Illuminating Dynamic Receptor Clustering in the Epidermal Growth Factor Receptor

描述（由申请人提供）：利用等离子体偶联在表皮生长因子受体信号转导通路中阐明动态受体聚集异常的表皮生长因子受体（EGFR）活性与恶性细胞的肿瘤形成和进展有关。对EGFR信号传导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的激活及其通过受体间外结构域相互作用介导仍然存在许多问题。EGFR信号转导途径中的操作形式不是单个的EGF受体，而是二聚体和潜在的高阶低聚物甚至更大的功能单位，称为簇。具有动态短、长序列的EGFR簇之间的瞬时相互作用可能在EGFR通路的调控中发挥重要作用。为了阐明受体细胞表面组织、受体动力学与单个egfr之间在1-100 nm长度范围内信号距离的激活、进展、衰减和治疗干预之间的联系，需要进行高时间分辨率的监测。这一距离范围属于常规荧光显微镜的“分辨间隙”，即一边是10 nm的空间荧光共振能量转移势垒，另一边是~300 nm的可见光衍射分辨极限。与使用荧光显微镜不同，本应用程序旨在利用金纳米颗粒标记的EGFR之间的等离子体耦合实时揭示活细胞中EGFR寡聚化和聚集的动力学。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褪色、它们的信号强度以及靠近粒子的等离子体耦合这一事实。这种等离子体耦合能够检测距离和距离变化显著超出空间FRET屏障，并使荧光显微镜的分辨率差距的距离测量。本项目的具体目标是：1。开发一种新的分子标尺，可以在1 - 100纳米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的短期和长期序列。2. 实验验证了egfr之间以纳米到几十纳米的距离组织成簇的假设。实时监测添加EGF后活细胞中EGFR簇大小和空间分布的变化。3. 监测簇内egfr间的距离，并通过实验验证EGF诱导egfr间距离变化的假设。

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.