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

描述（由申请人提供）：概述钙粘蛋白在几乎所有实体组织中表达，并在广泛的生理和病理过程中发挥关键作用。这些钙依赖性跨膜分子聚集在细胞-细胞接触的位点，在那里它们介导细胞粘附和信号传导，随后影响运动、分化和致癌。当形成细胞与细胞接触时，邻近细胞上表达的钙粘蛋白通过其细胞外结构域相互作用，而其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。本申请的中心假设是相邻细胞上的钙粘蛋白/钙粘蛋白相互作用通过特定蛋白质（例如1-连环蛋白）与钙粘蛋白的胞质结构域的结合来调节。这一假设在以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-在细胞质蛋白结合后从低亲和力转换为高亲和力构象。我们处于一个独特的位置，以测试这一假说的增强同型钙粘蛋白的亲和力介导的细胞质蛋白结合在活细胞中，因为我们有2个关键的工具，在我们的互补实验室开发：（I）细胞系，窝藏人类E-钙粘蛋白突变体来自患者遗传性弥漫性胃癌（HDGC）。我们最近的研究结果表明，这些致病突变阻止特定蛋白质结合到其胞质结构域，并与全球细胞-细胞粘附减弱和（II）全活细胞单分子力谱分析，可以探测钙粘蛋白/钙粘蛋白在两个相邻细胞之间的相互作用在单分子分辨率。拟议的研究是高风险的，因为-虽然预测了很长一段时间-在单个跨膜分子水平和活细胞中由细胞质蛋白介导的增强的受体-配体亲和力的直接证明以前没有显示，特别是，还没有提出钙粘蛋白。通过实现本文提出的目标，我们将有助于填补差距，了解E-钙粘蛋白失调与癌症发生和发展的机制。 公共卫生相关性：E-钙粘蛋白对维持组织结构至关重要，其缺失与癌症进展期间的侵袭和转移相关。对E-cadherin种系突变携带者的早期浸润性癌症的研究表明，其失调也是肿瘤发生的起始事件。当肿瘤细胞能够通过上皮基底膜侵入时，与上皮癌相关的死亡率显著增加。E-钙粘蛋白丢失在此过程中起着关键作用，被认为是癌进展和转移的临床转折点。正因为如此，旨在阐明E-钙粘蛋白功能和调节的研究已成为肿瘤生物学研究的关键相关性。实验证据支持这样的观点，即细胞粘附的失调是促进细胞侵袭的必要条件，因此通过解开控制E-钙粘蛋白依赖性细胞粘附的分子机制，可以鉴定侵袭性癌症的新分子靶点。

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.