Dynamics of Notch Signaling

Notch信号的动力学

• 批准号: 10686971
• 负责人: Stephen C. Blacklow
• 金额: $ 64.8万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10686971
• 关键词: Astrocytes; Binding; Biology; CRISPR/Cas technology; Cell Differentiation process; Cell Nucleus; Cell membrane; Cells; Chimeric Proteins; Coculture Techniques; Complex; Coupled; Cytoplasm; Disease; Emerging Technologies; Engineering; Ensure; Event; Excision; Family; Gene Expression; Genetic Transcription; Genome engineering; Genomics; Hour; Human; Imaging technology; Ions; Kinetics; Knock-in; Knowledge; Label; Laboratories; Life; Ligands; Light; Location; Malignant Neoplasms; Mammals; Mass Spectrum Analysis; Measures; Membrane; Microscopy; Modeling; Molecular; Monitor; Movement; Optics; Output; Pathogenesis; Pathogenicity; Pathway interactions; Physiological; Process; Proteins; Proteolysis; Proteomics; Receptor Activation; Resolution; Scanning Electron Microscopy; Series; Signal Transduction; Signal Transduction Pathway; Site; System; Technology; Therapeutic; Time; Travel; Visualization; Visualization software; Work; adaptive optics; endosome membrane; experimental study; extracellular; frontier; gamma secretase; in vivo; inhibitor; notch protein; optical lattices; receptor; recruit; response; spatiotemporal; stoichiometry; transmission process

Project Summary Signal transduction pathways underlie the very basis of life and are often critical targets for disease therapeutics. Yet, precise understanding of the intracellular dynamics, kinetics, and stoichiometry of how signals are transmitted – knowledge that is critical to capturing a holistic and more accurate view of signaling processes – has not been attainable for many pathways due to technological barriers to observing signaling events in real time in cells. Recent advances in technology, including proximity labeling approaches and light sheet microscopy, are finally now presenting solutions to knit together our fragmented view of signaling in vivo. Building on the expertise of the Blacklow laboratory in Notch signaling mechanism with the expertise of the Kirchhausen laboratory in advanced microscopy, we propose to use the Notch signaling system as a model to define precisely the series of events required for Notch signal activation in normal and pathogenic states, and in so doing, develop approaches and computational visualization tools that can be broadly applied to other pathways and systems. Notch signaling is an ideal model signaling system for this work because it exerts a critical influence on cell differentiation in all metazoans and its misregulation is associated with diverse diseases, including the pathogenesis of many human cancers. Moreover, fundamental facets of this signaling mechanism, including the dynamics of ligand and receptor molecules, the stoichiometry of ligand-receptor complexes at sites of activation, the timing and location of activating Notch proteolysis, and the path of Notch from plasma membrane to nucleus, remain incompletely understood. Here, we will address these gaps in knowledge by using APEX proximity labeling coupled with quantitative mass spectrometry to elucidate the pathway for passage of the Notch intracellular domain from plasma membrane to nucleus, and by implementing lattice light sheet microscopy to visualize the molecular events of Notch signal transduction. In preliminary work, we have used CRISPR/Cas9 genomic labeling in SVG-A immortalized astrocytes to create a Notch-APEX2 fusion protein for proximity labeling, and our preliminary analysis of a pilot experiment reveals dynamic changes in the labeling of proteins in different cellular compartments as a function of time, confirming the feasibility of this approach. We have also engineered fluorescently labeled receptor and ligand knockin proteins for LLSM. Now we will use this approach to quantify the number of receptor and ligand molecules that come together at the site of cell-cell contact as a function of time, and determine how many copies of each must be present to induce receptor cleavage and activate target gene expression. Successful completion of these aims will provide unprecedented resolution, in both space and time, of the fundamental events required for physiologic Notch signal transduction in living cells. We expect to not only answer long-standing questions about the molecular events involved in activation of Notch signals but to also open up a whole new realm of biology to mechanistic analysis.

项目摘要 信号转移途径是生命基础的基础，通常是疾病疗法的关键目标。 然而，对信号的细胞内动力学，动力学和化学计量计的精确理解 传输 - 知识对于捕获整体，更准确的信号过程观点至关重要 - 由于技术障碍可以观察到实际的信号事件，因此无法实现许多途径 在细胞中的时间。技术的最新进展，包括接近标签方法和轻纸 显微镜，最终提出了解决方案，以将我们在体内信号传导的碎片视图结合在一起。建筑 基于Notch信号机制的Blacklow实验室的专业知识，具有Kirchhausen的专业知识 在高级显微镜检查中，我们建议将Notch信号系统用作模型来确切定义 在正常和致病状态中，Notch信号激活所需的一系列事件，并且在这样做的过程中 可以广泛应用于其他途径和系统的方法和计算可视化工具。 Notch信号传导是这项工作的理想模型信号系统，因为它对细胞产生关键影响 所有后生动物的分化及其不调节都与潜水员疾病有关，包括 许多人类癌的发病机理。此外，该信号机制的基本方面，包括 配体和受体分子的动力学，在激活部位的配体 - 受体复合物的化学计量， 激活Notch蛋白水解的时间和位置，以及从质膜到核的Notch路径， 保持不完全理解。在这里，我们将通过使用Apex接近来解决知识中的这些差距 标记与定量质谱法结合以阐明通道的途径 细胞内结构域从质膜到核，并通过实现晶格光片显微镜 可视化Notch信号传递的分子事件。在初步工作中，我们使用了CRISPR/CAS9 SVG-A永生的星形胶质细胞中的基因组标记，以创建Notch-apex2融合蛋白的近端 标签，我们对试验实验的初步分析揭示了蛋白质标记的动态变化 在不同的细胞隔室随时间的函数中，请确认这种方法的可行性。我们也有 为LLSM设计了荧光标记的受体和配体敲击蛋白。现在我们将使用这种方法 量化在细胞 - 细胞接触部位聚集在一起的接收器和配体分子的数量 时间的功能，并确定必须存在多少份，以诱导接收器的裂解和 激活靶基因表达。这些目标的成功完成将提供前所未有的决议 生理细胞中生理缺口信号转导所需的基本事件的空间和时间。 我们期望不仅回答有关激活Notch涉及的分子事件的长期问题 信号，但还为机械分析开辟了一个全新的生物学领域。