Developing a novel, rapid-response biosensor and analyzing Notch signaling dynamics in cell fate decisions in vivo

开发新型快速响应生物传感器并分析体内细胞命运决定中的 Notch 信号动力学

• 批准号: 9910619
• 负责人: Justin Matthew Shaffer
• 金额: $ 4.55万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-12-16 至 2021-12-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9910619
• 关键词: ARNT gene; Address; Affect; Animal Model; Biosensor; Breathing; CRISPR/Cas technology; Caenorhabditis elegans; Cardiovascular Diseases; Cell Communication; Cell Nucleus; Cells; Cleaved cell; Color; Complex; Cytoplasm; Data; Defect; Development; Disease; Epidermal Growth Factor Receptor; Event; G-Protein-Coupled Receptors; Genetic; Genetic Transcription; Gonadal structure; Green Fluorescent Proteins; Histones; Human; Human Development; Image; Individual; Lateral; Ligands; Lobular Neoplasia; Maintenance; Malignant Neoplasms; Measures; Mediating; Membrane; Methods; Notch Signaling Pathway; Nuclear; Nuclear Export; Organism; Outcome; Pathway interactions; Peptide Hydrolases; Phosphorylation; Proteins; Protocols documentation; Reporter; Reporting; Role; Signal Transduction; Site; Speed; Stimulus; System; TEV protease; Testing; Therapeutic; Time; Tissues; Transgenes; Transgenic Organisms; Translations; Transmembrane Domain; Uterus; Work; experimental study; improved; in vivo; insight; notch protein; novel; precursor cell; protein complex; receptor; response; sensor; symptom treatment; targeted treatment; therapy development; tool

The Notch signaling pathway is remarkably important for the development and maintenance of most complex tissues in multicellular organisms and defective Notch signaling has been implicated in severe developmental defects and cancer. Understanding the basic mechanisms and functions of Notch is imperative to developing therapies for Notch-related diseases. Notch is a single-pass transmembrane receptor that, upon activation by a ligand, is cleaved in the transmembrane domain to release the intracellular domain into the cytoplasm. The free intracellular domain translocates to the nucleus where it forms a ternary protein complex to promote target gene transcription and influence cell fate and function. The Notch intracellular domain is difficult to visualize directly because the free intracellular domain is rapidly degraded, but manipulations affecting the stability or level of the intracellular domain cause cell fate transformations, complicating studies of signaling onset, duration, and dynamics. Thus, Notch activity has been assessed using target gene transcriptional reporters, which are limited by time lags due to transcription, translation, and fluorescent protein maturation, as well as perdurance of reporter proteins after signaling has ceased. Preliminary data shows that a Sensor Able to detect Lateral Signaling Activity, or SALSA, is a promising novel, rapid-response, in vivo biosensor of Notch activity. SALSA is a bipartite system consisting of a “switch” in which the Notch locus is endogenously tagged with a protease, and a cleavable, dual-fluorescent protein reporter. When Notch is activated, the protease- tagged intracellular domain translocates to the nucleus where the protease cleaves the reporter and that allows the individual fluorescent proteins to be localized to different cellular compartments. This project will 1) establish a protocol for quantifying the level of Notch activity using SALSA. This protocol will be established in model organism C. elegans because of the speed of generating transgenic lines, the ability to image the entire organism in vivo throughout its lifecycle, and the well conserved and characterized Notch signaling events. This project will then 2) use SALSA to determine how the timing and levels of Notch activity influence cell fate decisions in two paradigms for Notch signaling in C. elegans: the Vulval Precursor Cells and the Anchor Cell/Ventral Uterine precursor cell decision in the somatic gonad. Results from this project will establish a useful tool for studying many aspects of Notch signaling in a variety of contexts, and provide insight into a universal relationship between the timing and levels of Notch activity with cell fate decisions.

Notch信号通路对于大多数复杂的细胞的发育和维持非常重要。 多细胞生物体中的组织和缺陷的Notch信号传导已经涉及严重的发育障碍。 缺陷和癌症。了解Notch的基本机制和功能对于开发 Notch相关疾病的治疗。Notch是一种单通道跨膜受体，在被一种 配体在跨膜结构域中被切割以将胞内结构域释放到细胞质中。的 游离的胞内结构域易位到细胞核，在那里它形成三元蛋白复合物以促进靶向 基因转录并影响细胞命运和功能。Notch细胞内结构域难以可视化 直接因为游离的胞内结构域被迅速降解，但影响稳定性或 胞内结构域的水平导致细胞命运转变，使信号传导起始的研究复杂化， 持续时间和动态。因此，Notch活性已经使用靶基因转录报告基因进行了评估， 其受到由于转录、翻译和荧光蛋白成熟的时间滞后的限制，以及 报告蛋白在信号传导停止后的持久性。初步数据显示，传感器能够 检测侧向信号活性，或SALSA，是一种有前途的新型，快速响应，在体Notch生物传感器 活动SALSA是一个由“开关”组成的二分系统，其中Notch基因座被内源性标记 蛋白酶和可切割的双荧光蛋白报告基因。当Notch被激活时，蛋白酶- 标记的胞内结构域易位到细胞核，在那里蛋白酶切割报告分子， 单个荧光蛋白定位于不同的细胞区室。该项目将1） 建立使用SALSA定量Notch活性水平的方案。本方案将于 模式生物C.因为转基因株系的生成速度快， 在其整个生命周期中，Notch信号转导在生物体中的作用，以及良好保守和表征的Notch信号转导事件。 然后，该项目将使用SALSA来确定Notch活性的时间和水平如何影响细胞命运 决定在两个范例的Notch信号在C.秀丽隐杆线虫：外阴前体细胞和锚 细胞/生殖腺子宫前体细胞决定在体细胞性腺。该项目的成果将建立一个 这是一个有用的工具，用于研究Notch信号传导在各种背景下的许多方面，并提供了对Notch信号传导的深入了解。 Notch活性的时间和水平与细胞命运决定之间的普遍关系。