Quantifying Small Molecules in Cells of Live Organisms

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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.