Mechanisms of osmosensing and osmotic stress responses in tilapia

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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.