MCA: Cellular Responses to Thermal Stress in Antarctic Fishes: Dynamic Re-structuring of the Proteome in Extreme Stenotherms

第一部分：这个项目的重点是栖息在南极洲寒冷水域的一组具有重要生态意义的鱼类。它们是极地食物网的关键一环，因为它们是企鹅、海豹和齿鲸的猎物。这些鱼在持续的极端寒冷中进化了数百万年，因此对全球变暖导致的水温上升非常敏感。这些研究将通过检查这些鱼类对高温的反应能力或无法反应的能力，来调查增加的热暴露对这些鱼类生物学的影响。该项目将采用尖端技术来检查细胞水平上的反应，这可能有助于这些对环境敏感的鱼类适应全球变暖的挑战。主要目标是提高我们对极地生态系统在未来几十年可能受到的影响的集体理解。第二部分：拟议的研究旨在利用一种适应寒冷的南极鱼类的现有冷冻组织库来研究蛋白质水平对热应激的反应。这些样本是在PI早期在南极洲麦克默多站进行实地考察时收集的。四种组织（对照和热应激）将通过质谱分析来表征它们的蛋白质组，定义为样品中的整个蛋白质补体。这包括这些蛋白质的鉴定和定量。研究的目的是确定南极适应极冷的低温鱼类对温度升高的反应机制。后续分析将使用免疫印迹法（Western blotting），对蛋白质组学分析中显示的热反应性蛋白质亚组进行特异性抗体。由于这是一项中期职业发展奖，因此将支持PI在蛋白质组学分析方面的培训和指导，并在合作机构加州大学戴维斯分校度过时间。虽然越来越多地使用基因组技术来探测南极物种的基因表达谱，但很少有研究关注这些生物在暴露于热应激时蛋白质水平的变化。因此，拟议的研究将代表着我们对这些对环境敏感的物种在地球持续变暖和水温上升时如何在细胞水平上做出反应的理解上的一个巨大飞跃。当蛋白质在细胞中“工作”时，了解在压力下存在哪些蛋白质、数量以及“蛋白质组”的动态是至关重要的。拟议中的研究将利用现有的样本，为数千种蛋白质提供这些信息。这些发现将是全新的，并将使我们更好地了解在寒冷中进化了数百万年的动物是如何应对气候变化的。更广泛的影响PI已与几个区域K-12机构建立了关系，并将继续以课堂访问和课堂课程创建的形式提供外展服务。PI与俄勒冈海岸水族馆（Newport， OR）有一个持续的合作，为6-8年级制作新颖的教材。水族馆在周边学区有合作伙伴，并将帮助传播有关海洋生物学和气候变化的视频。根据这一建议，将创建有关极地物种的模块。将建立一个互动网站，展示南极食物网。所有在此奖项下产生的蛋白质组学分析和文库将公开提供给任何感兴趣的研究人员使用。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Part 1: This project focuses on a group of ecologically important species of fishes which inhabit the frigid waters of Antarctica. They represent a key link in the polar food web as they are prey for penguins, seals and toothed whales. These fish have evolved in the constant, extreme cold for millions of years and therefore, are very sensitive to the increasing water temperatures associated with global warming. These studies will investigate the impacts of incremental heat exposure on the biology of these fishes by examining their ability to respond, or inability to respond, to elevated temperatures. The project will employ cutting-edge technology to examine responses at the cellular level that may help these environmentally sensitive fishes adapt to the challenges of global warming. The primary goal is to increase our collective understanding of how polar ecosystems are likely to be impacted in the coming decades.Part 2: The proposed research is designed to use an existing bank of frozen tissues from a species of cold-adapted Antarctic fish to investigate protein-level responses to heat stress. These samples were collected earlier in the PI's career during fieldwork at McMurdo Station, Antarctica. Four tissues (control as well as heat- stressed) will be analyzed via mass spectrometry to characterize their proteome, defined as the entire complement of proteins in a sample. This includes both identification and quantification of these proteins. The goal is to determine what mechanisms of response to elevated temperature are available to the extremely cold-adapted, stenothermic fishes of Antarctica. Follow-up analyses will use immunoblotting (Western blotting) with antibodies specific to a sub-set of proteins revealed to be heat-responsive in the proteomic analyses. As this is a Mid-Career Advancement Award, training and mentorship in proteomic analyses for the PI will be supported, with time spent at the partner institution, the University of California, Davis. Intellectual MeritWhile there has been an increase in the use of genomic technologies to probe gene expression profiles in Antarctic species, few studies exist looking at protein level changes during exposure to heat stress in these organisms. Therefore, the proposed studies would represent a large leap forward in our understanding of how these environmentally sensitive species can, or cannot, respond at the cellular level as the Earth continues to warm and water temperatures rise. As proteins do the "work" in the cell, it's vital to understand which proteins are present and in what quantity and how dynamic this "proteome" is during stress. The proposed studies would provide this information for thousands of proteins, using already existing samples. The findings would be entirely novel and would allow us a much better picture of how animals that evolved in the cold for millions of years are likely to respond to climate change. Broader ImpactsThe PI has established relationships with several regional K-12 institutions and will continue to provide outreach in the form of classroom visits and the creation of classroom curricula. The PI has an on-going collaboration with the Oregon Coast Aquarium (Newport, OR) to create novel teaching materials for grades 6-8. The Aquarium has partners in surrounding school districts and will help disseminate videos about marine biology and climate change. Modules concerning polar species will be created under this proposal. An interactive website will be created demonstrating the Antarctic food web. All of the proteomic analyses and libraries generated under this award will be made publicly available for use by any interested researcher.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.