Dye-Based biosensors: simultaneous imaging of multiple protein activities

描述（由申请人提供）：控制细胞行为的蛋白质可以在细胞内的不同位置被激活（即磷酸化，经历构象变化）或以不同的动力学产生本质上相反的行为。破译信号的时空控制对于理解正常细胞稳态及其在许多疾病中的扰动至关重要。尽管我们在研究活细胞中单个蛋白质活性的能力方面取得了巨大的进步，但仍然很难表征多个活动的协调，这在快速形态变化和不同信号通路的相互作用中至关重要。基于环境感应染料的生物传感器比其他方法具有宝贵的优势，为使用基本活细胞成像显微镜上可用的设备进行多路成像提供了机会。基于环境感应染料的生物传感器由一个“识别元件”组成，这是一个小的蛋白质片段，只与目标蛋白质的激活状态结合，再加上一个明亮的荧光染料，当生物传感器与目标结合时，荧光就会改变。这种设计可以研究内源性的、未标记的靶蛋白，并且由于明亮的染料可以直接激发，因此具有高灵敏度。为了提高染料的亮度、光稳定性、水溶性等，这些染料的结构变化往往需要妥协，因为结构变化会影响一种性质，对另一种性质产生不利影响。我们研究了染料的光漂白机理和对溶剂极性的响应，并设计了新的方法来提高水溶性。在此基础上，我们将设计新一代生物传感器染料，改变其波长以允许多重成像，同时保持染料生物传感器的光物理特性。使用新染料，我们将构建“多路生物传感器”，以定量Cdc42或Src与RhoA或Rac1同时激活的激活。这些新的生物传感器将用于表征Src和Rho家族激活的时空协调性，因为它们在巨噬细胞增生和跨内皮迁移过程中产生细胞骨架变化。这一提议将开发新的方法来研究决定细胞如何对环境做出反应的细胞“电路”。这种电路由相互作用的蛋白质组成的复杂网络组成，这些蛋白质可以在细胞内的不同位置被激活，从而产生不同的细胞行为。目前很难研究同一细胞中多个这样的蛋白质的激活，特别是对于快速事件。这项新技术使我们能够更好地理解电路组件是如何相互作用的，通过实现多个电路组件的可视化，即使是快速激活事件。该技术开发后，将应用于研究细胞吞噬其他身体，这是一种在许多疾病中起重要作用的普遍反应（即免疫细胞吞噬入侵者，白细胞在炎症期间通过血管壁时被血管壁吞噬，细胞在血脑屏障上的移动也类似）。吞噬需要精确地协调蛋白质在时间和空间上的相互作用，这对新工具来说是一个理想的挑战。

DESCRIPTION (provided by applicant): Proteins that control cell behavior can be activated (i.e. phosphorylated, undergo conformational changes) in different locations within the cell or with different kinetics to produce essentially opposing behaviors. Deciphering the spatio-temporal control of signaling is essential to understanding normal cellular homeostasis and its perturbation in many diseases. Although we have made tremendous strides in our ability to study the activity of single proteins in living cells, it remains difficult to characterize the coordination of more than one activity, critically important in rapid morphological changes and in the interaction of different signaling pathways. Biosensors based on environment-sensing dyes, which have valuable advantages over other approaches, offer an opportunity for ready multiplex imaging using equipment available on even basic live cell imaging microscopes. Biosensors based on environment sensing dyes consist of a `recognition element', a small protein fragment that binds only to the activated state of the target protein, coupled to a bright fluorescent dye that changes fluorescence when the biosensor binds its target. This design enables study of endogenous, untagged target proteins, and provides high sensitivity because bright dyes can be directly excited. Structural changes in these dyes to enhance brightness, photostability, water solubility etc. often require compromises, as structural changes affecting one property adversely affect another. We have studied the mechanisms of dye photobleaching and response to solvent polarity, and devised novel approaches to enhance water solubility. Based on this we will design here a new generation of biosensor dyes, shifting their wavelengths to permit multiplex imaging, while maintaining the photophysical features that confer advantages on dye-based biosensors. Using the new dyes, we will build `multiplexing biosensors' to quantify activation of Cdc42 or Src simultaneously with activation of either RhoA or Rac1. These new biosensors will be used to characterize the spatio-temporal coordination of Src and Rho family activation as they generate cytoskeletal changes during macropinocytosis and transendothelial migration. This proposal will develop new methods to study the cellular `circuitry' that determines how a cell responds to its environment. Such circuits consist of complex networks of interacting proteins which can be activated in different positions within a cell to produce different cell behaviors. It is currently difficult to study the activation of more than one such protein in the same cell, especially for rapid events. The new technique enables us to better understand how circuit components interact by enabling visualization of multiple circuit components, even for rapid activation events. After the technique is developed, it will be applied to study cell engulfment of other bodies, a ubiquitous response that plays an important role in many diseases (i.e. immune cells engulf invaders, white blood cells are engulfed by blood vessel walls as they pass through them during inflammation, and cells move similarly across the blood brain barrier). Engulfment requires precise orchestration of protein interactions in time and space, an ideal challenge for the new tools.