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Methods for mapping cell adhesion receptors

绘制细胞粘附受体图谱的方法

基本信息

批准号：
10685306
负责人：
Sanjeevi Sivasankar
金额：
$ 33.25万
依托单位：
UNIVERSITY OF CALIFORNIA AT DAVIS
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-02-01 至 2025-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10685306
关键词：
Adhesions Adhesives Atomic Force Microscopy Benchmarking Binding Biochemical Biological Assay Biophysics Cadherins Cell Adhesion Cell Adhesion Molecules Cell Extracts Cell membrane Cell surface Cell-Cell Adhesion Cells Cellular Structures Chemicals Computer Simulation Coupling Cytoplasm Data Desmosomes Disease E-Cadherin Environment Enzymes Epidermis Epithelial Cells Exposure to Fluorescence Microscopy Funding Genes Goals Heart Heart Diseases Human In Situ Inherited Integral Membrane Protein Integrins Kinetics Label Ligands Maps Measurement Measures Mechanical Stress Mechanics Mediating Membrane Membrane Proteins Methods Molecular Molecular Conformation Monitor Mutation Organelles Pathology Play Positioning Attribute Proteins Receptor Protein-Tyrosine Kinases Role Signal Transduction Skin Structure Technology Testing Tissues Visualization adhesion receptor biophysical techniques biophysical tools cell type desmocollin experimental study extracellular insight instrumentation mutant new technology novel prototype recruit single molecule single-molecule FRET tool ultra high resolution

项目摘要

ABSTRACT Transmembrane proteins, which constitute 20%-30% of human genes, play essential roles in coupling cells and in sensing mechanical and biochemical signals from the environment. However, it is extremely challenging to map transmembrane protein interactions using traditional biochemical methods. The first goal of this proposal is to develop Binding Assay for Interacting Transmembrane proteins (BAIT), a molecular technology to discover novel transmembrane protein interactions in cells. BAIT will be performed in two steps. First, we will screen for transmembrane proteins that are located proximal to a target protein, by dual tagging both the extracellular and cytoplasmic region of the target with a proximity labeling enzyme. Next, we will directly test binding interactions between proximal proteins and the target protein using single molecule Atomic Force Microscopy (AFM) and also visualize co-localization of the target and binding partner using super-resolution fluorescence microscopy. We anticipate that BAIT will have a game changing impact in discovering novel transmembrane junctional proteins interactions on the cell surface. The second goal of our proposal is to use BAIT, along with other biophysical tools, to resolve the assembly and organization of desmosomes, an essential intercellular adhesive organelle that mediates the integrity of tissues like the epidermis and heart. While mutations in desmosomal proteins are common in hereditary heart diseases and in skin pathologies, the molecular mechanisms by which these proteins assemble at the plasma membrane are unknown. Since previous studies show that desmosome formation requires E- cadherin (Ecad), a ubiquitous cell-cell adhesion protein, we developed a prototype BAIT assay using Ecad as the target and discovered that two obligate desmosomal adhesive proteins, Desmocollin (Dsc) and Desmoglien (Dsg), bind to Ecad extracellular regions. Using biophysical experiments and cellular structure function studies we showed that Ecad recruits Dsg to intercellular contacts, and triggers desmosome formation. In Aim 2 of the proposal, we will characterize binding interfaces and kinetics of Ecad and Dsc/Dsg interactions and determine their binding conformations using single molecule AFM binding assays, single molecule Fluorescence Resonance Energy Transfer and computer simulations. We will also introduce mutant Ecad, Dsc and Dsg in epithelial cells and monitor desmosome assembly and ultrastructure using super-resolution fluorescence microscopy. These studies will provide key molecular insights into desmosomal integrity in both healthy tissues and in disease states.

摘要