Single-molecule analysis of cadherin-mediated cell-cell adhesion

钙粘蛋白介导的细胞间粘附的单分子分析

基本信息

批准号：
7772321
负责人：
Denis Wirtz
金额：
$ 15.08万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-03-01 至 2012-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7772321
关键词：
Actin-Binding Protein Actinin Actins Adhesions Adhesives Affect Affinity Architecture Binding Binding Proteins Biological Assay Biology Biophysics Cadherins Calcium Cancer Biology Cancer Etiology Carcinoma Cell Adhesion Cell Adhesion Molecules Cell Line Cell-Cell Adhesion Cell-Matrix Junction Cells Chemicals Chinese Hamster Ovary Cell Clinical Complex Cytoplasmic Protein Cytoplasmic Tail Cytoskeleton Data Diffuse Dissociation E-Cadherin Engineering Epithelial Epithelial Cells Event Extracellular Domain Family Family Dasypodidae Figs - dietary Funding Germ-Line Mutation Goals Human Individual Inherited Integrins Interdisciplinary Study Invaded Laboratories Life Ligands Link Maintenance Malignant Neoplasms Measures Mediating Membrane Missense Mutation Molecular Molecular Conformation Molecular Target Mutation Neoplasm Metastasis Oranges Pathologic Processes Patients Physiological Play Positioning Attribute Probability Process Proteins Public Health Regulation Research Resolution Role Science Signal Transduction Site Solid Spectrum Analysis Surface Technology Tensile Strength Testing Time Tissues Vinculin cancer initiation carcinogenesis cell motility cellular engineering cellular imaging disease-causing mutation extracellular high risk insight malignant stomach neoplasm molecular oncology mortality mutant nanoscale neoplastic cell prevent public health relevance receptor single bond single molecule tool tumor progression tumorigenesis

项目摘要

DESCRIPTION (provided by applicant): Summary Cadherins are expressed in virtually all solid tissues and play a key role in a wide range of physiological and pathological processes. These calcium-dependent transmembrane molecules cluster at sites of cell-cell contacts where they mediate cell adhesion and signaling, which subsequently influence motility, differentiation, and carcinogenesis. When a cell-cell contact is formed, cadherins expressed on neighboring cells interact through their extracellular domain while their cytoplasmic domain interacts with the cytoskeleton through the catenin family of armadillo cadherin- binding proteins. The central hypothesis of this application is that cadherin/cadherin interactions on adjoining cells are regulated by the binding of specific proteins, such as 1-catenin, to the cytoplasmic domain of cadherins. This hypothesis finds support in previous structural data suggesting that integrins - cell-matrix adhesion molecules - switch from a low-affinity to a high-affinity conformation following binding of cytoplasmic proteins. We are in a unique position to test this hypothesis of enhanced homotypic cadherin affinity mediated by binding of cytoplasmic proteins in living cells because we have available 2 key tools developed in our complementary laboratories: (I) cell lines harboring human E-cadherin mutants derived from patients with hereditary diffuse gastric cancer (HDGC). Our recent results show that these disease-causing mutations prevent binding of specific proteins to their cytoplasmic domain and correlate with weakened global cell-cell adhesion and (II) an all-live cell single-molecule force spectroscopy assay that can probe cadherin/cadherin interactions between two adjoining cells at single-molecule resolution. The proposed research is high-risk because - while predicted for a long time - a direct demonstration of enhanced receptor-ligand affinity mediated by cytoplasmic proteins at the level of a single transmembrane molecule and in live cells has not been shown previously, and in particular, has not been presented for cadherins. By achieving the goals here proposed, we will contribute to fill the gap toward the understating of the mechanisms linking E-cadherin deregulation to cancer initiation and progression. PUBLIC HEALTH RELEVANCE: E-cadherin is critical for the maintenance of tissue architecture and its loss is associated with invasion and metastasis during cancer progression. The study of early invasive cancers in carriers of E-cadherin germline mutations demonstrates that its deregulation is also an initiating event in tumorigenesis. The mortality rate associated to epithelial cancers strongly increases when tumor cells are able to invade through the epithelial basal membrane. E-cadherin loss plays a pivotal role in this process and is considered a clinical turning point in carcinoma progression and metastization. Because of that, studies aimed at elucidating E-cadherin function and regulation have become of critical relevance in oncobiology research. Experimental evidence supports the idea that deregulation of cell adhesion is a necessary condition to promote cell invasion, so that by unraveling the molecular mechanisms that govern E-cadherin-dependent cell adhesion new molecular targets for invasive cancers could be identified.

描述（由申请人提供）：总结钙粘蛋白几乎在所有实体组织中表达，并且在广泛的生理和病理过程中发挥关键作用。这些钙依赖性跨膜分子聚集在细胞与细胞接触的部位，介导细胞粘附和信号传导，从而影响运动、分化和癌变。当形成细胞-细胞接触时，邻近细胞上表达的钙粘蛋白通过其胞外结构域相互作用，而其胞质结构域通过犰狳钙粘蛋白结合蛋白的连环蛋白家族与细胞骨架相互作用。本申请的中心假设是，相邻细胞上的钙粘蛋白/钙粘蛋白相互作用是通过特定蛋白质（例如 1-连环蛋白）与钙粘蛋白胞质结构域的结合来调节的。这一假设在之前的结构数据中得到了支持，表明整合素（细胞基质粘附分子）在与细胞质蛋白结合后从低亲和力构象转变为高亲和力构象。我们处于独特的地位来测试活细胞中细胞质蛋白结合介导的同型钙粘蛋白亲和力增强的假设，因为我们有互补实验室开发的 2 个关键工具：(I) 含有源自以下细胞的人 E-钙粘蛋白突变体的细胞系：遗传性弥漫性胃癌（HDGC）患者。我们最近的结果表明，这些致病突变阻止特定蛋白质与其细胞质结构域的结合，并与整体细胞间粘附力减弱相关，以及（II）可探测钙粘蛋白/钙粘蛋白相互作用的全活细胞单分子力谱测定以单分子分辨率观察两个相邻细胞之间的情况。拟议的研究具有高风险，因为虽然预测了很长时间，但以前尚未直接证明在单个跨膜分子水平和活细胞中由细胞质蛋白介导的受体-配体亲和力增强。，尚未针对钙粘蛋白提出。通过实现此处提出的目标，我们将有助于填补理解 E-钙粘蛋白失调与癌症发生和进展之间联系机制的空白。公共健康相关性：E-钙粘蛋白对于维持组织结构至关重要，其丢失与癌症进展过程中的侵袭和转移有关。对 E-钙粘蛋白种系突变携带者的早期侵袭性癌症的研究表明，其失调也是肿瘤发生的起始事件。当肿瘤细胞能够侵入上皮基底膜时，与上皮癌相关的死亡率会急剧增加。 E-钙粘蛋白损失在此过程中起着关键作用，被认为是癌症进展和转移的临床转折点。因此，旨在阐明 E-钙粘蛋白功能和调节的研究在肿瘤生物学研究中变得至关重要。实验证据支持细胞粘附失调是促进细胞侵袭的必要条件的观点，因此通过揭示控制E-钙粘蛋白依赖性细胞粘附的分子机制，可以确定侵袭性癌症的新分子靶标。