Decoding mechanotransduction mechanisms of cell-surface receptors

项目摘要 最近，细胞微环境中的机械信号驱动细胞迁移已经变得很明显， 干细胞分化成不同的细胞类型，甚至是监视性T细胞如何被其正确的抗原触发， 巩固张力感应作为细胞功能的关键调节开关。毫不奇怪，改变 机械力是癌症等疾病中的一个新兴因素， 通常包括检测感觉比周围组织更硬和更硬的肿块。的确， 已经测量了正常和患病细胞/组织的可定量的“机械表型”。底层 这些正常和患病细胞微环境的细胞“机械表型”特征， 机械传感蛋白质，其在过程中将所感测的物理扰动转化为生化信号 称为机械传导。这些信号通路是新兴的“机械- 旨在纠正异常机械表型的“治疗”策略。戈登实验室的整体愿景 是创新技术，以确定潜在的疾病相关的机械表型的分子球员， 剖析它们的张力感应机制来治疗疾病。确定最大的挑战是 力感测的分子基础是测量由传感器感测的皮牛顿（pN）力的技术， 单个蛋白质在细胞中的作用仅仅出现在十年前，并且仍在不断发展中。 这削弱了识别参与特定细胞或疾病过程的新的机械传感蛋白 也留下了一个巨大的差距，在可测试的假设，关于如何力改变受体的构象， 引发生物反应我们的实验室已经建立了三个主要领域来解决这个问题， 技术开发和假设驱动的问题。方案一结合细胞成像，我们 开发和使用分子张力传感器（MTS）来测量由假设的机械传感所感知的力 蛋白质在细胞中的作用。我们计划将联合收割机MTS和CRISPR屏幕结合起来， 参与胶质母细胞瘤和T细胞迁移。方案二.第二，我们的目标是测试假设， 是一种传递机械刺激的机制。我们将利用结构生物物理学来进行新的研究 确定Notch样蛋白水解开关，并使用CRISPR标记和质谱法研究全球 受体蛋白质水解对外力的反应。方案三.最后，我们的实验室扩展到了第三个领域- HUH-内切核酸酶作为“HUH-标签”共价连接蛋白质和DNA的功能和应用。我们计划 HUH标签的工程序列特异性和RNA结合。我们准备用HUH标签来改进DNA 基于MTS和连接机械传感域的DNA纳米结构，以诱导蛋白质机械地 活化构象假设驱动下的蛋白质-DNA偶联新技术 研究推动了对生物医学科学和医学中重要问题的创造性和创新性方法。 1

PROJECT SUMMARY Recently, it has become apparent that mechanical cues in the cellular microenvironment drive cell migration, stem cell differentiation into distinct cell types and even how a surveilling T-cells is triggered by its correct antigen, solidifying tension-sensing as a key regulatory switch in cellular function. Not surprisingly, alteration of mechanical forces is an emerging factor in diseases like cancer, which makes intuitive sense given that diagnosis often involves detecting a lump that feels harder and stiffer than the surrounding tissue. Indeed, distinct and quantifiable “mechanical phenotypes” of normal and diseased cells/tissues have been measured. Underlying these cellular “mechanical phenotypes” characteristic of normal and diseased cellular microenvironments are mechanosensing proteins that convert sensed physical perturbations into biochemical signals in a process known as mechanotransduction. These signaling pathways are putative targets of emerging “mechano- therapeutic” strategies aimed to correct aberrant mechanical phenotypes. The overall vision of the Gordon lab is to innovate technology to identify the molecular players underlying disease-relevant mechanical-phenotypes, and dissect their tension-sensing mechanisms to cure disease. The greatest challenge to determining the molecular basis of force sensing is that the technology to measure picoNewton (pN) forces sensed by an individual protein in the context of the cell emerged only ten years ago, and is still under constant development. This has crippled identification of new mechanosensing proteins involved in a given cellular or disease process and also left a huge gap in testable hypotheses regarding how force alters the conformation of receptors to trigger a biological response. Our lab has established three major areas to tackle this problem that blend technology development and hypothesis driven questions. Program I. In combination with cellular imaging, we develop and use molecular tension sensors (MTS) to measure forces sensed by hypothesized mechanosensing proteins in the cellular context. We plan to combine MTS and CRISPR screens to identify mechanosensors involved in glioblastoma and T-cell migration. Program II. Second, we aim to test the hypothesis that proteolysis of receptors is a mechanism to convey mechanical stimuli. We will use structural biophysics to study newly identified Notch-like proteolytic switches and use CRISPR-tagging and mass spectrometry to study global receptor proteolysis in response to applied force. Program III. Finally, our lab has expanded into a third area- function and application of HUH-endonucleases as “HUH-tags” to covalently link proteins and DNA. We plan to engineer sequence specificity and RNA-binding of HUH-tags. We are poised to use HUH-tags to improve DNA- based MTS and to link mechanosensing-domains to DNA-nanostructures to coax proteins into mechanically activated conformations. The interleaving of new protein-DNA conjugation technology with hypothesis driven research drives creative and innovative approaches to important problems in biomedical science and medicine. 1