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

第一部分：这个项目关注的是生活在南极洲寒冷水域的一组重要的生态鱼类。它们代表着极地食物链中的关键一环，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，对与全球变暖相关的水温上升非常敏感。这些研究将通过检测这些鱼对高温的反应能力或无能来调查增加的热暴露对这些鱼生物学的影响。该项目将使用尖端技术来检查细胞水平的反应，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。主要目标是增加我们对极地生态系统在未来几十年可能受到影响的集体理解。第二部分：拟议的研究旨在利用一种适应寒冷的南极鱼类现有的冰冻组织库来研究蛋白质水平对热应激的反应。这些样本是在少年派职业生涯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(对照组织和热应激组织)将通过质谱仪进行分析，以确定其蛋白质组的特征，蛋白质组的定义是样本中的全部蛋白质。这包括对这些蛋白质的鉴定和定量。其目标是确定南极洲适应极端寒冷、低温的鱼类可以利用哪些对温度升高的反应机制。后续分析将使用免疫印迹(Western Blotting)，在蛋白质组分析中显示对热响应的蛋白质子集的特异性抗体。由于这是一个职业中期促进奖，将支持对PI进行蛋白质组学分析方面的培训和指导，并在伙伴机构加州大学戴维斯分校花费时间。智力价值虽然越来越多地使用基因组技术来探索南极物种的基因表达谱，但很少有研究关注这些生物在受热应激期间蛋白质水平的变化。因此，拟议的研究将代表着我们对这些环境敏感物种如何在细胞水平上做出反应的理解上的一大飞跃，因为地球继续变暖，水温上升。当蛋白质在细胞中进行“工作”时，了解哪些蛋白质存在，以多少数量存在，以及这种“蛋白质组”在应激过程中的动态程度是至关重要的。拟议中的研究将利用现有的样本，为数千种蛋白质提供这一信息。这一发现将是全新的，将让我们更好地了解在寒冷中进化了数百万年的动物可能会如何应对气候变化。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开设课堂课程的形式提供外联服务。PI与俄勒冈海岸水族馆(俄勒冈州纽波特)持续合作，为6-8年级制作新的教材。水族馆在周围的学区有合作伙伴，并将帮助传播有关海洋生物和气候变化的视频。根据这项提议，将建立关于极地物种的单元。将创建一个互动网站，展示南极食物网。根据该奖项产生的所有蛋白质组分析和文库将公开供任何感兴趣的研究人员使用。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.