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

钙粘蛋白几乎在所有实体组织中表达，在广泛的生理和病理过程中发挥关键作用。这些依赖钙的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，进而影响细胞的运动、分化和癌变。当细胞-细胞接触形成时，在邻近细胞上表达的钙粘蛋白通过胞外结构域相互作用，而它们的细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。该应用的中心假设是，钙粘蛋白/钙粘蛋白在相邻细胞上的相互作用是由特定蛋白质（如1-catenin）与钙粘蛋白的细胞质结构域结合而调节的。这一假设在先前的结构数据中得到了支持，这些数据表明整合素-细胞基质粘附分子-在与细胞质蛋白结合后从低亲和力转变为高亲和力构象。我们处于一个独特的位置来验证这一假说，即通过活细胞中细胞质蛋白的结合来增强同型钙粘蛋白的亲和力，因为我们有两个关键的工具，这些工具是在我们的互补实验室中开发的：(1)从遗传性弥散性胃癌（HDGC）患者身上提取的携带人类e -钙粘蛋白突变体的细胞系。我们最近的研究结果表明，这些致病突变阻止特定蛋白质与其细胞质结构域的结合，并与减弱的整体细胞-细胞粘附相关。（II）全活细胞单分子力谱分析可以在单分子分辨率下探测两个相邻细胞之间的钙粘蛋白/钙粘蛋白相互作用。拟议的研究是高风险的，因为-虽然预测很长一段时间-在单个跨膜分子水平和活细胞中，细胞质蛋白介导的受体-配体亲和力增强的直接证明，特别是没有提出钙粘蛋白。通过实现本文提出的目标，我们将有助于填补对e -钙粘蛋白解除管制与癌症发生和进展之间机制的理解空白。

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.