The Role of G Protein-coupled Receptors in Red Tide Dinoflagellate Bioluminescence

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Project Summary Red tide blooms significantly cause coastal ecosystem damage and a series of environmental diseases on people who live nearby the coast or eat the red tide contaminated seafoods. Despite their ecological and environmental significance, research in mechanistically understanding the red tide bloom occurrence is limited. Plasma membrane G protein-coupled receptor (GPCRs) are widely implicated in multi-cellular eukaryotic signal transductions, but their roles in the unicellular eukaryotic organism have not been well-explored. Recent studies implied the involvements of primitive GPCRs in the bioluminescent pathway of dinoflagellates, including Lingulodinium polyedra. L. polyedra is a prolific single-celled dinoflagellate implicated in red tide events and highly sensitive to wave turbulence and predator behavior. Gaq-type heterotrimeric G proteins, one of major signal pathways in the GPCR signaling, have been extensively reported for their associations with mechanochemical transducer. Our preliminary bioinformatic analysis indicate a set of L. polyedra GPCRs are implied in the bioluminescent pathway. The top 5 receptor candidates (RC1 through RC5) were subjected to anti-sense DNA oligonucleotide knockdown experiments. The knockdowns indicate that RC3 is a promising candidate involved in the bioluminescent signaling. Based on our preliminary data, this R21 project will further explore the role of RC3 in dinoflagellate L. polyedra bioluminescence, starting from confirmation its shear force- sensitivity, molecularly determining its role in bioluminescent signaling and biophysically characterizing the receptor and related signaling partners. The project’s three specific aims include the following: Aim 1, RT-qPCR will be used for evaluating the mRNA levels of RC3 and its effectors in the signaling pathway. CRISPR/Cas9 knockout strategy will also be used for validating the role of RC3 in shear-force elicited bioluminescence signaling. In Aim 2, we will first heterogeneously express RC3 in a yeast-based GPCR preparation system and then use monobromobiamine-based florescent spectroscopy to study its conformational responses to shear-forces and copepodamide lipids. In Aim 3, we will determine whether RC3 related bioluminescence signal pathway is Gaq- dependent, using both knockdown and knockout experiments along with in vitro measurements, including GTPase-GloTM assay and [3H]GDP based GDP release assay. The R21 project data will determine if the dinoflagellate derived GPCR—RC3 is associated with shear force-induced bioluminescence. The project’s completion will establish a priming foundation to completely study the molecular mechanism of GPCR signaling in the red tide blooms, with the far-reaching significance of controlling or modulating bloom occurrences.