Fluorescent copper probes for intracellular imaging

用于细胞内成像的荧光铜探针

• 批准号: 8324731
• 负责人: CHRISTOPH J FAHRNI
• 金额: $ 24.56万
• 依托单位: GEORGIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8324731
• 关键词: Address; Adopted; Affinity; Alzheimer' s Disease; Amines; Applications Grants; Binding; Biological; Buffers; Cell Cycle; Cell Line; Cell Proliferation; Cell division; Cell physiology; Cells; Cellular Structures; Communities; Consensus; Copper; Cytosol; DNA; Data; Defect; Development; Disease; Fluorescence; Fluorescence Microscopy; Fluorescent Probes; Gene Proteins; Glutathione; Goals; Golgi Apparatus; Grant; Health; Hepatolenticular Degeneration; Homeostasis; Human; Hydroxyl Radical; Image; Imaging Device; Imaging Techniques; Individual; Ions; Iron; Kinetics; Knowledge; Label; Life; Ligands; Light; Lipids; Location; Maps; Membrane; Menkes Kinky Hair Syndrome; Mitosis; Modality; Molecular; Molecular Chaperones; Monitor; Multimodal Imaging; Normal Cell; Optics; Organelles; Phase; Physiological; Play; Process; Production; Proteins; Psyche structure; Reaction; Reagent; Regulation; Research; Respiration; Roentgen Rays; Role; Series; Simulate; Site; Staging; Superoxides; Synchrotrons; Techniques; Technology; Testing; Thermodynamics; Time; Trace Elements; Transition Elements; Western Blotting; Xenobiotics; base; cell fixing; cofactor; combat; design; extracellular; fluorescence imaging; fluorophore; human disease; imaging modality; interest; light microscopy; mutant; novel diagnostics; novel therapeutics; public health relevance; sensor; small molecule; tool; trafficking; uptake

DESCRIPTION (provided by applicant): Copper is an essential trace element, which is critical to human health. It serves as a cofactor for many fundamental biological reactions, and is required in processes such as respiration, superoxide disproportionation, degradation of amines, or for iron mobilization and uptake. Because free copper ions catalyze the production of highly reactive hydroxyl radicals, which can damage lipids, proteins, DNA, and other biomolecules, copper uptake, distribution, and incorporation into proteins require a sophisticated machinery. Because the extracellular availability of copper may vary over time, cells are required to maintain buffer sites not only as a defense against deficiency, but also as protection from abnormally high levels. While great progress has been made in understanding the mechanisms of copper uptake, distribution, and regulation on a molecular level, still little is known about the subcellular location of such temporal storage sites and their redistribution during normal cell function or in specific disease states. To study these fundamental processes, new techniques are required for visualizing the dynamics of cellular copper in context of a live cell and to identify associated organelles or compartments. The goal of this grant application is to develop tools that will enable biologists to visualize cellular copper by combining two powerful imaging modalities, light and X-ray fluorescence microscopy. The former approach entails the development of membrane diffusible high-contrast- ratio copper-responsive fluorescent probes that will be suitable for interrogating the subcellular location and dynamics of kinetically labile copper pools in live cells. The second imaging modality is a highly sensitive synchrotron-based microanalytical technique that will provide quantitative information about the distribution of total copper and other transition elements in fixed cells. In order to bridge the two complementary techniques, we will develop a catalytically amplifiable xenobiotic fluorescent labeling approach, thus enabling direct correlative light and X-ray fluorescence microscopy of copper and associated cellular structures. Both imaging techniques will be applied to elucidate the role of the Golgi apparatus in copper redistribution and inheritance during mitosis and cell proliferation. The developed tools are expected to be of critical importance for elucidating other aspects of copper homeostasis and for the long-term development of novel diagnostic and therapeutic tools that will aid in combating copper related human diseases. PUBLIC HEALTH RELEVANCE: Copper is an essential trace element, which is critical to human health. An increasing number of diseases, including Wilson disease, Menkes syndrome, or Alzheimer's disease, are caused by impaired copper transport and regulation. We propose the development of sensitive probes and techniques that will be suitable to visualize copper by light and X-ray fluorescence microscopy, as these tools are expected to significantly aid and advance studies of copper storage and regulation, thus help unravel the fundamental mechanisms of these diseases.

说明(申请人提供)：铜是人体必需的微量元素，对人体健康至关重要。它是许多基本生物反应的辅因子，在呼吸、超氧化物歧化、胺的降解或铁的动员和吸收等过程中都是必需的。由于自由铜离子催化产生高活性的羟基自由基，这会损害脂质、蛋白质、DNA和其他生物分子，因此铜的吸收、分配和并入蛋白质需要复杂的机械。由于细胞外铜的可获得性可能会随着时间的推移而变化，因此细胞需要保持缓冲位置，不仅是为了防御缺乏，也是为了防止异常高水平的铜。虽然在分子水平上了解铜的吸收、分布和调控机制已经取得了很大的进展，但对于这些临时存储位置的亚细胞位置以及它们在正常细胞功能或特定疾病状态下的重新分布仍然知之甚少。为了研究这些基本过程，需要新的技术来可视化活细胞中细胞铜的动态，并识别相关的细胞器或隔室。这项赠款申请的目标是开发工具，使生物学家能够通过结合两种强大的成像方式--光显微镜和X射线荧光显微镜--来可视化细胞铜。前一种方法需要开发膜可扩散的高对比度铜响应荧光探针，这将适合于询问活细胞中动态不稳定的铜池的亚细胞位置和动力学。第二种成像方式是一种基于同步加速器的高灵敏度微量分析技术，它将提供关于固定细胞中总铜和其他过渡元素分布的定量信息。为了连接这两项互补技术，我们将开发一种可催化放大的外源荧光标记方法，从而实现铜及其相关细胞结构的直接相关光和X射线荧光显微镜。这两种成像技术将被用来阐明高尔基体在有丝分裂和细胞增殖过程中铜的再分配和遗传中的作用。预计所开发的工具将对阐明铜的动态平衡的其他方面以及帮助抗击与铜有关的人类疾病的新型诊断和治疗工具的长期发展具有至关重要的作用。 与公共健康相关：铜是一种基本的微量元素，对人类健康至关重要。越来越多的疾病，包括威尔逊病、门克斯综合征或阿尔茨海默病，是由铜运输和调节受损引起的。我们建议开发适用于通过光和X射线荧光显微镜观察铜的灵敏探针和技术，因为这些工具有望显著地帮助和推进铜储存和调节的研究，从而有助于揭开这些疾病的基本机制。