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或以不同的动力学被激活(即，磷酸化，经历构象变化)，从而产生基本上相反的行为。破译信号的时空控制对于理解正常的细胞内稳态及其在许多疾病中的扰动是至关重要的。虽然我们在研究活细胞中单个蛋白质的活性方面已经取得了巨大的进步，但仍然很难表征多个活性的协调，这在快速的形态变化和不同信号通路的相互作用中至关重要。与其他方法相比，基于环境敏感染料的生物传感器具有宝贵的优势，它提供了一种机会，即使在基本的活细胞成像显微镜上也可以使用设备进行即时多路成像。基于环境敏感染料的生物传感器由一个“识别元件”组成，这是一个只与目标蛋白的激活状态结合的小蛋白质片段，与一种明亮的荧光染料相结合，当生物传感器结合其目标时，该染料会改变荧光。这种设计使研究内源性、未标记的目标蛋白成为可能，并提供了高灵敏度，因为明亮的染料可以直接激发。这些染料的结构变化以提高亮度、光稳定性、水溶性等通常需要妥协，因为影响一种性质的结构变化会对另一种性质产生不利影响。我们研究了染料光漂白的机理和对溶剂极性的响应，并设计了新的方法来提高水的溶解度。基于此，我们将在这里设计新一代生物传感器染料，改变它们的波长以允许多路成像，同时保持赋予染料生物传感器优势的光物理特性。使用新的染料，我们将建立‘多路生物传感器’，以定量激活RhoA或rac1的同时激活的Cdc42或Src。这些新的生物传感器将被用来表征在巨噬细胞吞噬和跨内皮细胞迁移过程中，Src和Rho家族的激活产生细胞骨架变化的时空协调。这项提议将开发新的方法来研究决定细胞如何对其环境做出反应的细胞“电路”。这种回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。这项新技术通过实现多个电路组件的可视化，使我们能够更好地了解电路组件是如何相互作用的，甚至对于快速激活事件也是如此。这项技术开发出来后，将被应用于研究其他身体的细胞吞噬，这是一种普遍存在的反应，在许多疾病中发挥着重要作用(即免疫细胞吞噬入侵者，白血球在炎症期间穿过血管壁时被吞噬，细胞以类似的方式跨越血脑屏障)。吞噬需要精确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.