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

开发 CluMPS 报告基因以可视化受体酪氨酸激酶融合的聚集动态

• 批准号: 10321062
• 负责人: Lukasz Bugaj
• 金额: $ 3.37万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10321062
• 关键词: Biological Assay; Cancer cell line; Cell physiology; Cells; Disease; Drug Screening; Fluorescence; Funding; Goals; Methods; Morphologic artifacts; Normal Cell; Oncogenes; Pathologic; Pathology; Play; Preparation; Proteins; Receptor Protein-Tyrosine Kinases; Reporter; Role; Signal Transduction; Techniques; Variant; Visualization; Work; base; design; live cell microscopy; novel; overexpression; protein aggregation; receptor; sensor; success; therapeutic development

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.

摘要/摘要 蛋白质聚集在正常细胞生理学中起着重要作用，但异常聚集也是 许多疾病的病理。然而，研究这种病理性聚集通常具有挑战性，因为 我们无法看到病变细胞中的蛋白质聚集体。活细胞可视化的标准方法 包括荧光标记构建体的过表达或内源蛋白的直接标记 有荧光标记这些技术不仅具有显著的局限性， 过度表达伪影、弱信号和费力的制备，但病理性聚集体通常较小 因此难以通过常规的活细胞显微镜观察。我们最近开发了一种 提供内源性蛋白质聚集体的直接可视化的新类别或报告子的原理 和他们的动态。这一补充的目标将是应用这一报告策略，以可视化内源性 病变细胞中的病理性聚集体。具体来说，我们将靶向一类称为受体的癌基因， 酪氨酸激酶融合，其在细胞内形成蛋白质聚集体。我们将设计和验证报告基因变体 以最佳方式检测和可视化目标聚集体。然后，我们将可视化集群动态， 由相同受体融合驱动的独立癌细胞系。最后，我们将确定 我们的报告可以应用于可视化不同受体融合体的聚集动力学，其中超过50种具有 被识别。所描述的工作将验证我们用于观察细胞疾病的新报告策略， 将对治疗发展产生重要影响，例如通过允许多个 疾病状态，并建立一个新的测定荧光为基础的药物筛选。