Mechanisms of osmosensing and osmotic stress responses in tilapia

在理解鱼类和许多其他动物中的调节的效应器机制方面已经取得了显着进展。与此相反，很少有人知道的机制，这些效应器的调节，如何在环境中的渗透变化被认为是，以及如何通过细胞内信号转导通路的感觉信息的神经调节效应器。通过从一个强大的和易处理的鱼类调节效应器系统开始，以确定其调节元件接近这些问题，该项目解决了目前关于鱼类调节/液体和电解质稳态的知识中的一个很大的差距。 罗非鱼（Oreochromis mossambicus）是研究盐度敏感和渗透胁迫信号传导机制的极好模型，因为它们耐受的环境盐度范围极广。它们的基因组已被测序，蛋白质组也被很好地注释。 该奖项支持的研究将调查与肌醇生物合成途径的高渗诱导相关的机制和影响。 这个研究项目对生物学有着广泛的意义，因为肌醇和磷酸肌醇信号以及渗透胁迫反应对所有真核生物都是共同的。它对农业发展具有基本意义，因为研究与兼容的渗透剂合成途径的激活相关的机制和影响可能导致增加盐和干旱耐受性。了解肌醇在调节关键细胞内信号传导途径和能量稳态中的作用在应激相关疾病中也很重要。PI实验室在过去已经产生了大量的资源，以推进罗非鱼的感官和神经调节机制的研究，包括定量蛋白质组学工作流程和几个高度耐受性的细胞系。初步数据显示，这两种酶参与这一途径，肌醇磷酸合酶（MIPS）和肌醇单磷酸酶1（IMPase 1），以及肌醇水平在高渗胁迫期间在多个罗非鱼组织和细胞系中非常高度上调。因此，该途径代表了用于拟定研究的稳健系统。该项目的目的是确定MIPS和IMPase 1基因中的高渗响应顺式元件，并测试这样的元件是肌醇生物合成途径的高渗诱导所必需的假设。此外，将检验MIPS和IMPase 1影响细胞膜调节的假设，即肌醇是相容的渗透剂。具体而言，提出MIPS和IMPase 1直接与参与细胞保护的其他蛋白质相互作用，并且它们的调节间接影响细胞磷酸肌醇信号传导（PI 3 K/ PTEN/Akt和PLC/PKC/IP 3途径）和能量代谢（通过螯合葡萄糖-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获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着提高我们的理解渗透压信号传导机制，并提供新的洞察力的进化驱动力，塑造了肌醇作为一个关键的代谢产物在大多数生物体。该项目的活动和成果的传播将通过同行评审的出版物，会议演示，研讨会和更广泛的宣传渠道进行，包括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，以展示研究和推广活动。 两名研究生将接受培训，每人将监督一名本科实习生。 将优先招收代表性不足的少数民族学生。

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.