Developing CluMPS reporters to visualize aggregation dynamics of receptor tyrosine kinase fusions

摘要/摘要 蛋白质聚集在正常细胞生理中起很大作用，但异常聚集也是基础。 无数的疾病病理。然而，研究这种病理性聚集通常是具有挑战性的，因为 我们无法在患病的细胞中看到蛋白质聚集体。活细胞可视化的标准方法 包括荧光标记的构建体的过表达或内源性蛋白的直接标记 带着一个荧光标签。这些技术不仅有很大的局限性，还包括 过度表达的伪影、微弱的信号和费力的准备，但病理聚集通常很小 因此很难通过传统的活细胞显微镜观察到。我们最近开发了一种证明- 提供内源蛋白质聚集的直观可视化的新类别或报告程序的原理 以及它们的动态。本增刊的目标是应用这一记者策略来可视化内生 病变细胞中的病理性聚集体。具体地说，我们将针对一类被称为受体的癌基因 酪氨酸激酶融合，在细胞内形成蛋白质聚集体。我们将设计和验证记者的变体 以最佳方式检测和可视化目标聚集体。然后，我们将可视化以下方面的群集动态 由相同受体融合驱动的独立癌细胞系。最后，我们将确定在多大程度上 我们的记者可以用来可视化不同受体融合的聚集动力学，其中超过50个 已被确认身份。所描述的工作将验证我们新的记者观察细胞疾病和 将对治疗开发具有重要意义，例如，通过允许可视化多个 疾病状态和建立一种新的基于荧光的药物筛选方法。

Summary/Abstract Protein aggregation plays a large role in normal cell physiology, but aberrant aggregation also underlies numerous disease pathologies. It is often challenging to study such pathological aggregation, however, because of our inability to visualize protein aggregates in diseased cells. Standard methods for visualization in live cells include either overexpression of a fluorescently tagged construct or direct tagging of the endogenous protein with a fluorescent tag. Not only do these techniques have significant limitations techniques including overexpression artefacts, weak signal, and laborious preparation, but pathological aggregates are often small and thus difficult to visualize through conventional live-cell microscopy. We have recently developed proof-of- principle of a new class or reporter to provide straightforward visualization of endogenous protein aggregates and their dynamics. The goal of this supplement will be to apply this reporter strategy to visualize endogenous pathological aggregates in diseased cells. Specifically, we will target a class of oncogenes called receptor tyrosine kinase fusions, which form protein aggregates within cells. We will design and validate reporter variants that optimally detect and visualize the target aggregates. We will then visualize cluster dynamics across independent cancer cell lines driven by the same receptor fusion. Finally, we will determine the extent to which our reporter can be applied to visualize aggregation dynamics of distinct receptor fusions, of which over 50 have been identified. The described work will validate our new reporter strategy for observation of cellular disease and will have important implications for therapeutic development, for example by allowing visualization of multiple disease states and by establishing a new assay for fluorescence-based drug screens.