Live Cell Fluorescence Imaging Using Molecular Switches

使用分子开关进行活细胞荧光成像

• 批准号: 7022699
• 负责人: MARC A OSTERMEIER
• 金额: $ 11.97万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-06-01 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7022699
• 关键词: binding proteins; bioimaging /biomedical imaging; biomedical equipment development; biosensor device; fluorescence; gene mutation; genetic recombination; genetic screening; maltose; metal metabolism; molecular /cellular imaging; neurons; time resolved data; tissue /cell culture; zinc

DESCRIPTION (provided by applicant): Many ongoing research programs focus on the development and utilization of new genomics and proteomic tools for studying cellular function. In contrast, relatively few research programs seek to analogously address the metabolome. The ability to temporally and spatially address the presence, identity, concentrations and flux of metabolites, small molecules and second messengers in live cells, tissues and organisms would have wide applicability to the study of basic biological and biomedical problems. Such abilities would play a major role in understanding the underlying pathways involved in cellular homeostasis, development and aging. Existing approaches predominantly are highly-invasive, toxic and lack the desired sensitivity, specificity, temporal resolution and sub-cellular targeting. Furthermore, most approaches are specific to a particular analyte and are not easily generalizable. This proposal seeks to develop a versatile platform for developing protein biosensors for identifying and quantifying cellular metabolites and second messengers and their fluxes at high anatomical, spatial, and temporal resolution in situ. We propose to create novel protein switches that couple small-molecule levels to beta-lactamase enzyme activity and to protein fluorescence. This will be accomplished through the in vitro recombination of existing proteins with the perquisite small-molecule binding and catalytic/fluorescent properties by a technology recently developed by the PI. Specifically, we plan to develop biosensors for Zn(ll) as a proof of principle of our approach. In addition to its structural and catalytic roles, zinc plays diverse roles in biological processes including the regulation of enzymes, the regulation of gene expression, apoptosis and cell-to-cell signaling. In addition, abnormal patterns of intracellular Zn(ll) accumulation have been found in patients with Alzhemier's disease, diabetes and cancer. Despite its diversity of roles, its importance has only recently become clear and the mechanistic details surrounding intracellular zinc accumulation, trafficking and function are poorly defined. Although we propose to develop sensors for Zn(ll) that will have important applications for understanding zinc's role in biology, the broader and more significant goal is to demonstrate our general strategy for making sensors for live cell fluorescence imaging of small molecules, metabolites and second messengers.

描述（由申请人提供）：许多正在进行的研究项目侧重于开发和利用新的基因组学和蛋白质组学工具来研究细胞功能。相比之下，相对较少的研究项目试图类似地解决代谢组问题。在时间和空间上解决活细胞、组织和生物体中代谢物、小分子和第二信使的存在、身份、浓度和通量的能力将广泛适用于基本生物学和生物医学问题的研究。这种能力将在理解细胞稳态、发育和衰老的潜在途径方面发挥重要作用。现有的方法主要是高度侵入性的、有毒的，并且缺乏所需的敏感性、特异性、时间分辨率和亚细胞靶向。此外，大多数方法都是针对特定分析物的，并且不容易推广。该提案旨在开发一个多功能平台，用于开发蛋白质生物传感器，用于在原位以高解剖、空间和时间分辨率识别和量化细胞代谢物和第二信使及其通量。我们建议创建新型蛋白质开关，将小分子水平与 β-内酰胺酶活性和蛋白质荧光耦合。这将通过 PI 最近开发的技术，通过现有蛋白质的体外重组以及必要的小分子结合和催化/荧光特性来实现。具体来说，我们计划开发 Zn(ll) 生物传感器作为我们方法原理的证明。除了其结构和催化作用外，锌还在生物过程中发挥多种作用，包括酶的调节、基因表达的调节、细胞凋亡和细胞间信号传导。此外，在阿尔茨海默病、糖尿病和癌症患者中也发现了细胞内 Zn(II) 积累的异常模式。尽管其作用多种多样，但其重要性直到最近才变得清晰，并且围绕细胞内锌积累、运输和功能的机制细节尚不清楚。尽管我们建议开发 Zn(II) 传感器，这对于了解锌在生物学中的作用具有重要的应用，但更广泛和更重要的目标是展示我们制造用于小分子、代谢物和第二信使活细胞荧光成像的传感器的总体策略。