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彼此之间以及与它们的环境及其环境之间进行通信的过程。 调节对于维持组织、器官和生物体水平的体内平衡至关重要。因为 异常的信号转导是大多数疾病的发病机制的基础，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学 信令受到多种技术限制。例如，传统上分析信号通路 使用同时对数千个细胞进行平均测量的生物化学方法， 提供了一个全球信号格局的印象，忽略了潜在的细胞间变异性，以及 作为分子介质的动态定位和移位。虽然荧光显微镜具有 通过实时观察亚临界温度下的快速分子事件来克服这一限制的潜力， 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的 单分子事件。最近，我们对细胞过程成像的能力已经被单- 由于荧光量子点探针和生物正交标记化学的进步， 与此同时，先进的细胞工程工具，如CRISPR/Cas9和微图案，现在允许我们 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， 上下文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 作为一套紧密的工具，用于精确绘制和分析细胞信号。因此，这一目标 建议是开发和验证三种技术，这些技术相结合将使细胞内单- 分子分析，包括（1）用于分子过程的细胞内成像的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 在不同的时间尺度上空间映射微图案化细胞中的过程， 地点我们预计，通过同时推进这些技术，我们将创建一个新的平台， 用单分子分辨率实时研究活细胞中的细胞信号。我们将实现我们的 通过由安德鲁·史密斯博士整合的多学科团队的协作工作， 光学探头工程和成像专家，以及巴勃罗佩雷斯-皮涅拉博士，他在以下方面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在合作， 本申请中描述的工作。从概念上讲，该平台是分析细胞的革命性方法 因此，它不仅将提高我们对基本生物过程的理解，而且可以 也使得能够以前所未有的精确度开发靶向这些途径的治疗方法， 功效