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 信号转导通路是生命的基础，通常是疾病治疗的关键靶点。 然而，对信号如何发生的细胞内动力学、动力学和化学计量学的精确理解 传输的信息-对于全面、更准确地了解信号传递过程至关重要的知识， 由于在真实的环境中观察信号事件的技术障碍， 时间在细胞技术的最新进展，包括邻近标记方法和光片 显微镜，现在终于提出了解决方案，编织在一起，我们在体内信号的碎片视图。建筑 Blacklow实验室在Notch信号机制方面的专业知识与Kirchhausen的专业知识 在先进的显微镜实验室，我们建议使用Notch信号系统作为模型，以精确定义 在正常和致病状态下Notch信号激活所需的一系列事件，并在此过程中， 方法和计算可视化工具，可以广泛应用于其他途径和系统。 Notch信号转导系统对细胞的生长、分化、分化和分化有着重要的影响，是研究Notch信号转导系统的理想模型 所有后生动物的分化及其失调与多种疾病有关，包括 许多人类癌症的发病机制。此外，这种信号机制的基本方面，包括 配体和受体分子的动力学，活化位点处配体-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Notch从质膜到核的路径， 仍然不完全理解。在这里，我们将通过使用APEX邻近度来解决这些知识差距 结合定量质谱的标记以阐明Notch通过的途径 细胞内结构域从质膜到细胞核，并通过实施点阵光片显微镜， 可视化Notch信号转导的分子事件。在初步工作中，我们使用了CRISPR/Cas9 SVG-A永生化星形胶质细胞中的基因组标记，以产生Notch-APEX 2融合蛋白，用于邻近 标记，我们的初步分析的试点实验揭示了动态变化的标记蛋白质 在不同的细胞隔室中作为时间的函数，证实了这种方法的可行性。我们还 用于LLSM的工程化荧光标记的受体和配体敲入蛋白。现在我们将使用这种方法 为了定量在细胞-细胞接触位点聚集在一起的受体和配体分子的数量， 时间的函数，并确定必须存在多少拷贝的每一个来诱导受体裂解， 激活靶基因表达。成功地实现这些目标将提供前所未有的解决办法， 在活细胞中生理Notch信号转导所需的基本事件的空间和时间。 我们希望不仅能回答长期存在的关于Notch激活的分子事件的问题， 信号，同时也为机械分析开辟了一个全新的生物学领域。