RUI: MCB: the effect of stretch on giant cytoskeletal protein structure/function

RUI：MCB：拉伸对巨细胞骨架蛋白结构/功能的影响

• 批准号: 1607024
• 负责人: Nathan Wright
• 金额: $ 29.16万
• 依托单位: James Madison University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2021-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1607024&HistoricalAwards=false
• 关键词: RUI; MCB; effect; stretch; giant

Cells live in a world full of motion and physical strain, and therefore they must have mechanisms to react to these stresses. One platform cells use to sense and respond to stretch is the cytoskeleton. This meshwork of multiple different proteins gives a cell its shape, yet is constructed in a way that allows flexibility and motion. This research is focused on the role that the cytoskeletal protein obscurin plays in stretch response and recognition. Obscurin acts like a tether, connecting far-away segments of the cell to each other. Due to its shape, obscurin has the capacity to expand and contract. There are two possibilities of how obscurin could move like this. Obscurin could behave like a rope, and only resist stretch when significantly elongated, or obscurin could behave like a spring, and resist an ever-increasing amount of force as it is stretched. Obscurin also can propagate biochemical signals, and there is circumstantial evidence that this function could be activated by stretch. The PI will test both facets of obscurin function - stretch response and signaling. Together, these studies will provide insight into how cells passively and actively respond to physical stretch. This work will be conducted primarily by undergraduate students, in an effort to train the next generation of scientists. The data accrued here will be incorporated into a free education website, where others who do not have access to significant research support can also learn the technical skills of how to do this kind of research. Additionally, the PI will develop a scientific ethics curriculum for undergraduates. Specifically, the investigator will study how obscurin reacts to physical stretch using both cellular and in vitro models. Obscurin is composed of multiple stand-alone domains. Many of these individual domains have been characterized extensively. In Aim 1, the PI will characterize how these domains act in groups of two or three. Using protein NMR techniques, the cross-talk between neighboring domains will be measured. Small angle X-ray scattering (SAXS) provide a dynamic model of obscurin's overall shape. These experimental approaches will be complimented through computer simulations to test how these multi-domain systems respond to stretch. Together, these experiments will detail how obscurin resists force. In Aim two, the PI will test what effect obscurin has on the whole cell when the cell is stretched. Cells with and without obscurin will be stretched, and the biochemical consequences of obscurin's presence in these conditions will be analyzed. Together, these two aims will help define how obscurin behaves as a stretch resistor, and will answer the question of whether or not obscurin is a mechanosensor.

细胞生活在一个充满运动和物理压力的世界里，因此它们必须有对这些压力作出反应的机制。细胞用来感知和响应拉伸的一个平台是细胞骨架。这种由多种不同蛋白质组成的网状结构构成了细胞的形状，但其构造方式却允许细胞具有灵活性和运动性。本研究的重点是细胞骨架蛋白在拉伸反应和识别中的作用。暗盲蛋白的作用就像一根绳，将细胞的不同片段彼此连接起来。由于它的形状，它具有扩张和收缩的能力。有两种可能来解释为什么暗波林会这样移动。Obscurin可以表现得像一根绳子，只有在明显拉长时才能抵抗拉伸，或者Obscurin可以表现得像一个弹簧，在拉伸时能够抵抗不断增加的力。暗素还可以传播生化信号，有间接证据表明，这种功能可以通过拉伸激活。PI将测试蒙皮蛋白功能的两个方面——拉伸反应和信号。总之，这些研究将深入了解细胞是如何被动和主动地对身体拉伸做出反应的。这项工作将主要由本科生进行，以培养下一代科学家。这里积累的数据将被纳入一个免费的教育网站，在那里，其他无法获得重要研究支持的人也可以学习如何进行这类研究的技术技能。此外，PI将为本科生开发科学伦理课程。具体来说，研究者将使用细胞和体外模型来研究奥布林对物理拉伸的反应。Obscurin由多个独立域组成。许多这些单独的领域已经被广泛地描述了。在Aim 1中，PI将描述这些结构域如何以两个或三个为一组进行作用。利用蛋白质核磁共振技术，相邻结构域之间的串扰将被测量。小角x射线散射（SAXS）提供了一种微球整体形状的动态模型。这些实验方法将通过计算机模拟加以补充，以测试这些多域系统如何响应拉伸。总之，这些实验将详细说明模糊蛋白是如何抵抗力的。在第二项测试中，PI将测试当细胞被拉伸时，暗色蛋白对整个细胞的影响。有和不含奥布林的细胞将被拉伸，并分析在这些条件下奥布林存在的生化后果。总之，这两个目标将有助于定义obscurin作为拉伸电阻的行为，并将回答obscurin是否为机械传感器的问题。