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），以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细胞内稳态及其在许多疾病中的扰动至关重要。虽然我们已经取得了巨大的进步，在我们的能力，研究活细胞中的单个蛋白质的活动，它仍然很难表征的协调多个活动，在快速的形态变化和不同的信号通路的相互作用至关重要。基于环境传感染料的生物传感器，比其他方法具有宝贵的优势，提供了一个机会，使用设备，甚至基本的活细胞成像显微镜准备多重成像。基于环境传感染料的生物传感器由“识别元件”组成，该“识别元件”是一种小的蛋白质片段，其仅与靶蛋白的活化状态结合，该小的蛋白质片段与亮的荧光染料偶联，当生物传感器结合其靶时，该荧光染料改变荧光。这种设计使得能够研究内源性的、未标记的靶蛋白，并提供高灵敏度，因为明亮的染料可以被直接激发。这些染料中的结构变化以增强亮度、光稳定性、水溶性等通常需要折衷，因为影响一种性质的结构变化不利地影响另一种性质。我们已经研究了染料光漂白和响应溶剂极性的机制，并设计了新的方法来提高水溶性。基于此，我们将在这里设计新一代的生物传感器染料，改变其波长，允许多重成像，同时保持生物物理特性，赋予基于染料的生物传感器的优势。使用新的染料，我们将建立“多路复用生物传感器”，以量化Cdc42或Src的激活，同时激活RhoA或Rac1。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，因为它们在巨胞饮和跨内皮迁移期间产生细胞骨架变化。这项提议将开发新的方法来研究决定细胞如何对其环境作出反应的细胞“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种此类蛋白质的激活，尤其是对于快速事件。这项新技术使我们能够更好地了解电路组件如何通过可视化多个电路组件进行交互，即使是快速激活事件。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，在许多疾病中起着重要作用（即免疫细胞吞噬入侵者，白色血细胞在炎症期间穿过血管壁时被血管壁吞噬，细胞以类似的方式穿过血脑屏障）。吞噬需要在时间和空间上精确协调蛋白质相互作用，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.