Abstract
Mitogen-activated protein kinases (MAPKs) are key regulators of numerous biological processes in plants. To better understand the mechanisms by which these kinases function, high resolution measurement of MAPK activation kinetics in different biological contexts would be beneficial. One method to measure MAPK activation in plants is via fluorescence-based genetically-encoded biosensors, which can provide real-time readouts of the temporal and spatial dynamics of kinase activation in living tissue. Although fluorescent biosensors have been widely used to study MAPK dynamics in animal cells, there is currently only one MAPK biosensor that has been described for use in plants. To facilitate creation of additional plant-specific MAPK fluorescent biosensors, we report the development of two new tools: an in vitro assay for efficiently characterizing MAPK docking domains and a translocation-based kinase biosensor for use in plants. The implementation of these two methods has allowed us to expand the available pool of plant MAPK biosensors, while also providing a means to generate more specific and selective MAPK biosensors in the future. Biosensors developed using these methods have the potential to enhance our understanding of the roles MAPKs play in diverse plant signaling networks affecting growth, development, and stress response.
Highlights
Conserved across all eukaryotes [1], mitogen-activated protein kinases (MAPKs) have been shown to play critical roles in a wide array of biological processes
In order to establish a pipeline for efficiently developing new plant MAPK fluorescent biosensors, we first set out to establish an in vitro assay for efficiently identifying new plant MAPK docking domains
The important functional domains of EKAREV are the YPet yellow fluorescent protein domain [56], the mTurquoise-GL cyan fluorescent protein domain [57], the EV linker [48], the FHA1 phosphoamino acid binding domain [58,59], a 14 amino acid phosphoacceptor domain from the mammalian Cdc25C protein [60], and a docking domain recognized by the human extracellular signal-regulated kinases (ERKs) [61] (Figure 1A), which are a type of MAPK
Summary
Conserved across all eukaryotes [1], mitogen-activated protein kinases (MAPKs) have been shown to play critical roles in a wide array of biological processes. Biochemical methods to measure MAPK signaling have provided valuable insight into MAPK function within these pathways, these methods generally report the average MAPK activity within a population of cells and cannot provide cell-level resolution of the spatiotemporal dynamics these kinases display in different biological contexts. One approach that can allow one to measure MAPK activation with greater resolution involves the use of fluorescence-based genetically-encoded biosensors [26,27]. Fluorescent biosensors provide an quantifiable, real-time readout of a targeted parameter in living cells, elucidating the roles of different biomolecules in the complex dynamics that underlie cellular networks [28]. A variety of fluorescent biosensors have been developed to shed light on the mechanisms by which MAPKs function and are regulated in animal cells [27]; the most common of these biosensors are Förster resonance energy transfer (FRET) biosensors [26,29,30,31,32,33,34,35,36]
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