Mechanisms of osmosensing and osmotic stress responses in tilapia

在理解鱼类和许多其他动物渗透调节的效应机制方面已经取得了显著的进展。相比之下，对这些效应器的调节机制、环境中的渗透变化是如何被感知的，以及渗透感觉信息是如何通过细胞内信号通路传递到渗透调节效应器的知之甚少。通过从强健和易处理的渗透压调节效应器系统开始探讨这些问题，以确定其调节元件，该项目解决了目前关于鱼类渗透调节/液体和电解质动态平衡的知识中的一大空白。罗非鱼(Oreochromis Mossambius)是研究渗透感受和渗透胁迫信号机制的极佳模型，因为它们能耐受极大范围的环境盐度。它们的基因组已经被测序，蛋白质组也得到了很好的注释。该奖项支持的研究将调查与高渗诱导肌醇生物合成途径相关的机制和意义。这个研究项目具有广泛的生物学意义，因为肌醇和磷酸肌苷信号以及渗透胁迫反应在所有真核生物中都是常见的。这对农业发展具有基本意义，因为研究与激活亲和渗透分子合成途径相关的机制和影响可能导致提高耐盐性和抗旱性。了解肌醇在调节关键的细胞内信号通路和能量动态平衡中的作用，对于应激相关的疾病也具有重要意义。PIS实验室在过去已经产生了大量的资源来推进罗非鱼渗透感觉和渗透调节机制的研究，包括定量蛋白质组学工作流程和几个高渗透压耐受细胞系。初步数据表明，在罗非鱼组织和细胞系中，参与这一途径的两种酶，即肌醇磷酸合成酶(MIPS)和肌醇单磷酸酶1(IMPase 1)，以及肌醇水平在高渗胁迫下都高度上调。因此，对于拟议的研究来说，这一途径代表了一个强大的系统。该项目旨在确定MIPS和IMPase1基因中的渗透压响应顺式元件，并验证这些元件是高渗诱导肌醇生物合成途径所必需的假说。此外，MIPS和IMPase1影响细胞渗透调节的假说不只是肌醇是一种相容的渗透调节物质，这一假设将得到验证。具体地说，MIPS和IMPase 1直接与其他参与渗透保护的蛋白质相互作用，它们的调节间接影响细胞磷脂信号转导(PI3K/PTEN/Akt和PLC/PKC/IP3通路)和能量代谢(通过隔离葡萄糖-6-磷酸)。该项目使用复杂的蛋白质组学工具和工作流程，以前所未有的方式捕捉与渗透胁迫信号相关的分子表型。这种独特的资源组合将显著增强我们对渗透胁迫信号机制的理解，并为进化驱动力提供新的见解，这些驱动力将肌醇塑造为大多数生物体中的关键代谢物。该项目的活动和成果的传播将通过同行评议出版物、会议报告、研讨会和更广泛的外联途径进行，包括让K12学生和教育工作者、水产养殖生产者、社区团体和保护组织参与研究。将开发一个公共实验室网站，以展示研究和外联活动。将培训两名研究生，每人指导一名本科生实习生。将优先招收代表性不足的少数族裔学生。

Remarkable progress has been made in understanding the effector mechanisms of osmoregulation in fishes and many other animals. In contrast, less is known about the mechanisms by which these effectors are regulated, how osmotic changes in the environment are perceived, and how osmosensory information is transduced via intracellular signaling pathways to the osmoregulatory effectors. By approaching these questions starting from a robust and tractable osmoregulatory effector system to identify its regulatory elements, the project addresses a large gap in the current knowledge about fish osmoregulation/ fluid and electrolyte homeostasis. Tilapia (Oreochromis mossambicus) represent a superb model for studying mechanisms of osmosensing and osmotic stress signaling because they tolerate an extremely wide range of environmental salinity. Their genome has been sequenced and proteome well-annotated. The research supported by this award will investigate the mechanisms and implications associated with hyperosmotic induction of the myo-inositol biosynthesis pathway. This research project has broad implications for biology because myo-inositol and phosphoinosite signaling as well as osmotic stress responses are common to all eukaryotes. It has basic implications for agricultural development because studying mechanisms and implications associated with activation of compatible osmolyte synthesis pathways could lead to increasing salt and drought tolerance. Understanding the role of myo-inositol in the regulation of key intracellular signaling pathways and energy homeostasis is also significant in the light of stress-related disorders. The PIs lab has generated significant resources in the past to advance the study of osmosensory and osmoregulatory mechanisms in tilapia, including quantitative proteomics workflows and several highly osmotolerant cell lines. Preliminary data show that both enzymes involved in this pathway, myo-inositol phosphate synthase (MIPS) and inositol monophosphatase 1 (IMPase 1), as well as myo-inositol levels are extremely highly upregulated during hyperosmotic stress in multiple tilapia tissues and cell lines. Thus, this pathway represents a robust system for the proposed studies. The project aims to identify osmoresponsive cis elements in the MIPS and IMPase1 genes and test the hypothesis that such elements are necessary for hyperosmotic induction of the myo-inositol biosynthesis pathway. Moreover, the hypothesis that MIPS and IMPase1 influence cellular osmoregulation beyond myo-inositol being a compatible osmolyte will be tested. Specifically, it is proposed that MIPS and IMPase 1 directly interact with other proteins involved in osmoprotection and that their regulation indirectly affects cellular phosphoinosite signaling (PI3K/ PTEN/Akt and PLC/PKC/IP3 pathways) and energy metabolism (by sequestering glucose-6-phosphate). The project uses sophisticated proteomics tools and workflows to capture molecular phenotypes associated with osmotic stress signaling in an unprecedented fashion. This unique combination of resources will significantly enhance our understanding of osmotic stress signaling mechanisms and provide novel insight into evolutionary driving forces that have shaped myo-inositol as a key metabolite in most organisms. Dissemination of activities and results of this project will be done via peer-reviewed publications, conference presentations, seminars, and broader outreach avenues, including engagement of K12 students and educators, aquaculture producers, community groups, and conservation organizations in research. A public lab website will be developed to showcase research and outreach activities. Two graduate students will be trained and each of them will supervise an undergraduate intern. Priority will be given to recruit students from underrepresented minorities.