Novel ratiometric luminescence reporters for intracellular free calcium

描述(由申请人提供)：概述游离钙离子通量(Ca++)是动物、植物和微生物细胞中生物过程和信号的通用调节器。这些钙离子通量的量化对于理解这些中央调控和信号通路至关重要。目前，基于荧光的探针是测量钙离子水平的首选方法，但这些方法存在与荧光激发相关的问题，如自发荧光、光毒性、光漂白和组织穿透性差。发光方法是避免这些问题的另一种方法。该项目将开发和表征可遗传编码的新型发光探针，并按比例定量测定[Ca++]。这将通过在荧光素酶和金星荧光团之间使用生物发光共振能量转移(BRET)来实现，该荧光素酶受钙敏感连接体的调节，因此发射光谱取决于钙离子浓度。这种钙离子探针非常适用于自发荧光、光漂白、组织穿透以及不良的光传导和光毒性问题。使用一个非常灵敏的CCD摄像机，通过双视图连接到显微镜上，可以从各种组织和细胞中获得钙离子通量的图像。除了开发和表征这种新一代的发光钙离子探针外，这些报告的使用还将被应用于生物时钟(昼夜节律)中钙离子通量的研究。昼夜节律是人类心理和身体健康的重要组成部分，它调节睡眠、警觉性、激素和许多其他生物过程。这些节律与某些类型的抑郁症和睡眠障碍(如睡眠过多)密切相关。新的钙离子探针将用于研究钙离子通量的昼夜振荡。该项目适合于探索/发展R21生物工程计划，因为它建议开发新的分子探针，用于测量和成像与无处不在的钙++信号相关的功能。公共卫生：该项目将开发新的钙离子通量探测器，在目前可用的方法学有限的条件下，这种探测器将是最佳的。作为测试案例，这些探头将应用于对心理和身体健康有重要影响的生物钟主题。

DESCRIPTION (provided by applicant): Summary fluxes of free calcium ions (Ca++) are a universal regulator of biological processes and signaling in animal, plant, and microbial cells. Quantification of these Ca++ fluxes is crucial for understanding these central regulatory & signaling pathways. Currently, fluorescence-based probes are the preferred methods for measuring Ca++ levels, however these methods suffer from problems associated with fluorescence excitation, such as autofluorescence, phototoxicity, photobleaching, and poor penetration of tissue. Luminescence methods are an alternative approach that avoids these problems. This project will develop and characterize novel luminescence probes that are genetically encodable and quantify [Ca++] ratiometrically. This will be accomplished by using Bioluminescence Resonance Energy Transfer (BRET) between a luciferase and the Venus fluorophore that is modulated by Ca++-sensitive linkers so that the spectrum of emission is dependent upon Ca++ concentration. This Ca++ probe will be ideal for applications where autofluorescence, photobleaching, tissue penetration, and undesirable phototransduction & phototoxicity are problems. Using a very sensitive CCD camera coupled to a microscope through a Dual-View, Ca++-fluxes will be imaged from a variety of tissues and cells. In addition to developing and characterizing this new generation of luminescent Ca++ probes, the use of these reporters will be applied to the study of Ca++-fluxes in biological clocks (circadian rhythms). Circadian (daily) rhythms are a crucial component of human mental and physical health that regulates sleep, alertness, hormones, and many other biological processes. These rhythms are strongly implicated in some types of depression and in sleep disorders such as hypersomnia. The new Ca++ probes will be used to study circadian oscillations of Ca++ fluxes. This project is appropriate for the Exploratory/Developmental R21 Bioengineering Program because it proposes to develop new molecular probes for measurement and imaging of function as related to ubiquitous Ca++ signaling. Public Health: This project will develop new probes for Ca++ fluxes that will be optimal for conditions under which currently available methodology is limited. As a test case, these probes will be applied to the topic of biological clocks that have an important influence over mental and physical health.