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.

项目概要 细胞信号传导是细胞之间以及细胞与环境及其环境进行通信的过程。 调节对于维持组织、器官和生物体水平的稳态至关重要。因为 异常的信号转导是大多数疾病发病机制的基础，细胞信号转导的研究已经 成为细胞和分子生物学研究的核心部分。然而，目前分析细胞的方法 信令受到多种技术限制。例如，传统上分析信号通路 使用生化方法对数千个细胞同时获得的测量值进行平均， 提供了忽略潜在细胞间变异性的全球信号传导格局的印象 作为分子介质的动态定位和易位。虽然荧光显微镜已 通过实时观察亚子的快速分子事件，有可能克服这一限制 微米分辨率，这些方法不能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，我们对细胞过程进行成像的能力已经通过单 由于荧光量子点探针和生物正交标记化学的进步，分子成像得以实现。 同时，先进的细胞工程工具（例如 CRISPR/Cas9 和微图案化）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个蛋白质的成像 语境。这些技术已经各自成熟，我们建议它们现在已经准备好应用 作为一套紧密结合的工具，用于精确绘制和分析细胞信号传导。因此，本次活动的目标 该提案旨在开发和验证三种技术，这些技术结合起来将能够实现细胞内单 分子分析包括 (1) 用于分子过程细胞内成像的 QD 标记，(2) 天然蛋白质 通过基因编辑进行标记以实现高效缀合，以及 (3) 优化的自动图像分析算法 空间映射不同时间尺度的微图案细胞中的过程并记录细胞内 地点。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 以单分子分辨率实时研究活细胞中的细胞信号传导。我们将完成我们的 通过安德鲁史密斯博士整合的多学科团队的协作工作来实现目标，他是 光学探针工程和成像方面的专家，Pablo Perez-Pinera 博士在以下领域拥有丰富的专业知识： 基因编辑和基因组工程。他们的实验室多年来一直合作启动 本申请中描述的工作。从概念上讲，该平台是一种革命性的细胞分析方法 因此，它不仅可以提高我们对基本生物过程的理解，而且可以 还能够以前所未有的精度开发针对这些途径的疗法 功效。