Methods for mapping cell adhesion receptors

摘要 跨膜蛋白占人类基因的20%-30%，在偶联过程中起着至关重要的作用 在感知来自环境的机械和生物化学信号方面也是如此。然而，它是极端的 使用传统的生物化学方法绘制跨膜蛋白相互作用图具有挑战性。的第一个目标 这项建议是为了发展相互作用跨膜蛋白(诱饵)的结合分析，这是一项分子技术 发现细胞内新的跨膜蛋白相互作用。诱饵将分两步进行。首先，我们将 筛选位于目标蛋白近端的跨膜蛋白，通过双重标记 用邻近标记酶标记靶标的胞外和胞浆区域。接下来，我们将直接测试 单分子原子力作用下近端蛋白质与靶蛋白质的结合作用 显微镜(AFM)，并使用超分辨率可视化目标和结合伙伴的共同定位 荧光显微镜。我们预计，诱饵将在发现小说方面产生改变游戏规则的影响 跨膜连接蛋白在细胞表面的相互作用。 我们提案的第二个目标是使用诱饵以及其他生物物理工具来解决 桥粒的组装和组织，这是一种重要的细胞间黏附细胞器，介导 表皮和心脏等组织的完整性。而桥粒蛋白的突变在 遗传性心脏病和皮肤病理中，这些蛋白组装的分子机制 在质膜上是未知的。由于先前的研究表明桥粒的形成需要E- 钙粘附素(ECAD)，一种普遍存在的细胞间黏附蛋白，我们建立了一种以ECAD为模板的诱饵检测方法 并发现两种专有的桥粒黏附蛋白，桥粒粘附素(Desmocolin，DSC)和桥粒粘附素 (DSG)，与ECAD胞外区结合。利用生物物理实验和细胞结构功能研究 我们发现，ECAD招募DSG进行细胞间接触，并触发桥粒的形成。在目标2中， 我们将表征ECAD和DSC/DSG相互作用的结合界面和动力学，并确定 用单分子AFM结合分析、单分子荧光法测定其结合构象 共振能量转移和计算机模拟。我们还将引入突变体ECAD、DSC和DSG 利用超分辨荧光技术监测上皮细胞桥粒组装和超微结构 显微镜。这些研究将为这两种健康组织的桥粒完整性提供关键的分子洞察力 在疾病状态下。

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.