Quantifying Small Molecules in Cells of Live Organisms

定量活体细胞中的小分子

• 批准号: 1045256
• 负责人: Joanne Chory
• 金额: $ 30万
• 依托单位: The Salk Institute for Biological Studies
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2013-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1045256&HistoricalAwards=false
• 关键词: Quantifying; Small; Molecules; Cells; Live

Small molecules form part of almost every biologically interesting signaling pathway. While they are commonly thought of as freely diffusible signals, it is likely that their localization is as tightly regulated as it is for most other components of signal transduction pathways. For example, although neurotransmitters are small, easily diffusible molecules, they remain highly localized to one part of a neuron, the synaptic cleft. On a larger scale, the developmentally important plant hormone auxin undergoes active transport within the plant that directs its localization to appropriate sites of action. Fluorescent protein tagging or immunofluorescence microscopy allows imaging of almost any protein of interest. In contrast to proteins, however, small molecules cannot be optically tagged or detected by antibodies, and so direct measurements of their localization and concentration in live, growing organisms are currently impossible in most cases. The goal of this proposal is to address these limitations by developing a generalizable procedure for quantitative real-time optical sensing of biologically active small molecules in living cells and whole organisms. Localized sensing of auxin and brassinosteroids (BRs) in Arabidopsis thaliana will serve as a proof-of-principle in addition to providing new insights into the interactions between these two hormone-signaling pathways. Once validated, this approach to detecting small molecules in living cells should be easily applicable to any small molecule of interest in any model organism, opening up many new approaches to the study of signal transduction. In a set of complementary experiments that are specific to plants, reporter plants that monitor BR or auxin receptor activation will be developed and made available to the community. Together with the first approach, these tools for hormone signaling will contribute to a deeper knowledge of plant growth and development, which can be used to develop predictive models for how plants cope with various environmental stresses. This project will also provide the opportunity to train excellent postdoctoral fellows in a multidisciplinary and forward-thinking area of biology.

小分子构成了几乎所有生物学上有趣的信号通路的一部分。虽然它们通常被认为是自由扩散的信号，但它们的定位很可能与信号转导途径的大多数其他成分一样受到严格调节。例如，虽然神经递质是小而容易扩散的分子，但它们仍然高度局限于神经元的一个部分，即突触间隙。在更大的范围内，植物发育中重要的植物激素生长素在植物内部进行主动运输，将其定位到适当的作用位点。荧光蛋白标记或免疫荧光显微镜允许成像几乎任何感兴趣的蛋白质。然而，与蛋白质相比，小分子不能被光学标记或被抗体检测，因此在大多数情况下，直接测量它们在活的、生长的生物体中的定位和浓度是不可能的。本提案的目标是通过开发一种可用于活细胞和整个生物体中生物活性小分子定量实时光学传感的通用程序来解决这些限制。拟南芥中生长素和油菜素内固醇（BRs）的局部感知除了为这两种激素信号通路之间的相互作用提供新的见解外，还将作为原理证明。一旦得到验证，这种检测活细胞小分子的方法应该很容易适用于任何模式生物中感兴趣的任何小分子，为信号转导的研究开辟了许多新的方法。在一系列针对植物的补充实验中，监测BR或生长素受体激活的报告植物将被开发并提供给社区。与第一种方法一起，这些激素信号传导工具将有助于更深入地了解植物的生长和发育，这可以用来开发植物如何应对各种环境胁迫的预测模型。该项目还将为培养生物学多学科和前瞻性领域的优秀博士后提供机会。