FastTrack GECI: Development of novel fast calcium indicators for intracellular, extracellular and in vivo imaging

为了理解和明确目标过程发生在生物体中，显微镜技术是有用的。使用光发射作为神经冲动的指示器或显示分子或离子等成分的运动是一种具有最小侵入性的有吸引力的方法。为了提供信息，需要设计特异性的指示分子，并以最大的保真度报告生物过程。钙离子是刺激与效应之间的重要中介。它作为信使，控制着从肌肉收缩、心跳到学习和形成记忆的一系列功能。神经传导和肌肉收缩是快速的过程，在千分之一秒内启动。适当的分子指示剂应该同样快。我们的建议解决了已制定的指标比进程慢得多的问题。通过了解决定指示剂响应速度的分子属性，我们对分子结构进行了一些修改，并证明了我们可以增加响应时间。接下来，我们将把同样的策略应用于更新、更亮但仍然缓慢的一系列指标。这些改进探针的应用将使神经和心脏活动的可视化更加真实。然而，即使是快速探针也有限制其使用的特性，它们需要一定水平的钙来检测它，而且无论钙含量高多少，它们都会显示出相同的亮度。为了解决这个问题，我们提出了一套探头，其信号将随着钙的水平成比例地增加。钙不仅是细胞内的信使，也是细胞间空间的信使。这里的钙含量是细胞内的一万多倍，监测这样的水平需要指示剂对钙的亲和力较低。我们的第三组探针通过上面提到的合理设计解决了这个问题。最后，利用现有的先进显微镜技术，我们不仅可以监测细胞内钙的活动，还可以监测细胞特定部位钙的活动。这样做的能力对于更详细地了解细胞如何工作以及在疾病条件下识别异常钙水平和时间过程具有很大的潜力。钙浓度是精确定时和局部改变的，有很多证据表明，在许多疾病中，细胞精确调节钙水平的能力受到损害。这与衰老、癫痫、痴呆和记忆障碍有关，也会导致心脏问题。我们提出的探针将能够更好地了解潜在的条件和在长期点治疗目标。

To understand and specifically target processes that occur in living organisms, microscopy techniques are useful. Using light emission as indicator of nerve impulses or showing the movements of components such as molecules or ions is an attractive approach with minimum invasion. To be informative, indicator molecules need to be designed that are specific and report biological processes with maximum fidelity. An important mediator between stimulus and effect is the calcium ion. It acts as a messenger for and controls a wide range of functions from muscle contraction and heart beat to learning and forming memories. Nerve conduction and muscle contraction are rapid processes, switched on in 1/1000 of a second. Appropriate molecular indicators should be just as fast. Our proposal addresses the issue that the indicators that have been developed are much slower than the processes. By understanding the molecular attributes that determine how fast the indicator's response is, we applied a number of modifications to the molecular structure and demonstrated that we can increase the response times. We will next apply the same strategy to a newer, brighter but still slow family of indicators. The application of these improved probes will allow more faithful visualisation of nerve and heart activity. However, even the fast probes have a property which limits their use, they require a certain level of calcium to detect it and furthermore will show the same brightness regardless of any higher calcium levels. To counter this problem, we propose a set of probes whose signal will increase with the level of calcium proportionately. Calcium is a messenger not only within the cell but also in the space between cells. Here the calcium levels are over 10000-fold higher than in the cell and monitoring such levels requires that the indicator has low affinity for calcium. Our third set of probes addresses this issues by the rational design referred to above. Finally, with the advanced microscopy techniques that are available, we can monitor the actions of calcium not only in cells but also in specific parts of the cell. The ability to do so has great potential for more detailed understanding of how the cell works and also to identify abnormal calcium levels and time courses in disease conditions. Calcium concentrations are precisely timed and locally changed and there is much evidence that in many diseases the ability of the cell to precisely adjust calcium levels is damaged. This is relevant in aging, epilepsy, dementia and memory disorders and also causes problems in the heart.Our proposed probes will enable the better understanding of the underlying condition and in the long-term point to treatment targets.