Dissecting signaling in vivo via precise control and visualization of protein activity

通过蛋白质活性的精确控制和可视化剖析体内信号传导

• 批准号: 10626879
• 负责人: Klaus M. Hahn
• 金额: $ 81.03万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2027-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10626879
• 关键词: Active Sites; Adhesions; Behavior; Binding Sites; Biological Models; Biosensor; Blood Vessels; Cell physiology; Cells; Cellular biology; Charge; Chemicals; Collagen; Complex; Computer Models; Cytoskeleton; Data; Elements; Engineering; Event; Feedback; Frequencies; Frustration; Geometry; Image Analysis; Immune; Individual; Kinetics; Light; Macrophage; Mechanical Stress; Methods; Microscopy; Molecular Conformation; Neoplasm Metastasis; Pattern; Phagocytes; Phagocytosis; Process; Proteins; Regulation; Reproducibility; Role; Shapes; Signal Transduction; Signaling Protein; Stimulus; Stretching; Surface; Techniques; Time; Traction; Variant; Visualization; cancer cell; extracellular; in vivo; mathematical model; mechanical force; multiplexed imaging; novel; novel strategies; organizational structure; quantitative imaging; small molecule; spatiotemporal; tool

Abstract An ongoing revolution in microscopy has revealed previously inaccessible aspects of signaling. These include organization of signaling proteins on dynamic cytoskeletal elements, subtle but important variations in activation kinetics, and information encoded in the oscillation frequencies of signaling circuits. We will explore these regulatory mechanisms by developing novel approaches to visualize and control protein activity, and by using them together in the same cell. Novel image analysis methods and computational modeling applied to multiplexed imaging data will reveal causal connections between signaling events, including those dependent on nested feedback loops. We will pursue new methods to produce biosensors bright enough to study the conformational changes of individual molecules in living cells. On podosomes and adhesion complexes, where important dynamic structures are organized on a scale below the diffraction limit, we will use these new methods to examine the opening of stretch-activated binding sites in real time. Proteins will be controlled with light or small molecules using engineered domains inserted away from the active site, where they can allosterically control activity without perturbing normal interactions. We will study three model systems that are well suited to extract basic principles re the spatio-temporal regulation of signaling: 1) In 'frustrated phagocytosis' macrophages attempt to engulf micropatterned circles. The phagocytic apparatus they build around the circles has precise, reproducible geometry, which will enable us to use quantitative image analysis and simultaneous protein control/visualization to develop mathematical models. 2) We will study how cancer cells move on aligned collagen "superhighways" to find blood vessels. There we will ask how cells use localized signaling to sense and respond to anisotropic mechanical stresses. 3) Finally, cell protrusion/retraction will be used to examine the integration of chemical gradients, mechanical force, and cytoskeletal dynamics. We hope that the behavior of these model systems will reveal ubiquitous signaling mechanisms, and that the new tools can help others explore diverse questions in cell biology.

抽象的 一场持续的显微镜革命揭示了先前无法访问的信号传导方面。这些包括 在动态细胞骨架元素上组织信号蛋白，细微但重要的变化 激活动力学和在信号电路的振荡频率中编码的信息。我们将探索 这些调节机制通过开发新的方法来可视化和控制蛋白质活性，以及 在同一单元格中一起使用它们。新颖的图像分析方法和计算建模应用于 多路复用成像数据将揭示信号事件之间的因果关系，包括那些依赖性 在嵌套的反馈回路上。我们将采用新的方法来生产足够明亮的生物传感器，以研究 活细胞中个体分子的构象变化。关于足体和粘附复合物，其中 重要的动态结构以低于衍射极限的比例组织，我们将使用这些新的 实时检查拉伸激活结合位点开放的方法。蛋白质将通过 使用从活动地点插入的工程域的光或小分子，可以在其中 变构控制活动而不会扰动正常相互作用。我们将研究三个模型系统 非常适合提取基本原则是信号传导的时空调节：1） 吞噬作用的巨噬细胞试图吞噬微图案。它们建造的吞噬仪 圆圈周围具有精确的可重现几何形状，这将使我们能够使用定量图像分析 并同时进行蛋白质控制/可视化以开发数学模型。 2）我们将研究癌症 细胞在对齐的胶原蛋白上移动以找到血管。在那里我们将询问细胞如何使用 局部信号传导以感知并响应各向异性机械应力。 3）最后，单元格 突出/缩回将用于检查化学梯度，机械力和 细胞骨架动力学。我们希望这些模型系统的行为能够揭示无处不在的信号传导 机制，新工具可以帮助其他人探索细胞生物学中的各种问题。