UTSW-UNC Center for Cell Signaling Analysis

摘要 细胞行为受不同的信号机制调节，这些机制通常依赖于分子动力学。 在空间和时间上有精确的组织。分子事件的瞬时定位和动力学大量丢失 生化分析和单细胞蛋白质组学。理解这些因素需要可视化和 对活细胞和组织中的分子事件进行定量分析，现在可以通过将分子 探头设计、高分辨率活细胞显微镜和计算图像分析。然而，将这些结合在一起 将多种技术整合为一个整体，用于对活细胞信号网络进行定量分析 很有挑战性。拟议的细胞信号分析中心旨在使先进的方法民主化， 把这些工具交到那些没有致力于成像事业的科学家手中。我们建议开发 并传播一个用户友好的集成管道，该管道结合了生物传感器、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，以及3)图像分析和 用于推导信令网络体系结构的计算建模，包括 关系。该中心将集中精力在3个技术开发项目之间进行整合： 一)以替代方法为基础的光遗传学、化学遗传学和生物传感器 功能和减少信令的扰动。 Ii)多个模块化、高性价比和高分辨率3D光片显微镜，可以 由非专家快速组装，可提供~220 nm和~450 nm的横向和轴向分辨率。这些 将设计为最大限度地利用计算机视觉工作流、生物传感器和 光遗传学/化学遗传学。 三)斐济开放源码外挂程序库，用于分析2D亚细胞信号转导 以及使用统计时间序列分析中的先进方法的3D活细胞时间推移数据。 通过我们的合作驱动生物项目(DBP)，我们将反复完善和改进我们的探测器， 成像平台和图像分析方法。我们已经确定了8个针对免疫细胞主题的DBP 迁移、免疫突触功能、机械感觉、肌节形成、肿瘤扩散和 耐药、突触可塑性和腹侧沟内侵。我们建议大力传播 最大限度地利用现有基础设施的组件，包括拥有3,000个用户的成像设施 在美国各地，被广泛采用的斐济软件生态系统Addgene、GitHub和 科学仪器。我们将提供广泛的培训(面对面、远程和以主题为导向的课程) 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.