Advanced Molecular Probes and Cell Engineering Tools for Accurate Single-Molecule Analysis of Signaling in Individual Cells

用于对单个细胞信号传导进行精确单分子分析的先进分子探针和细胞工程工具

• 批准号: 10363683
• 负责人: Andrew Michael Smith
• 金额: $ 24.26万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-03-01 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10363683
• 关键词: 3-Dimensional; Adaptor Signaling Protein; Algorithmic Analysis; Algorithms; Biochemical; Biological; Biological Models; Biological Process; Biology; CRISPR/Cas technology; Cell physiology; Cells; Cellular biology; Chemistry; Cytokine Receptors; Disease; Dyes; Engineering; Ensure; Environment; Epidermal Growth Factor; Event; Exhibits; Fluorescence Microscopy; Genes; Genome engineering; Genotype; Geometry; Goals; Health; Homeostasis; Hour; Human; Human Development; Image; Imaging technology; Individual; Label; Laboratories; Light; Location; Maps; Measurement; Mediator of activation protein; Methodology; Methods; Molecular; Molecular Biology; Molecular Probes; Morphologic artifacts; Morphology; Optics; Organ; Organism; Pathogenesis; Pathology; Pathway interactions; Pattern; Phototoxicity; Physiology; Process; Proteins; Quantum Dots; Regulation; Reproducibility; Research; Research Personnel; Resolution; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Signaling Molecule; Stochastic Processes; Structure; System; TNF gene; Techniques; Technology; Time; Tissues; Transgenic Organisms; Work; automated image analysis; cell behavior; cell type; cellular engineering; cellular imaging; cohesion; cytokine; impression; improved; millisecond; molecular imaging; multidisciplinary; new technology; novel; overexpression; protein function; single molecule; spatiotemporal; submicron; technology research and development; therapeutic development; therapeutic target; tool; transcription factor; two-dimensional

PROJECT SUMMARY Cell signaling is the process by which cells communicate with each other and with their environments and its regulation is critically important to maintaining homeostasis at the tissue, organ, and organism level. Because aberrant signal transduction underlies the pathogenesis of most diseases, the study of cell signaling has become a central part of cell and molecular biology research. However, current methodologies to analyze cell signaling suffer from multiple technical limitations. For example, signaling pathways are traditionally analyzed using biochemical methods that average measurements obtained across thousands of cells simultaneously, providing an impression of the global signaling landscape that ignores underlying cell-to-cell variability, as well as dynamic localizations and translocations of the molecular mediators. While fluorescence microscopy has the potential to overcome this limitation by enabling real-time observations of rapid molecular events at sub- micron resolution, these methods do not provide sufficient sensitivity or signal stability to observe discrete single-molecule events. Recently, our ability to image cellular processes has been transformed by single- molecule imaging due to advances in fluorescent quantum dot probes and bioorthogonal labeling chemistries. Simultaneously, advanced cell engineering tools like CRISPR/Cas9 and micropatterning now allow us to precisely control cellular genotype and morphology to facilitate imaging of single proteins in a native cellular context. These technologies have matured individually and we propose that they are now primed to be applied as a cohesive suite of tools for precise mapping and analysis of cell signaling. As such, the goal of this proposal is to develop and validate three technologies that in combination will enable intracellular single- molecule analysis including (1) QD labels for intracellular imaging of molecular processes, (2) native protein tagging through gene editing for efficient conjugation, and (3) automated image analysis algorithms optimized to spatially map processes in micropatterned cells across different time scales and registered intracellular locations. We anticipate that by simultaneously advancing these technologies, we will create a novel platform to study cell signaling in living cells with single-molecule resolution in real-time. We will accomplish our objectives through the collaborative work of a multidisciplinary team integrated by Dr. Andrew Smith, who is an expert in optical probe engineering and imaging, and Dr. Pablo Perez-Pinera, who has extensive expertise in gene editing and genome engineering. Their laboratories have been working together for years to initiate the work described in this application. Conceptually, this platform is a revolutionary method to analyze cell signaling and, therefore, it will not only improve our understanding of essential biological processes, but can also enable the development of therapeutics that target these pathways with unprecedented precision and efficacy.

项目摘要 细胞信号传导是细胞相互通信及其环境及其环境的过程 调节对于维持组织，器官和生物水平的稳态至关重要。因为 异常信号转导基础是大多数疾病的发病机理，细胞信号的研究已有 成为细胞和分子生物学研究的中心部分。但是，当前分析细胞的方法 信号受到多种技术限制。例如，传统分析信号通路 使用生化方法，同时在数千个细胞上获得的平均测量值， 还提供了忽略基本单元间可变性的全局信号景观的印象 作为分子介质的动态定位和易位。而荧光显微镜具有 通过实现在亚下快速分子事件的实时观察来克服这一限制的潜力 微分分辨率，这些方法没有提供足够的灵敏度或信号稳定性来观察离散 单分子事件。最近，我们成像细胞过程的能力已通过单个形象转化 分子成像由于荧光量子点探针的进展和生物正交标记化学作用而导致的成像。 同时，高级细胞工程工具（例如CRISPR/CAS9和Micropaterning）现在使我们能够 精确控制细胞基因型和形态，以促进天然细胞中单蛋白的成像 语境。这些技术已经单独成熟，我们建议它们现在已应用于应用 作为用于精确映射和分析细胞信号的凝聚力套件。因此，目标的目标 提案是开发和验证三种技术将使细胞内单个技术 分子分析包括（1）QD标记用于分子过程的细胞内成像，（2）天然蛋白 通过基因编辑进行标记以进行有效的结合，（3）自动图像分析算法优化了 在不同时间尺度和注册的细胞内绘制微图案细胞中的空间映射过程 位置。我们预计，通过同时推进这些技术，我们将创建一个新颖的平台 实时研究具有单分子分辨率的活细胞中的细胞信号。我们将完成我们的 通过由安德鲁·史密斯（Andrew Smith）博士集成的跨学科团队的合作工作的目标，他是一个 光学探测工程和成像专家，以及Perez-Pinera博士，他在广泛的专业知识 基因编辑和基因组工程。他们的实验室已经共同努力了多年 此应用程序中描述的工作。从概念上讲，该平台是分析细胞的革命性方法 信号传导，因此，它不仅会提高我们对基本生物过程的理解，还可以 还可以以前所未有的精度和 功效。