Mechanisms of Heat Shock Protein Regulation and Function in Muscles of a Model Teleost

肌肉细胞表达许多小的应激反应蛋白，称为小热休克蛋白，已知对细胞的生存、生长和修复至关重要。然而，这些蛋白质是如何完成它们的任务的还不清楚。拟议的研究将检查这些蛋白质在肌肉细胞中对损伤、运动和冬眠期间发生的低氧条件的反应的调节和功能。这项工作将以斑马鱼幼体作为模型系统进行，因为它们可以用来研究活组织中荧光标记蛋白质的组织和功能。这些研究的数据有望为肌肉细胞如何应对压力的潜在机制提供新的见解。这样的见解对理解或修改各种生物体的肌肉表现、工程、适应和修复的极限具有重要意义。除了产生新的信息外，该项目还将通过促进教学、培训和学习以及通过扩大未被充分代表的群体对科学的参与来造福社会。将通过开展斑马鱼幼体实践研究项目，发展和加强与地区高中的外联教育讲习班。将通过研讨会前和研讨会后的评估来评估学生的概念理解。还将为高中生创造机会，让他们参观华盛顿州立大学和邻近的爱达荷州大学的活跃调查人员的实验室并在其中工作。每年暑期将为该地区的高中科学教师提供实习机会，他们将学习适用于K-12课堂环境的斑马鱼饲养方法和斑马鱼胚胎发育分析。研究的总体目标是检验热休克诱导转录因子HSF1调节小热休克蛋白表达的假设，热休克蛋白以磷酸化依赖的方式与缺氧肌肉细胞中的细胞骨架细丝动态关联，以保持细胞骨骼的完整性和肌肉功能。该提案包括三个具体目标。首先，我们将评估HSF1在低氧反应中调节肌肉特异性小热休克蛋白表达的作用。为了实现这一点，我们将对转录因子HSF1的功能进行调控，并分析其对低氧诱导的小分子热休克蛋白表达的影响。还将对小的热休克蛋白启动子进行突变，以确定低氧诱导表达所需的基序。其次，将分析低氧肌肉细胞中小分子热休克蛋白与细胞骨架的依赖于磷酸化的相互作用动力学。将对丝氨酸/苏氨酸残基进行突变，并建立对低氧诱导的磷酸化的影响。野生型和磷酸化位点突变的热休克蛋白与细胞骨架的动态相互作用将在对照和低氧肌肉中进行检测。最后，小的热休克蛋白在肌肉中的表达将发生改变，并将评估肌肉细胞对缺氧的反应的细胞结构和功能。为了实现这一目标，将创造出在骨骼肌中过度和低表达小热休克蛋白的斑马鱼品系。在对照条件下和暴露在低氧水平后，将对幼虫和成虫的肌肉细胞结构和功能进行分析。

Muscle cells express a number of small stress responsive proteins called small heat shock proteins that are known to be critical to cell survival, growth, and repair. However, how these proteins accomplish their tasks is not understood. Proposed studies will examine the regulation and function of these proteins in muscle cells responding to low oxygen conditions that occur during injury, exercise and hibernation. The work will be conducted using zebrafish larvae as a model system because they can be used to study the organization and function of fluorescently labeled proteins in living tissues. Data resulting from these studies are expected to provide novel insight into the mechanisms underlying how muscle cells respond to stress. Such insights have implications for understanding or modifying the limits of muscle performance, engineering, adaptation and repair in a wide variety of organisms. In addition to the new information generated, the project will also benefit society by promoting teaching, training and learning, and by broadening the participation of underrepresented groups in science. Outreach educational workshops with area high schools will be developed and strengthened through conducting hands-on research projects with zebrafish larvae. Student conceptual understanding will be assessed through the use of pre-and post-workshop evaluations. Opportunities will also be created for high school students to visit and work in laboratories of active investigators at Washington State University and the neighboring University of Idaho. A summer internship will be provided annually to area high school science teachers who will learn methods for zebrafish husbandry and analysis of zebrafish embryonic development applicable to a K-12 classroom environment.The overall goal of the studies is to test the hypothesis that the heat shock inducible transcription factor, HSF1, regulates expression of small heat shock proteins, which dynamically associate with cytoskeletal filaments in a phosphorylation-dependent manner in hypoxic muscle cells to preserve cytoskeletal integrity and muscle function. The proposal includes three specific aims. First the role of HSF1 in regulating muscle specific small heat shock protein expression in response to hypoxia will be assessed. To accomplish this, the function of transcription factor HSF1 will be manipulated and effects on hypoxia inducible small heat shock protein expression analyzed. Mutagenesis of small heat shock protein promoters will also be conducted to identify motifs required for hypoxia-induced expression. Second, phosphorylation-dependent interaction kinetics of small heat shock proteins with the cytoskeleton will be analyzed in hypoxic muscle cells. Mutagenesis of serine/threonine residues will be conducted and effects on hypoxia inducible phosphorylation established. Dynamic interactions of wild-type and phosphorylation site mutant heat shock proteins with the cytoskeleton and each other will be examined in control and hypoxic muscles. Finally, small heat shock protein expression will be altered in muscles, and cytoarchitecture and function of muscle cells responding to hypoxia will be assessed. To accomplish this aim, zebrafish lines that over-, and under-express small heat shock proteins in skeletal muscle will be created. Muscle cell architecture and function will be assayed in larvae and adults under control conditions and after exposure to low oxygen levels.