Decoding mechanotransduction mechanisms of cell-surface receptors

项目总结 最近，细胞微环境中的机械线索驱动细胞迁移已经变得明显， 干细胞分化成不同的细胞类型，甚至监视T细胞是如何被其正确的抗原触发的， 巩固了张力传感作为细胞功能的关键调节开关。不足为奇的是， 机械力是癌症等疾病的一个新兴因素，考虑到这种诊断，这是直觉上的道理 通常包括检测到比周围组织感觉更硬和更硬的肿块。事实上，独特的和 测量了正常和病变细胞/组织的可量化的“机械表型”。潜在的 这些细胞“机械表型”是正常和疾病细胞微环境的特征 在一个过程中将感知的物理扰动转化为生化信号的机械传感蛋白质 称为机械转导。这些信号通路可能是新出现的“机械性”的靶点。 治疗“策略旨在纠正异常的机械表型。戈登实验室的总体愿景 是创新技术以确定与疾病相关的机械表型背后的分子作用者， 并剖析了它们治疗疾病的张力感应机制。确定最大的挑战 力传感的分子基础是测量微微牛顿(Pn)力的技术 细胞背景下的单个蛋白质十年前才出现，目前仍在不断发展中。 这削弱了对特定细胞或疾病过程中涉及的新的机械传感蛋白的识别。 这也在关于力如何改变受体构象的可检验假说中留下了巨大的空白 引发一种生物反应。我们的实验室建立了三个主要领域来解决这个混合的问题 技术发展和假设驱动的问题。方案一，结合细胞成像，我们 开发和使用分子张力传感器(MTS)来测量通过虚拟机械传感传感的力 细胞环境中的蛋白质。我们计划将MTS和CRISPR屏幕结合起来，以识别机械传感器 参与胶质母细胞瘤和T细胞迁移。计划II。第二，我们的目标是检验蛋白质分解作用的假设 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研究新的 鉴定Notch样蛋白水解性开关并使用CRISPR标记和质谱学研究全球 在外力作用下，受体蛋白的降解。计划III。最后，我们的实验室已经扩展到第三个领域-- HH-内切酶作为蛋白质和DNA共价连接的“HH-标签”的功能和应用。我们计划 HH-tag的工程序列特异性和RNA结合。我们准备使用HH标签来改进DNA- 并将机械传感结构域连接到DNA纳米结构，以将蛋白质机械地诱骗到 激活的构象。假说驱动下蛋白质-DNA偶联新技术的交叉 研究推动对生物医学科学和医学中的重要问题采取创造性和创新性的方法。 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