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.

描述（由申请人提供）：铜是必不可少的痕量元素，对人类健康至关重要。它是许多基本生物学反应的辅助因子，在呼吸，超氧化物相比，胺降解或铁动员和摄取等过程中是必需的。因为游离铜离子会催化高反应性羟基自由基的产生，从而损害脂质，蛋白质，DNA和其他生物分子，铜摄取，分布，并掺入蛋白质中需要复杂的机械。由于铜的细胞外供应可能会随着时间的流逝而变化，因此需要细胞维持缓冲位点，不仅是防御缺乏症的防御，而且还可以保护异常高水平。尽管在分子水平上了解铜摄取，分布和调节的机制方面取得了巨大进展，但对于这种时间储存位点的亚细胞位置及其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，需要新技术来可视化活细胞中细胞铜的动力学，并识别相关的细胞器或隔室。该赠款应用的目的是开发工具，以使生物学家通过结合两个强大的成像方式，即光和X射线荧光显微镜来可视化细胞铜。前者的方法需要开发膜扩散的高对比度比率铜响应荧光探针，该探针适用于在活细胞中询问动力学不稳定铜池的亚细胞位置和动态。第二个成像方式是一种高度敏感的基于同步加速器的微分析技术，它将提供有关固定细胞中总铜和其他过渡元件的分布的定量信息。为了弥合两种互补技术，我们将开发一种可催化的可扩增的异种生物荧光标记方法，从而实现铜和相关细胞结构的直接相关光和X射线荧光显微镜。这两种成像技术都将用于阐明高尔基体在有丝分裂和细胞增殖过程中铜重新分布和遗传中的作用。预计开发的工具对于阐明铜稳态的其他方面以及新型诊断和治疗工具的长期开发至关重要，这些工具将有助于打击相关的与铜相关的人类疾病。 公共卫生相关性：铜是必不可少的痕量元素，对人类健康至关重要。越来越多的疾病，包括威尔逊疾病，Menkes综合征或阿尔茨海默氏病，是由铜运输和调节受损引起的。我们提出了敏感的探针和技术的开发，这些探针和技术适合通过光和X射线荧光显微镜可视化铜，因为这些工具有望大大帮助并提高对铜的储存和调节的研究，从而有助于揭开这些疾病的基本机制。