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

摘要 现代显微镜和图像分析，加上荧光探针技术，已经进化到量化 在活细胞和动物中以几秒和微米的分辨率发出信号。最近，光遗传学和 化学遗传学使在体内控制信号成为可能，从而探索因果关系。 信号分子之间的相互作用，因为它们受到时空动力学的调节。我们在这里建议将 在同一细胞中进行蛋白质可视化和控制，在研究Rho如何 GTPase信号通过反馈和前馈关系来协调。以产生受控制的蛋白质 对于轻分子或小分子，我们将使用新的方法，提供对许多不同的 结构，最大限度地减少对活细胞的干扰。这些包括基于染料的内源性生物传感器 蛋白质构象，光抑制或激活的工程变构控制，以及其用途 可以作为内源蛋白质替代品的光响应蛋白质类似物。我们会研究 “受挫吞噬作用”，这是一种系统，在该系统中，驱动吞噬的复杂动力学被保留下来，但 仅限于两个维度，并出现在由图案化衬底生成的精确几何形状中。我们会 使用单分子显微镜检查空间受限的信号区之间的通信 蛋白质构象变化。将使用对激活梯度、动力学和定位的精确控制来 告知数学模型，检查如何精确地维护隔离的信令域。在一个 第二个项目，我们将与我们的合作者Janelia Farm的Eric Betzig和Leong Chew合作，以适应 生物传感器和光遗传技术到晶格光片显微镜，用于可视化和控制 巨核细胞在产生血小板时会经历复杂的形态变化。这就是我们的最终目标 将通过光遗传修饰信号来增强血小板的生成。我们将专注于支持 产生最小扰动的生物传感器和光遗传工具的技术，可以被其他 广泛领域的研究人员。

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.