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：
10297678
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/10297678
关键词：
Adhesions Adhesives Atomic Force Microscopy Benchmarking Binding Biochemical Biological Assay Biophysics Cell Adhesion Cell Adhesion Molecules Cell Extracts Cell membrane Cell surface Cell-Cell Adhesion Cells Cellular Structures Chemicals Computer Simulation Coupling Data Desmosomes Disease E-Cadherin Environment Enzymes Epidermis Epithelial Cells Exposure to Fluorescence Microscopy Foundations Funding Genes Goals Heart Heart Diseases Human In Situ Inherited Integral Membrane Protein Integrins Kinetics Label Ligands Longitudinal Studies Maps Measurement Measures Mechanical Stress Mechanics Mediating Membrane Membrane Proteins Methods Molecular Molecular Conformation Monitor Mutation Organelles Pathology Play Positioning Attribute Protein Tyrosine Kinase Proteins Resolution Role Signal Transduction Skin Structure Technology Testing Tissues adhesion receptor base biophysical techniques biophysical tools cell type desmocollin experimental study extracellular insight instrumentation mutant new technology novel prototype receptor recruit single molecule single-molecule FRET tool

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.

抽象的构成人类基因的20％-30％的跨膜蛋白在耦合中起着重要作用细胞以及来自环境的机械和生化信号。但是，这是非常使用传统的生化方法来绘制跨膜蛋白相互作用的挑战。第一个目标该建议是开发与分子技术相互作用的跨膜蛋白（诱饵）相互作用的结合测定发现细胞中新型的跨膜蛋白相互作用。诱饵将分两个步骤进行。首先，我们会的通过双重标记，筛选位于靶蛋白近端的跨膜蛋白靶标的细胞外和细胞质区域，带有邻近标记酶。接下来，我们将直接测试近端蛋白与靶蛋白之间使用单分子原子力之间的结合相互作用显微镜（AFM），还可以使用超分辨率可视化目标和结合伙伴的共定位荧光显微镜。我们预计诱饵会在发现小说时会改变游戏的影响跨膜连接蛋白在细胞表面上的相互作用。我们建议的第二个目标是将诱饵与其他生物物理工具一起解决脱糖体的组装和组织，这是一种必不可少的细胞间粘合剂细胞器，可介导表皮和心脏等组织的完整性。虽然脱乳小蛋白中的突变在遗传性心脏病和皮肤病理中，这些蛋白质组装的分子机制在质膜上是未知的。由于先前的研究表明，向染色体的形成需要E- 钙粘蛋白（ECAD）是一种无处不在的细胞 - 细胞粘附蛋白，我们使用ECAD AS开发了一种原型诱饵测定法目标并发现两种强制性脱蓝色粘合剂蛋白，Desmocollin（DSC）和Desmoglien （DSG），与ECAD细胞外区域结合。使用生物物理实验和细胞结构功能研究我们表明ECAD将DSG招募到细胞间接触，并触发脱骨体形成。在目标2中提案，我们将表征ECAD和DSC/DSG相互作用的结合界面和动力学它们使用单分子AFM结合测定的结合构象，单分子荧光共振能量传输和计算机模拟。我们还将在IN中引入突变ECAD，DSC和DSG 上皮细胞并使用超分辨率荧光监测脱骨体组装和超微结构显微镜。这些研究将为两个健康组织中的脱斑小体完整性提供关键的分子见解和疾病状态。