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

细胞生活在一个充满运动和生理压力的世界里，因此它们必须有对这些压力做出反应的机制。细胞用来感知和对拉伸做出反应的一个平台是细胞骨架。这种由多个不同蛋白质组成的网络赋予细胞其形状，但其构建方式允许灵活性和运动性。本研究的重点是细胞骨架蛋白Oblcurin在拉伸反应和识别中的作用。Obscurin的作用就像一根系绳，将细胞遥远的部分相互连接起来。由于其形状，暗黑蛋白具有膨胀和收缩的能力。有两种可能的暗黑物质会像这样移动。Obscurin可以像绳子一样工作，只有在显著拉长的情况下才能抵抗拉伸，或者Obscurin可以像弹簧一样工作，并在拉伸时抵抗不断增加的力。蛇床子素还可以传播生化信号，有间接证据表明，拉伸可以激活这一功能。PI将测试Oblcurin功能的两个方面--拉伸反应和信号传递。总之，这些研究将为细胞如何被动和主动地对物理拉伸做出反应提供洞察力。这项工作将主要由本科生进行，以努力培养下一代科学家。这里收集的数据将被纳入一个免费教育网站，在那里，其他没有获得重大研究支持的人也可以学习如何进行此类研究的技术技能。此外，PI还将为本科生开发一门科学伦理课程。具体地说，研究人员将使用细胞和体外模型研究暗黑蛋白对物理拉伸的反应。Obscurin由多个独立的域组成。其中许多单独的领域已经被广泛地描述了。在目标1中，PI将描述这些域如何以两个或三个为一组的方式起作用。利用蛋白质核磁共振技术，将测量相邻结构域之间的串扰。小角X射线散射(SAXS)提供了暗影整体形状的动态模型。这些实验方法将通过计算机模拟来补充，以测试这些多域系统如何响应拉伸。总而言之，这些实验将详细说明暗黑蛋白是如何抵抗力量的。在第二个目标中，PI将测试当细胞被拉伸时，暗黑蛋白对整个细胞的影响。有无暗黑蛋白的细胞将被拉伸，并将分析在这些条件下暗黑蛋白存在的生化后果。这两个目标合在一起，将有助于定义暗黑蛋白作为拉伸电阻的行为，并将回答暗黑蛋白是否是机械传感器的问题。

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.