UTSW-UNC Center for Cell Signaling Analysis

摘要 细胞的行为是由不同的信号机制，往往取决于分子动力学调节 在空间和时间上精确地组织起来。分子事件的瞬时定位和动力学在批量中丢失 生物化学分析和单细胞蛋白质组学。理解这些因素需要可视化， 定量分析活细胞和组织中的分子事件，现在可以通过结合分子生物学方法， 探针设计、高分辨率活细胞显微镜和计算图像分析。然而，结合这些 将多种技术整合成一个工作整体，用于活细胞信号网络的定量分析， 挑战性拟议中的细胞信号分析中心旨在使先进的方法民主化， 把这些工具交给那些没有把自己的职业生涯奉献给成像的科学家。我们建议发展 并传播一个用户友好的综合管道，结合1）生物传感器，光遗传学和 化学遗传学2）模块化，高速和高分辨率光片显微镜，和3）图像分析和 计算建模，推导出信令网络架构，包括因果关系和动力学 连接.该中心将集中在3个技术开发项目之间的整合工作： i）光遗传学、化学遗传学和生物传感器，基于具有互补性的替代方法 能力和减少的信号干扰。 ii）多个模块化、高性价比和高分辨率的3D光片显微镜， 由非专业人员快速组装，提供约220和约450 nm的横向和轴向分辨率。这些 将最大限度地利用计算机视觉工作流程，生物传感器， 光遗传学/化学遗传学。 iii）基于斐济的开源插件库，用于分析二维亚细胞信号转导 和3D活细胞时间推移数据使用先进的统计时间序列分析方法。 通过我们的合作驱动生物项目（DBPs），我们将反复完善和改进我们的探针， 成像平台和图像分析方法。我们已经确定了8个解决免疫细胞主题的DBP 迁移、免疫突触功能、机械感觉、肌节形成、癌扩散和 耐药性、突触可塑性和腹侧沟内移。我们建议大力传播 最大限度地利用现有基础架构的组件，包括用户数超过3，000的映像设施 位于美国各地，广泛采用的斐济软件生态系统，Addgene，GitHub和Applied 科学仪器。我们将提供广泛的培训（亲自，远程和主题驱动的课程）， 在一个全面和不断更新的网站上集中组织中心的产出。

ABSTRACT Cellular behavior is regulated by diverse signaling mechanisms that often depend on molecular dynamics precisely organized in space and time. The transient positioning and kinetics of molecular events is lost in bulk biochemical analysis and in single cell proteomics. Understanding such factors requires visualization and quantitative analysis of molecular events in living cells and tissues, now made possible by combining molecular probe design, high-resolution live cell microscopy and computational image analysis. However, combining these diverse technology into a working whole for the quantitative analysis of live cell signaling networks remains challenging. The proposed Center for Cell Signaling Analysis aims to democratize advanced methods that put these tools in the hands of scientists who have not devoted their careers to imaging. We propose to develop and disseminate a user-friendly and integrated pipeline that combines 1) biosensors, optogenetics and chemogenetics 2) modular, high-speed, and high-resolution light-sheet microscopes, and 3) image analysis and computational modeling to derive signaling network architecture, including the causality and kinetics of connections. The Center will concentrate its integration effort between 3 Technology Development Projects: i) Optogenetics, chemogenetics, and biosensors based on alternate approaches with complementary capabilities and reduced perturbation of signaling. ii) Multiple modular, cost-effective, and high-resolution 3D light-sheet microscopes that can be assembled rapidly by non-experts and deliver ~220 and ~450 nm lateral and axial resolution. These will be designed to maximally leverage computer vision workflows, biosensors, and optogenetics/chemogenetics. iii) A library of open-source Fiji-based plugins for the analysis of subcellular signal transduction in 2D and 3D live cell time-lapse data using advanced methods in statistical time series analysis. Through our collaborative Driving Biological Projects (DBPs), we will iteratively refine and improve our probes, imaging platforms and image analysis methods. We have identified 8 DBPs that address topics in immune cell migration, immunological synapse function, mechanosensation, sarcomere formation, cancer dissemination and drug resistance, synaptic plasticity, and ventral furrow ingression. We propose a strong dissemination component that maximally leverages existing infrastructure, including imaging facilities with >3,000 users located throughout the United States, the widely adopted Fiji software ecosystem, Addgene, GitHub, and Applied Scientific Instrumentation. We will provide extensive training (in person, remote, and topic-driven courses) and centrally organize the Center outputs on a comprehensive and continuously updated website.