Novel ratiometric luminescence reporters for intracellular free calcium

概述：游离钙离子（Ca++）通量是动物、植物和微生物细胞中生物过程和信号传导的普遍调节剂。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Ca++水平的首选方法，然而，这些方法存在与荧光激发相关的问题，如自身荧光、光毒性、光漂白和组织穿透性差。发光法是避免这些问题的另一种方法。该项目将开发和表征新型发光探针，这些探针是基因可编码的，并以比例计量方式量化[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，该荧光团由Ca++敏感连接剂调制，因此发射光谱依赖于Ca++浓度。这种Ca++探针将是理想的应用，在自身荧光，光漂白，组织渗透，以及不良的光导和光毒性的问题。使用一个非常灵敏的CCD相机耦合到一个显微镜通过双视图，钙++通量将从各种组织和细胞成像。除了开发和表征这种新一代发光钙离子探针外，这些报告器的使用将应用于研究生物钟（昼夜节律）中的钙离子通量。昼夜节律是人类身心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（如嗜睡症）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。该项目适合于探索性/发展性R21生物工程项目，因为它提出了开发新的分子探针，用于测量和成像与无处不在的Ca++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针，这将是目前可用方法有限的条件下的最佳探针。作为一个测试案例，这些探针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.