Illuminating Dynamic Receptor Clustering in the Epidermal Growth Factor Receptor

描述(由申请人提供)：利用血浆偶联异常的表皮生长因子受体(EGFR)活性阐明表皮生长因子受体信号转导通路中的动态受体聚集与恶性肿瘤的形成和进展有关。对EGFR信号机制的分子理解为开发有效的抗癌治疗策略提供了机会。然而，EGFR的激活及其受体胞外结构域相互作用的介导仍然存在许多问题。EGFR信号转导通路中的作用形式不是单个的EGF受体，而是二聚体和潜在的更高阶低聚物或甚至更大的功能单位，称为簇。在具有动态短程和长程有序的团簇中，EGFR之间的瞬时相互作用的影响可能在EGFR途径的调节中发挥重要作用。为了阐明受体细胞表面组织、受体动力学与1-100 nm尺度上单个EGFR之间信号距离的激活、进展、衰减和治疗干预之间的关系，需要以高时间分辨率监测。这一距离范围位于常规荧光显微镜的“分辨率间隙”内，该“分辨率间隙”由空间荧光共振能量传递势垒10 nm和可见光范围~300 nm的衍射分辨极限来定义。这项应用不是使用荧光显微镜，而是旨在通过实时揭示活细胞中标记的金纳米颗粒之间的等离子体耦合来揭示EGFR寡聚和聚集的动力学。等离子体耦合显微镜的优势在于探针的光物理稳定性，贵金属纳米颗粒不会闪烁或漂白，它们的信号强度，以及等离子体子在粒子附近耦合的事实。这种等离子激元耦合使得能够在空间FRET屏障之外探测到距离和距离的显著变化，并且使得能够在荧光显微镜的分辨率间隙中进行距离测量。本项目的具体目标是：1.开发一种新的分子尺子，可以在1-100 nm的长度范围内实时监测活细胞上EGFR之间的距离。这项新技术将使我们能够探测活细胞表面EGFR的短程和长程有序。2.从实验上验证了EGFR是以纳米到几十纳米的距离组织成簇的假说。实时监测加入EGF后活细胞中EGFR簇的大小和空间分布的变化。3.监测集群内的EGFR间距离，并从实验上验证EGF导致EGFR间距离变化的假设。 公共卫生相关性：利用等离子体偶联阐明表皮生长因子受体信号转导通路中的动态受体聚集拟议的项目使用纳米技术来探索癌细胞中表皮生长因子(EGFR)异常行为的潜在机制。揭示EGFR信号的调控机制对公众健康具有重要意义。表皮生长因子(EGFR)在许多癌症中过度表达，是抗癌治疗的重要靶点。从分子水平上了解EGFR的激活机制将为癌症的早期诊断和改进现有的抗癌治疗策略提供新的机遇。

DESCRIPTION (provided by applicant): Illuminating Dynamic Receptor Clustering in the Epidermal Growth Factor Receptor Signal Transduction Pathway Using Plasmon Coupling Aberrant Epidermal Growth Factor Receptor (EGFR) activity has been linked to tumor formation and progression in malignant cells. A molecular understanding of the EGFR signaling mechanism offers opportunities for the development of efficient anti-cancer therapeutic strategies. The EGFR activation and its mediation by interreceptor ectodomain interactions, however, still pose many questions. The operational form in the EGFR signal transduction pathway is not the individual EGF receptor, but dimers and potentially higher order oligomers or even larger functional units, referred to as clusters. The influence of transient interactions between EGFRs in clusters with dynamic short- and long-range orders could play a prominent role in the regulation of the EGFR pathway. To elucidate the connections between receptor cell surface organization, receptor dynamics, and the activation, progression, attenuation and therapeutic intervention of signaling distances between individual EGFRs on the 1-100 nm length scale need to be monitored with high temporal resolution. This distance range lies in the "resolution gap" of conventional fluorescent microscopy which is defined by the spatial Fluorescence Resonance Energy Transfer barrier of 10 nm on one side and the diffraction resolution limit in the visible of ~300 nm on the other side. Instead of using a fluorescence microscopy this application aims to unravel the dynamics of EGFR oligomerization and clustering using plasmon coupling between gold nanoparticle labeled EGFRs in living cells in real time. The advantages of plasmon coupling microscopy are given by the photophysical stability of the probes, noble metal nanoparticles don't blink or bleach, their signal intensity, and the fact that plasmons in close by particles couple. This plasmon coupling enables to detect distances and distance changes significantly beyond the spatial FRET barrier and enables distance measurements in the resolution gap of fluorescence microscopy. The specific aims of this project are: 1. Develop a new molecular ruler that allows monitoring distances between EGFRs on living cells in real time on length scales between 1 - 100 nm. This new technology will enable us to probe both the short- and long-range order of EGFRs on the surface of living cells. 2. Experimentally verify the hypothesis that EGFRs are organized in clusters with inter-EGFR distances ranging from nanometers to tens of nanometers. Monitor changes in the size and spatial distribution of EGFR clusters in living cells in real time upon addition of EGF. 3. Monitor inter-EGFR distances within the clusters and experimentally verify the hypothesis that EGF induces changes in the inter-EGFR distances. PUBLIC HEALTH RELEVANCE: Illuminating Dynamic Receptor Clustering in the Epidermal Growth Factor Receptor Signal Transduction Pathway Using Plasmon Coupling The proposed project uses nanotechnology to probe the underlying mechanisms of the abnormal behavior of epidermal growth factors (EGFRs) in cancerous cells. Revealing the control mechanisms of EGFR signaling is of high relevance to public health. Epidermal growth factors (EGFRs) are overexpressed in many cancers and are prominent targets for anti-cancer therapies. A molecular understanding of the EGFR-activation mechanism will provide new opportunities for the early cancer diagnostics and for the improvement of current therapeutic anti-cancer strategies.