Quantifying Small Molecules in Cells of Live Organisms

小分子构成了几乎所有生物学上感兴趣的信号通路的一部分。虽然它们通常被认为是可自由扩散的信号，但它们的定位可能与信号转导途径的大多数其他组分一样受到严格调控。例如，虽然神经递质是小的、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，发育重要的植物激素生长素在植物内进行主动运输，将其定位到适当的作用部位。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，与蛋白质相反，小分子不能被光学标记或被抗体检测到，因此在大多数情况下，直接测量它们在活的生长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推广的程序来解决这些限制，该程序用于活细胞和整个生物体中生物活性小分子的定量实时光学传感。在拟南芥中生长素和油菜素甾醇（BR）的局部传感将作为一个证明的原则，除了提供新的见解这两个信号通路之间的相互作用。一旦得到验证，这种检测活细胞中小分子的方法应该很容易适用于任何模式生物中的任何感兴趣的小分子，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，将开发监测BR或生长素受体激活的报告植物，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生长和发育，可用于开发植物如何科普各种环境压力的预测模型。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研究员提供机会。

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.