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

摘要 现代显微镜和图像分析，以及荧光探针技术，已经发展到量化， 在活细胞和动物中的信号传导具有秒和微米分辨率。最近，光遗传学和 化学遗传学使得在体内控制信号传导成为可能，从而探索因果关系 在信号分子之间，因为它们受到时空动力学的调节。我们在此建议将联合收割机 在同一个细胞中进行蛋白质可视化和控制，在研究Rho如何 通过反馈和前馈关系来协调GTdR信号。为了产生蛋白质， 通过光或小分子，我们将使用新的方法， 结构和最小化活细胞的扰动。这些包括基于染料的内源性生物传感器， 蛋白质构象、用于光抑制或激活的工程化变构控制以及它们的用途 光响应蛋白类似物，可以作为内源性蛋白的替代品。我们将研究 “吞噬作用受挫”，一个系统，其中驱动吞噬作用的复杂动力学被保留，但 限制为二维，并且以由图案化衬底产生的精确几何形状出现。我们将 使用单分子显微镜检查信号的空间限制区之间的通信， 蛋白质构象变化将使用激活梯度、动力学和定位的精确控制， 通知检查如何精确地保持分离的信令域的数学模型。中 第二个项目，我们将与我们的合作者埃里克贝齐格和梁洲的珍妮利亚农场，以适应 生物传感器和光遗传学技术，晶格光片显微镜，可视化和控制的 巨核细胞在产生血小板时经历复杂的形态学变化。我们的终极目标 将是信号的光遗传修饰以增强血小板的产生。我们将致力于使 产生最小干扰生物传感器和光遗传学工具的技术， 研究人员在广泛的领域。

ABSTRACT Modern microscopy and image analysis, together with fluorescent probe technology, has evolved to quantify signaling in living cells and animals with seconds and microns resolution. More recently, optogenetics and chemogenetics have made it possible to control signaling in vivo, and thereby explore causal relationships among signaling molecules as they are regulated by spatio-temporal dynamics. We propose here to combine protein visualization and control in the same cell, for unprecedented quantitative accuracy in studying how Rho GTPase signals are coordinated by feed-back and feed-forward relationships. To generate proteins controlled by light or small molecules, we will use novel approaches that provide ready access to many different structures and minimize perturbation of living cells. These include dye-based biosensors of endogenous protein conformation, engineered allosteric control for inhibition or activation by light, and the use of photoresponsive protein analogs that can serve as substitutes for endogenous proteins. We will study ‘frustrated phagocytosis’, a system where the complex dynamics driving phagocytosis are preserved, but are restricted to two dimensions and occur in precise geometries generated by patterned substrates. We will examine communication between spatially restricted zones of signaling using single molecule microscopy of protein conformational change. Precise control of activation gradients, kinetics and localization will be used to inform mathematical models examining how precisely segregated signaling domains are maintained. In a second project, we will work with our collaborators Eric Betzig and Leong Chew of Janelia Farm to adapt biosensor and optogenetic technologies to lattice light sheet microscopy, for visualization and control of the complex morphological changes megakaryocytes undergo as they produce platelets. There our ultimate goal will be optogenetic modification of signaling to enhance platelet production. We will focus on enabling technologies to generate minimally perturbing biosensors and optogenetic tools that can be applied by other researchers in a wide range of fields.