Decoding mechanotransduction mechanisms of cell-surface receptors

项目概要 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，甚至监视 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，考虑到诊断结果，这具有直观意义 通常涉及检测感觉比周围组织更硬的肿块。确实，独特且 正常和患病细胞/组织的可量化“机械表型”已被测量。底层证券 正常和患病细胞微环境的这些细胞“机械表型”特征是 机械传感蛋白将感知到的物理扰动转化为过程中的生化信号 称为机械传导。这些信号通路是新兴“机械- 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 是创新技术来识别疾病相关机械表型的分子参与者， 并剖析它们治疗疾病的张力感应机制。确定的最大挑战 力传感的分子基础是测量皮牛顿 (pN) 力的技术 细胞中的单个蛋白质仅在十年前出现，并且仍在不断发展中。 这削弱了对特定细胞或疾病过程中涉及的新机械传感蛋白的识别 并且在关于力如何改变受体构象的可检验假设方面也留下了巨大的空白 引发生物反应。我们的实验室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 开发并使用分子张力传感器 (MTS) 来测量通过假设的机械传感感应到的力 细胞环境中的蛋白质。我们计划结合 MTS 和 CRISPR 筛选来识别机械传感器 参与胶质母细胞瘤和 T 细胞迁移。计划二。其次，我们的目的是检验蛋白质水解作用的假设 受体是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 鉴定出 Notch 样蛋白水解开关，并使用 CRISPR 标记和质谱分析来研究全局 受体因施加的力而发生蛋白水解。计划三。最后，我们的实验室扩展到了第三个领域—— HUH 核酸内切酶作为“HUH 标签”共价连接蛋白质和 DNA 的功能和应用。我们计划 设计 HUH 标签的序列特异性和 RNA 结合。我们准备使用 HUH 标签来改进 DNA- 基于 MTS 并将机械传感域连接到 DNA 纳米结构，以机械方式诱导蛋白质 激活的构象。新蛋白质-DNA 缀合技术与假设驱动的交叉 研究推动了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