Fluorescent copper probes for intracellular imaging

描述（申请人提供）：铜是一种必需的微量元素，对人体健康至关重要。它作为许多基本生物反应的辅因子，在呼吸、超氧化物歧化、胺降解或铁动员和吸收等过程中是必需的。由于游离铜离子催化高活性羟基自由基的产生，这可能会破坏脂质，蛋白质，DNA和其他生物分子，铜的吸收，分布和掺入蛋白质需要一个复杂的机制。由于铜的细胞外可用性可能随时间而变化，因此细胞需要维持缓冲位点，不仅作为对缺陷的防御，而且作为对异常高水平的保护。虽然已经取得了很大的进展，在分子水平上理解铜的吸收，分布和调节的机制，仍然知之甚少的亚细胞的位置，这样的临时存储网站和它们的重新分配在正常细胞功能或在特定的疾病状态。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。这项资助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扩散高对比度铜响应荧光探针，这将是适合于询问在活细胞中的亚细胞位置和动力学不稳定的铜池。第二种成像方式是一种高灵敏度的基于同步加速器的微量分析技术，它将提供有关固定细胞中总铜和其他过渡元素分布的定量信息。为了弥合这两个互补的技术，我们将开发一种催化生物异源荧光标记的方法，从而使直接相关的光和X射线荧光显微镜的铜和相关的细胞结构。这两种成像技术将被应用于阐明铜在有丝分裂和细胞增殖过程中的重新分配和遗传的高尔基体的作用。预计开发的工具对于阐明铜稳态的其他方面以及长期开发有助于对抗铜相关人类疾病的新型诊断和治疗工具至关重要。 铜是一种必需的微量元素，对人体健康至关重要。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，都是由铜转运和调节受损引起的。我们建议开发灵敏的探针和技术，这些探针和技术将适合通过光和X射线荧光显微镜观察铜，因为这些工具有望显著帮助和推进铜储存和调节的研究，从而有助于揭示这些疾病的基本机制。

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.