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

• 批准号: 7588535
• 负责人: Denis Wirtz
• 金额: $ 20.65万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-03-01 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7588535
• 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

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)：钙粘附素在几乎所有固体组织中都有表达，并在广泛的生理和病理过程中发挥关键作用。这些钙依赖的跨膜分子聚集在细胞与细胞接触的位置，在那里它们介导细胞黏附和信号，从而影响运动、分化和癌症的发生。当细胞-细胞接触形成时，邻近细胞上表达的钙粘附素通过它们的胞外结构域相互作用，而它们的胞浆结构域通过联结蛋白家族与细胞骨架相互作用。这一应用的中心假设是，相邻细胞上的钙粘蛋白/钙粘蛋白相互作用受特定蛋白的调节，如1-连环蛋白与钙粘蛋白的细胞质结构域的结合。这一假说在以前的结构数据中得到了支持，即整合素-细胞-基质黏附分子-在细胞质蛋白质结合后从低亲和力构象转换到高亲和力构象。我们处于独特的位置来测试这一假设，即通过活细胞中细胞质蛋白的结合来增强同型钙粘素亲和力，因为我们在互补的实验室中开发了两个关键工具：(I)携带来自遗传性弥漫性胃癌(HDGC)患者的人E-钙粘素突变的细胞系。我们最近的结果表明，这些致病突变阻止了特定蛋白质与其细胞质结构域的结合，并与全球细胞-细胞黏附减弱有关，以及(Ii)全活细胞单分子力谱分析，可以单分子分辨率探测相邻两个细胞之间的钙粘蛋白/钙粘素相互作用。这项拟议的研究是高风险的，因为虽然预测了很长一段时间，但以前还没有在单个跨膜分子水平和活细胞中直接证明细胞质蛋白介导的受体-配体亲和力增强，尤其是钙粘附素。通过实现这里提出的目标，我们将有助于填补对E-钙粘素去调控与癌症发生和进展联系机制的低估的空白。 公共卫生相关性：E-钙粘附素对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-钙粘蛋白胚系突变携带者的早期浸润性癌症的研究表明，E-钙粘蛋白胚系突变的解除也是肿瘤发生的始动事件。当肿瘤细胞能够通过上皮基底膜侵袭时，与上皮性癌症相关的死亡率显著增加。E-钙粘附素丢失在这一过程中起着关键作用，被认为是肿瘤进展和转移的临床转折点。正因为如此，旨在阐明E-钙粘蛋白功能和调控的研究在肿瘤生物学研究中具有重要意义。实验证据支持这一观点，即放松对细胞黏附的调控是促进细胞侵袭的必要条件，因此，通过解开控制E-钙粘素依赖的细胞黏附的分子机制，可以识别侵袭性癌症的新分子靶点。