Illuminating Dynamic Receptor Clustering in the Epidermal Growth Factor Receptor

项目摘要/摘要 照亮表皮生长因子受体信号中的动态受体聚集 利用等离子激元耦合的信号转导途径 表皮生长因子受体(EGFR)活性异常与肿瘤的形成和发展有关 恶性细胞的进展。对EGFR信号机制的分子理解提供了 发展有效的抗癌治疗策略的机会。表皮生长因子受体的激活及其机制 然而，受体间胞外结构域相互作用的中介作用仍然存在许多问题。运营形式 在EGFR信号转导途径中，不是单个的EGF受体，而是二聚体和潜在性更高的 订购齐聚物或甚至更大的功能单元，称为簇合物。瞬变相互作用的影响 具有动态短程和长程有序的星系团中的EGFR之间可能在 EGFR途径的调控。为了阐明受体细胞表面组织之间的联系， 受体动力学与信号的激活、进展、衰减和治疗干预 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进行监测 决议。这一距离范围位于传统荧光显微镜的“分辨率间隙”，即 由单边10 nm的空间荧光共振能量转移势垒和衍射定义 分辨率限制在可见光范围内，另一侧为~300 nm。这不是使用荧光显微镜，而是 该提案旨在通过等离子体偶联来揭开EGFR寡聚和聚集的动力学 在活细胞中实时标记的金纳米颗粒之间的EGFR。等离子体激元耦合的优势 显微镜下给出的是光物理稳定性的探针，贵金属纳米颗粒不眨眼或 漂白剂，它们的信号强度，以及靠近粒子的等离子体耦合的事实。这种等离子激元耦合 允许检测距离和距离变化，大大超出空间FRET障碍，并启用 荧光显微镜分辨率间隙中的距离测量。该项目的具体目标是： 1.开发一种新的分子尺子，可以实时监测活细胞上EGFR之间的距离 长度上的时间在1-100 nm之间。这项新技术将使我们能够探测到短期- 活细胞表面EGFR的长程有序排列。 2.从实验上验证了EGFR是以EGFR间距离组织成簇的假设 从纳米到几十纳米不等。监控大小和空间分布的变化 当加入EGF时，EGFR在活细胞中实时聚集。 3.监测集群内的EGFR间距离，并从实验上验证EGF 引起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.