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

项目总结 细胞信号是细胞相互之间以及与它们的环境和环境进行交流的过程 调节对于维持组织、器官和生物体水平的动态平衡至关重要。因为 异常的信号转导是大多数疾病发病的基础，对细胞信号转导的研究 成为细胞和分子生物学研究的中心部分。然而，当前分析细胞的方法 信令受到多个技术限制。例如，传统上对信号通路进行分析 使用生化方法对同时获得的数千个细胞的测量值进行平均， 还提供了一种全球信号环境的印象，忽略了潜在的细胞到细胞的可变性 作为分子介体的动态局部化和易位。而荧光显微镜则有 克服这一限制的可能性是，通过能够实时观测亚高压区的快速分子事件 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，我们成像细胞过程的能力已经被单一的- 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。 与此同时，先进的细胞工程工具，如CRISPR/CAS9和微图案化，现在允许我们 精确控制细胞的基因型和形态，以便于在天然细胞中对单个蛋白质进行成像 背景。这些技术已经单独成熟，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。因此，这一目标是 提案是开发和验证三项技术，这些技术结合在一起将使细胞内单一- 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有效的接合，以及(3)优化的自动图像分析算法 在不同时间尺度和注册的细胞内绘制微图案化细胞中的过程的空间图谱 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 以单分子分辨率实时研究活细胞中的细胞信号。我们将完成我们的使命 目标通过安德鲁·史密斯博士整合的多学科团队的协作工作，安德鲁·史密斯博士是 光学探测器工程和成像方面的专家，以及巴勃罗·佩雷斯-皮涅拉博士，他在 基因编辑和基因组工程。他们的实验室多年来一直在合作，以启动 此应用程序中描述的工作。从概念上讲，该平台是一种革命性的细胞分析方法 因此，它不仅将提高我们对基本生物过程的理解，而且可以 也使得针对这些途径的治疗方法的开发具有前所未有的精确度和 功效。

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.