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

总结 表皮生长因子受体（EGFR）是一种模型酪氨酸激酶，其过表达在肿瘤中常见。 各种癌症，包括基底样乳腺癌（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， 了解时空调控机制。受体在细胞膜上的不均匀分布 质膜导致EGFR在膜区域中的富集，所述膜区域具有数十至 数百纳米，其中受体二聚化和寡聚化是有利的，由于高局部 受体浓度。这些集群不是静态的，而是经历连续的结构波动。的 这种动态结构和局部信号激活之间的关系还没有得到充分的理解， 由于缺乏适当的光学工具来映射具有高时间分辨率的亚衍射极限动力学， 带宽和长观测时间。本申请的目标1利用了独特的物理特性， 等离子体纳米粒子（NP）的特性，可提供大而稳定的光学信号，并编码 关于NP在其远场光谱中深亚衍射极限分离的信息，以表征 单个EGFR簇的横向扩散和结构动力学具有高时间分辨率， 观察时间的物理限制。等离子体激元耦合显微镜（PCM）的先前实现利用 在暗视野照明下检测NP光谱以鉴定EGFR簇集。暗场探测需要 然而，大（~40 nm）NP标记。在目标1中，我们将实现一种新的干涉PCM（iPCM），用于检测 5 nm（探测EGFR-EGFR接触）和10 nm（探测EGFR寡聚化和聚簇）金NP标记。 iPCM将增加相关分析，以量化等离子体耦合中的连续波动。这 技术将被应用于测试的假设，即EGF结合的结果，在减少集群内的动力学 和EGFR磷酸化的增加。结构性监管的另一个未被充分理解的因素是， 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 第二信使释放目的2将阐明EGFR聚集如何影响NP-EGF诱导的活性氧 ROS的形成和ROS介导的EGFR活化。在该方法中，EGF-官能化的NP（NP-1）是一种天然的纳米颗粒。 EGF）不是用于绘制EGFR的亚衍射极限簇的简单成像工具， 而是代表局部EGFR活化的定量单位。在目标3中，将应用纳米缀合的EGF 作为探针来量化EGFR活化和一氧化氮（NO）形成之间的串扰。As NO合成 在空间和时间上受到严格调控，其浓度取决于EGFR活化，其随时间变化。 NP-EGF的浓度。目的3将验证NP-EGF诱导NO产生的假设， 是BLBC细胞中c-Jun N-末端激酶（JNK）介导的凋亡的调节因子。

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.