Illuminating Dynamic Receptor Clustering in the Epidermal Growth Factor Receptor

项目总结/摘要 阐明表皮生长因子受体信号中的动态受体聚集 利用等离子体耦合的转导途径 异常表皮生长因子受体（EGFR）活性与肿瘤形成有关， 恶性细胞的进展。对EGFR信号传导机制的分子理解提供了 为开发有效的抗癌治疗策略提供了机会。EGFR的激活及其 然而，通过受体间胞外域相互作用的介导仍然存在许多问题。操作形式 在EGFR信号转导通路中不是单个EGF受体，而是二聚体和潜在的更高的EGF受体。 有序低聚物或甚至更大的功能单元，称为簇。瞬态相互作用的影响 在具有动态短程和长程有序的簇中，EGFR之间的相互作用可能在 EGFR通路的调节。为了阐明受体细胞表面组织之间的联系， 受体动力学以及信号传导的激活、进展、减弱和治疗干预 在1-100 nm长度尺度上的单个EGFR之间的距离需要以高时间间隔监测 分辨率该距离范围位于常规荧光显微镜的“分辨率间隙”中， 由一侧10 nm的空间荧光共振能量转移势垒和衍射定义 另一侧的可见光分辨率极限为~300 nm。而不是使用荧光显微镜， 一项提案旨在利用等离子体偶联来阐明EGFR寡聚化和聚集的动力学 金纳米颗粒标记的EGFR在活细胞中的真实的时间。等离子体激元耦合的优点 由于探针的物理稳定性，贵金属纳米颗粒不会闪烁， 漂白，它们的信号强度，以及等离子体激元与粒子耦合的事实。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， 在荧光显微镜的分辨率差距的距离测量。该项目的具体目标是： 1.开发一种新的分子尺子，可以真实的监测活细胞上EGFR之间的距离 长度上的时间尺度在1 - 100 nm之间。这项新技术将使我们能够探测短- 以及活细胞表面EGFR的长程有序性。 2.通过实验验证EGFR以EGFR间距离成簇组织的假设 范围从纳米到几十纳米。监测的大小和空间分布的变化， 加入EGF后，EGFR在活细胞中以真实的时间聚集。 3.监测簇内的EGFR间距离，并通过实验验证EGF 诱导EGFR间距离的变化。

PROJECT SUMMARY/ABSTRACT 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 proposal 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.