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: 7244060
• 负责人: MARC A OSTERMEIER
• 金额: $ 11.63万
• 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 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7244060
• 关键词: Address; Aging; Alzheimer' s Disease; Apoptosis; Binding; Biological; Biological Process; Biology; Biosensor; Cell physiology; Cells; Code; Coupling; Cultured Cells; Detection; Development; Diabetes Mellitus; Disease; Enzymes; Fluorescence; Gene Expression Regulation; Genes; Genetic Recombination; Genomics; Goals; Homeostasis; Hybrids; Image; In Situ; In Vitro; Invasive; Lactamase; Life; Malignant Neoplasms; Maltose; Methodology; Molecular; Monitor; Mutation; National Institute of General Medical Sciences; Neurons; Organism; Pathway interactions; Patients; Pattern; Play; Property; Proteins; Proteomics; Regulation; Research; Research Personnel; Research Proposals; Resolution; Risk; Role; Second Messenger Systems; Sensitivity and Specificity; Signal Transduction; Techniques; Technology; Time; Tissues; United States National Institutes of Health; Zinc; beta-Lactamase; cellular targeting; desire; enzyme activity; fluorescence imaging; genetic selection; innovation; maltose-binding protein; metabolomics; novel; programs; protein B; protein function; response; second messenger; sensor; small molecule; technology development; tool; trafficking; zinc-binding protein

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计划着重于研究细胞功能的新基因组学和蛋白质组学工具的开发和利用。相比之下，相对较少的研究计划试图类似地解决该代谢组。在活细胞，组织和生物中，时间和空间上解决代谢产物，小分子和第二个使者的存在，身份，浓度和通量的能力将在研究基本生物学和生物医学问题上具有广泛的适用性。这种能力将在理解细胞稳态，发展和衰老所涉及的潜在途径方面发挥重要作用。现有方法主要是高度侵入性，有毒的，并且缺乏所需的灵敏度，特异性，时间分辨率和细胞亚靶向。此外，大多数方法是特定于特定分析物的，并且不容易概括。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，以识别和量化细胞代谢物和第二信使及其在高解剖，空间和时间分辨率的原位下的通量。我们建议创建新型的蛋白质开关，使小分子水平与β-内酰胺酶活性和蛋白质荧光。这将通过现有蛋白质的体外重组，并通过PI最近开发的技术具有可行的小分子结合和催化/荧光性能。具体而言，我们计划开发Zn（LL）的生物传感器，以证明我们方法的原理。除了其结构和催化作用外，锌还在包括酶调节，基因表达的调节，凋亡和细胞间信号传导的调节中起着不同的作用。此外，在阿尔茨米尔氏病，糖尿病和癌症患者中发现了细胞内锌（LL）积累的异常模式。尽管角色多样化，但它的重要性直到最近才变得清晰，并且围绕细胞内锌积累，运输和功能的机械细节却很差。尽管我们建议开发Zn（LL）的传感器，这些传感器将在理解锌在生物学中的作用中具有重要的应用，但更广泛，更重要的目标是展示我们为小分子，代谢物和第二个使者的实时细胞荧光成像制造传感器的一般策略。