Illuminating Dynamic Receptor Clustering in the Epidermal Growth Factor Receptor

描述(申请人提供)：表皮生长因子(EGF)受体(EGFR)家族成员的失调与肿瘤的发生和生长有关。由于其与癌症发展的相关性，EGFR是癌症药物发现的重要靶点，已经开发了几种EGFR靶向治疗方法。然而，他们的临床成功往往不大，这加强了对EGFR信号通路更完整了解的必要性。这项建议特别关注大规模的EGFR同工(“簇”)在信号启动和转导中所起的作用还未被充分了解。虽然我很早就知道配体诱导的二聚化在受体信号转导中起着关键作用，但越来越多的证据表明，这个教科书模型需要扩充，以解释受体在质膜中的异质性侧向分布。“微域”或“纳米簇”中受体的局部富集会强烈地影响合作受体。 相互作用并通过局部集中效应改变局部EGFR关联平衡。用常规荧光显微镜对大规模受体组织的基本机制进行实验研究仍然具有挑战性，因为该方法在吞吐量、空间和时间分辨率以及最长观察时间方面存在局限性。等离子体耦合显微镜(PCM)是一种基于非荧光的新方法，它利用贵金属纳米颗粒(NPs)之间的电磁相互作用来研究亚衍射极限距离(但超过荧光共振能量转移(FRET)的空间势垒)上的受体聚集。NP不会眨眼或漂白，非常明亮，可以在传统的宽视场显微镜下成像。因此，PCM有助于同时监测多个单个细胞中的EGFR聚集，而不受观察时间的限制。这一竞争性更新建立在上一个资金周期开发的基于等离子激元耦合的工具的基础上，并概述了一项强有力的研究计划，以阐明动态EGFR集群的结构起源。然后将应用PCM来检验受体聚集调节信号的方式和强度的假设，并阐明信号强度和结果的空间调节的潜在机制。所获得的洞察力将提高对广泛受体的空间调节机制的理解。贵金属纳米粒子不仅是表征EGFR聚集在细胞膜上的优良光学标记，而且在与EGF共价结合后，它们也是恢复和增强负的EGFR信号的潜在治疗工具。这一假设在这一提议中得到了实验验证。如果成功，这一策略将为克服癌症中的细胞凋亡逃避提供一种新的方法。

DESCRIPTION (provided by applicant): Dysregulation of members of the epidermal growth factor (EGF) receptor (EGFR) family is associated with oncogenesis and tumor growth. Due to its relevance in cancer development, EGFR is an important target in cancer drug discovery, and several EGFR targeted therapies have already been developed. Their clinical success has, however, often been modest, reinforcing the need for a more complete understanding of the EGFR signaling pathway. This proposal focuses on the insufficiently understood role of large-scale EGFR associates ("clusters") in signaling initiation and transduction, in particular. While i has long been known that ligand induced dimerization plays a critical role in receptor signaling, there is growing evidence that this textbook model needs to be augmented to account for the heterogenous lateral distribution of the receptor in the plasma membrane. The local enrichment of the receptors in ''micro-domains" or ''nanoclusters'' could strongly affect cooperative receptor interactions and shift the local EGFR association equilibria through a local concentration effect. The experimental investigation of the fundamental mechanisms underlying the large-scale receptor organization with conventional fluorescence microscopy remains challenging, due to the method's limitation with regard to throughput, spatial and temporal resolution, and maximum observation time. Plasmon Coupling Microscopy (PCM) is a novel non-fluorescence based approach that uses electromagnetic interactions between noble metal nanoparticles (NPs) to investigate receptor clustering on subdiffraction limit distances (but beyond the spatial barrier o Fluorescence Resonance Energy Transfer, FRET). NP's do not blink or bleach, are very bright, and can be imaged in a conventional widefield microscope. Consequently, PCM facilitates the monitoring of EGFR clustering without limitations in observation time in many individual cells simultaneously. This competitive renewal builds upon the plasmon coupling based tools developed in the previous funding cycle and outlines a vigorous research plan to elucidate the structural origin of dynamic EGFR clustering. PCM will then be applied to test the hypothesis that receptor clustering regulates the mode and strength of signaling and to elucidate the mechanisms underlying a spatial regulation of signaling intensity and outcome. The obtained insight will improve the understanding of spatial regulation mechanisms for a broad range of receptors. Noble metal NPs are not only superb optical labels for characterizing EGFR clustering in the plasma membrane, but they also represent potential therapeutic tools to restore and enhance negative EGFR signaling after covalent attachment to EGF. This hypothesis is experimentally tested in this proposal. If successful, this strategy would provide a new approach for overcoming apoptosis evasion in cancer.