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

项目摘要 赤潮暴发严重破坏沿海生态系统，引发一系列环境疾病 居住在海岸附近或食用受赤潮污染的海鲜的人。尽管它们的生态和 对于赤潮发生的环境意义，从机理上认识赤潮发生的研究是有限的。 质膜G蛋白偶联受体(GPCRs)广泛参与多细胞真核信号 转导，但它们在单细胞真核生物中的作用还没有得到很好的研究。最新研究 暗示原始GPCRs参与了甲藻的生物发光途径，包括 多叶灵芝Linguloddium polyedra.多叶藻是一种多产的单细胞甲藻，与赤潮事件和 对波浪湍流和捕食者行为高度敏感。Gaq型异三聚体G蛋白是主要的 GPCR信号中的信号通路已被广泛报道，因为它们与 机械力化学换能器我们的初步生物信息学分析表明，一组多粒乳杆菌的GPCR是 隐含在生物发光途径中。前5个候选受体(Rc1到Rc5)受到 反义DNA寡核苷酸敲除实验。击倒表明RC3是一种很有前途的 参与生物发光信号传递的候选人。根据我们的初步数据，这个R21项目将进一步 从确认RC3的剪切力入手，探讨RC3在甲藻生物发光中的作用。 敏感性，从分子上确定它在生物发光信号中的作用，并从生物物理学的角度表征 受体和相关信号伙伴。该项目的三个具体目标包括：目标1、RT-qPCR 将用于评估RC3及其效应分子在信号通路中的mRNA水平。CRISPR/CAS9 基因敲除策略也将用于验证RC3在剪切力诱导的生物发光信号中的作用。 在目标2中，我们将首先在基于酵母的gpr制备系统中异源表达rc3，然后使用 基于单溴双胺的荧光光谱研究其构象对剪切力和 可波达胺类脂类。在目标3中，我们将确定RC3相关的生物发光信号通路是否为Gaq- 依赖，使用基因敲除和基因敲除实验以及体外测量，包括 GTPase-GloTM法和基于[~3H]GDP的GDP释放法。R21项目数据将决定是否 甲藻来源的GPCR-RC3与剪切力诱导的生物发光有关。该项目的 该研究的完成将为全面研究gpr信号的分子机制奠定基础。 赤潮中的水华，对控制或调控水华的发生具有深远的意义。

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.