating, and some structures of individual proteins and domains have been solved. However, there is a lack of a concerted effort to integrate available genomic (evolutionary) and structural information to rigorously solve the hierarchical structural organization of these multi-protein complexes at the atomic, meso and macro scale. The composition and sub-cellular locations of cell adhesions are highly dynamic and integral to signaling, but a dearth of structural information prevents the development of tools to explore adhesion dynamics in situ. A further barrier to progress is the availability of purified proteins for multiprotein complex reconstitution experiments, since classical approaches are not feasible due to the unstable nature of complex protein assemblies.
The Consortium will leverage high-throughput expression and structures of large sets of target families of proteins and signaling networks to understand the structural and functional organization of cell-cell and cell-ECM adhesion complexes. The Consortium will integrate expertise in structural biology (biophysical and structural techniques, cryo-EM/tomography of complexes in situ), biochemistry and cell biology (in vitro protein complex reconstitution), chemistry and live-cell imaging (bio-sensors and caged proteins). The Consortium goals are to provide structure/function maps for multi-protein complexes linking cell adhesion membrane receptors to signaling networks and the cytoskeleton, and thereby understand how diversity in functional output is built into different combinations of structurally similar proteins in cell-cell and cell-ECM adhesion complexes.
Specific Aim 1: Define multi-protein-protein interactions, stoichiometries and affinities in solution. Collaborate with PSI/HT Centers to optimize crystallization “hit-rate” from adhesion proteins and complexes. Reconstitute multi-protein assembly in solution; define protein stoichiometries and affinities; provide direct information on domain organization of proteins within complexes, and investigate role of phosphorylation on complex interactions.
Specific Aim 2: Reconstitute multi-protein complexes on biological membranes. Reconstitute multiprotein assembly on different cell adhesion complexes bound to biological membranes, phospholipid bilayers, and model protein mimics of the cytoplasmic face and define conditions for assembly and organization of the cytoskeleton (actin, microtubule and intermediate filaments).
Specific Aim 3: Define structures of multi-protein complexes in situ in a physiological environment. Provide three-dimensional maps of cell adhesion complexes in a cellular context to establish a realistic structural platform for defining native protein-protein interaction using atomic level structures for domains, proteins and protein complexes.
Specific Aim 4: Analyze dynamic protein-protein interactions and assembly in live cells. Develop probes, caged proteins and image analysis methods for direct analysis of protein activity and interactions in living cells to place in vitro structural and reconstitution results in a dynamic, real-time context.
This is a critical area of human health. Cell adhesion complexes are important in all aspects of normal cell and organ function, and are commonly defective in genetic and acquired diseases of all organ systems. Results will provide new understanding of how defects disrupt normal function and contribute to disease, and the identification of new therapeutic targets.