Illuminating Dynamic Receptor Clustering in the Epidermal Growth Factor Receptor Signal Transduction Pathway Using Plasmon Coupling

摘要 表皮生长因子受体(EGFR)是一种模型酪氨酸激酶，其过度表达在 各种癌症，包括基底细胞样乳腺癌(BLBC)。越来越清楚的是，EGFR是 不仅由配体-受体结合诱导的常规生化调节，而且也远不是很好 了解空间和时间调节机制。受体的异质性分布在 质膜导致膜区域内EGFR的丰富，其侧向尺寸为数十到 数百纳米，由于高局域性，受体二聚和寡聚被看好 受体的浓度。这些星系团不是静态的，而是经历了持续的结构波动。这个 这种动态结构和局部信号激活之间的关系在一定程度上还没有被充分了解 由于缺乏适当的光学工具来绘制具有高时间间隔的亚衍射极限动力学 带宽大，观测时间长。这个应用程序的目标1利用了独特的光物理 等离子体纳米粒子(NPs)的性质，提供大而稳定的光信号，并还编码 关于NPs远场光谱中深亚衍射极限分离的信息以表征 高时间分辨率和非时间分辨率单个EGFR团簇的横向扩散和结构动力学 观察时间的物理限制。利用等离子体激元耦合显微镜(PCM)的先前实现 在暗场照明下检测到的NP光谱来识别EGFR聚集性。暗场探测需要， 然而，大(~40 nm)的NP标记。在目标1中，我们将实现一种新的干涉PCM(IPCM)来检测 5 nm(用于探测EGFR-EGFR接触)和10 nm(用于探测EGFR寡聚和聚集)金NP标记。 IPCM将增加相关分析，以量化等离子体激元耦合中的连续波动。这 将应用技术来检验EGF结合导致簇内动力学降低的假设 EGFR磷酸化增加。另一个对结构性监管认识不足的因素是 与EGFR聚集相关的是通过受体间接触或EGF诱导的横向信号传播 第二次信使释放。目标2将阐明EGFR聚集性如何影响NP-EGF诱导的活性氧 物种(ROS)的形成和ROS介导的EGFR激活。在这种方法中，EGF功能化的NP(NP- 具有已知EGF负载的EGF)不是用于绘制EGFR的亚衍射极限簇的简单成像工具， 而是可量化地代表局部EGFR激活的单位。在AIM 3中，将应用纳米连接的EGF 作为定量EGFR激活和一氧化氮(NO)形成之间的串扰的探针。因为没有合成 在空间和时间上受到严格调控，其浓度取决于EGFR的激活，EGFR的激活随时间的变化而变化 局部NP-EGF浓度的函数关系。目标3将检验NP-EGF诱导NO生成的假设 是c-jun氨基末端激酶(JNK)介导的BLBC细胞凋亡的调节因子。

SUMMARY The epidermal growth factor receptor (EGFR) is a model tyrosine kinase whose overexpression is common in various cancers, including basal-like breast cancer (BLBC). It is becoming increasingly clear that EGFR underlies not only conventional biochemical regulation induced by ligand-receptor binding but also much less well understood spatial and temporal regulation mechanisms. A heterogeneous distribution of the receptor at the plasma membrane results in an enrichment of EGFR in membrane regions with lateral dimensions of tens to hundreds of nanometers where receptor dimerization and oligomerization is favored due to a high local concentration of receptors. These clusters are not static but undergo continuous structural fluctuations. The relationship between this dynamic structure and the local signaling activation are insufficiently understood, partly because of a lack of appropriate optical tools for mapping subdiffraction limit dynamics with high temporal bandwidth and long observation time. Aim 1 of this application takes advantage of the unique photophysical properties of plasmonic nanoparticles (NPs) that provide large and stable optical signals and also encode information about deeply subdiffraction limit separations between NPs in their far-field spectrum to characterize the lateral diffusion and structural dynamics of individual EGFR clusters with high temporal resolution and without physical limitation in observation time. Previous implementations of plasmon coupling microscopy (PCM) utilized the NP spectrum detected under darkfield illumination to identify EGFR clustering. Dark-field detection requires, however, large (~40 nm) NP labels. In Aim 1, we will implement a new interferometric PCM (iPCM) for detecting 5 nm (to probe EGFR-EGFR contacts) and 10 nm (to probe EGFR oligomerization and clustering) gold NP labels. iPCM will be augmented with a correlation analysis to quantify continuous fluctuations in plasmon coupling. This technology will be applied to test the hypothesis that EGF binding results in a decrease of intracluster dynamics and an increase in EGFR phosphorylation. Another insufficiently understood element of structural regulation that is associated with EGFR clustering is lateral signal propagation through inter-receptor contacts or EGF-induced second messenger release. Aim 2 will elucidate how EGFR clustering impacts NP-EGF-induced reactive oxygen species (ROS) formation and ROS-mediated EGFR activation. In this approach EGF-functionalized NPs (NP- EGF) with known EGF loading are not simple imaging tools for mapping sub-diffraction limit clusters of EGFR, but instead, represent quantifiably units of local EGFR activation. In Aim 3 nanoconjugated EGF will be applied as probe to quantify the cross-talk between EGFR activation and nitric oxide (NO) formation. As NO synthesis is spatially and temporally strictly regulated, its concentration depends on EGFR activation, which varies as function of the local NP-EGF concentration. Aim 3 will test the hypothesis that NP-EGF-induced NO generation is a regulatory factor for c-Jun N-terminal kinase (JNK)-mediated apoptosis in BLBC cells.