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.
All organisms—from bacteria to plants to humans— biosynthesize and use small molecules for a variety of purposes. These benefits include control of metabolism, regulation of growth and development, response to biotic and abiotic factors in the environment and modulation of nervous system function. While such signaling is ubiquitous in nature, we currently have no way to non-invasively track these molecules in living organisms at a cellular or sub-cellular level. The goal of this project was to develop a method to optically sense the concentration of small molecules in living organisms using signal amplification and fluorescence imaging. Fluorescent proteins, originally discovered in jellyfish and corals, have been developed into multicolored tools that allow us to visualize processes in living cells. Using fluorescent proteins coupled to engineered protein domains that respond to the binding of specific small molecules, our plan was to develop a system that will turn a chemical signal (the concentration of a specific small-molecule compound) into an optical signal (a change in the color of fluorescence emission from the cells). Our initial goal was the detection of two hormones important for plant growth and development: auxin and brassinosteroids. We used the wild mustard Arabidopsis thaliana, a standard model plant, as the system in which to test our sensors. Because concentrations of the desired hormones are likely in the nanomolar to sub-nanomolar range, we are using mammalian and bacterial kinases as amplifiers for our sensing system. As a target for a proof-of-concept sensor, we used the human estrogen receptor alpha, which conditionally interacts with the LXXLL motif of coactivators upon estrogen binding. Our preliminary data indicate that it may be possible to modulate the action of a human src tyrosine kinase on a FRET sensor via estrogen. The next steps were focused on obtaining binding domains that recognize auxin and BL with specificity and affinity appropriate to physiological concentrations of these hormones. Specifically, we wanted to make mutant forms of the human estrogen receptor that could bind brassinosteroids or auxin. The human receptor for estrogen is not found in the plant kingdom, so we reasoned that expressing it in plant cells would not interfere with native pathways. With the Evans lab, we have screened human nuclear receptors for response to these hormones, but to our chagrin, we found none that would respond in their wild type configuration. Conclusions: Expression of the estrogen receptor results in simple single cell systems gave rise to low signal to noise read-outs. Therefore, estrogen receptor is unlikely to work well in a complex organism such as plants and our results are not reliable enough to evolve these proteins in E.coli. In retrospect, we built a "top of the line" sensor. It might have been too much to ask for within a single year. We are now taking a step back and working on more primitive sensors.
