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

摘要 显微镜的一场持续革命揭示了以前无法触及的信号方面。这些包括 组织的信号蛋白的动态细胞骨架元素，微妙的，但重要的变化， 激活动力学和信号电路的振荡频率中编码的信息。我们将探讨 通过开发新的方法来可视化和控制蛋白质活性， 在同一个牢房里一起使用新的图像分析方法和计算建模应用于 多重成像数据将揭示信号事件之间的因果关系，包括那些依赖于 嵌套的反馈循环我们将寻求新的方法来生产生物传感器， 活细胞中单个分子的构象变化。在podosomes和粘附复合体上， 重要的动态结构是在衍射极限以下的尺度上组织的，我们将使用这些新的 真实的检查拉伸激活结合位点的打开的方法。蛋白质将被控制， 轻分子或小分子使用远离活性位点插入的工程结构域，在活性位点处它们可以 变构控制活性而不干扰正常相互作用。我们将研究三个模型系统， 非常适合于提取信号的时空调节的基本原理：1）在'沮丧 吞噬作用的巨噬细胞试图吞噬微图案的圆圈。它们建造的吞噬器 具有精确的、可重复的几何形状，这将使我们能够使用定量图像分析， 和同时蛋白质控制/可视化以开发数学模型。2)我们将研究癌症 细胞在排列整齐的胶原蛋白“高速公路”上移动以找到血管。在那里我们会问细胞是如何利用 局部信号传导以感测和响应各向异性机械应力。3)最后，Cell 突出/回缩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将揭示无处不在的信号 这些新工具可以帮助其他人探索细胞生物学中的各种问题。

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.