Imaging heme based mitochondrial-cell signaling networks in cell and animal models of heavy metal toxicity

重金属毒性细胞和动物模型中基于血红素的线粒体细胞信号网络成像

基本信息

批准号：
9059090
负责人：
Amit Ram Reddi
金额：
$ 21.78万
依托单位：
GEORGIA INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-05-01 至 2017-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9059090
关键词：
Animal Model Animals Antioxidants Applications Grants Arsenic Binding Biological Cadmium Caenorhabditis elegans Cell Line Cell Nucleus Cell Proliferation Cell model Cell physiology Cells Code Color Cytosol Drug Metabolic Detoxication Energy Metabolism Enterocytes Environmental Hazards Environmental Pollutants Eukaryota Fluorescent Probes Future Gases Goals Health Heavy Metals Heme Hemeproteins Histocompatibility Testing Homeostasis Image Imaging Device Institutes Iron Lead Lead Poisoning Life Mammalian Cell Maryland Measures Membrane Mercury Metabolism Methods MicroRNAs Mitochondria Mitochondrial Matrix Modeling Monitor Nature Neurons Nuclear Organ Organelles Organism Oxygen Palate Pathway interactions Phase Physiological Processes Process Proteins Public Health Regulation Reporter Research Role Saccharomyces cerevisiae Signal Transduction Signaling Molecule Site Stress Technology Thermodynamics Tissues Toxic Environmental Substances Toxic effect Translations Universities Whole Organism Xenobiotics Yeasts base biological adaptation to stress biophysical properties chemical genetics chemical property circadian pacemaker cofactor cytotoxic cytotoxicity detection of nutrient fluorescence imaging genetic manipulation heme a heme biosynthesis innovation metal poisoning novel oxidant stress oxidation quantitative imaging ratiometric response sensor stressor tool trafficking transcription factor transmission process

项目摘要

DESCRIPTION (provided by applicant): As mitochondria are central hubs for energy metabolism and formation of intermediate metabolites, they must be able to transmit signals that communicate alterations in its functioning to the nucleus so that cells can remodel metabolism in response. Mitochondrial-nuclear signaling is particularly important when mitochondrial function is compromised by environmental toxicants, including heavy metals. However, the nature and dynamics of these inter-organelle signals and the mechanisms by which they are transduced are poorly understood. Given the importance of heme-regulated transcription factors that control diverse cellular processes from energy metabolism to the anti-oxidant stress response to cell proliferation, and the exquisite sensitivity of mitochondrial heme homeostasis to heavy metals, we propose that mitochondrial- derived heme signals the cellular response to heavy metal toxicity through mitochondrial-nuclear retrograde regulation. However, the chemical and genetic tools available to study the transmission of heme-based signals do not exist. In the current grant application, the focus is to generate and apply a genetically encoded ratiometric fluorescent heme sensors in cell and animal models to study the transduction of mitochondrial-derived intra- and inter-organ heme signals in response to heavy metal toxicity. During the R21 phase, the intent is develop and apply the first ratiometric heme sensors for quantitative heme imaging in yeast and mammalian cell models of heavy metal toxicity. Finally, the goal is to (a) characterize the thermodynamics of heme binding to the heme sensors and apply them site-specifically in the yeast mitochondrial matrix, inter-membrane space, cytosol, and nucleus to quantitatively image heme signals in response to lead toxicity; and (b) apply sensors in a compartment specific manner in mammalian cell models of lead toxicity. Successful completion of these aims will provide the impetus to begin the R33 phase in which studies will be expanded to develop heme sensors that can be utilized for simultaneous imaging of heme between cellular compartments, and that are oxidation state-specific, and apply them to animal models of Pb toxicity. In this phase the focus is to (a) diversify the color palate of the heme sensors for simultaneous fluorescence imaging between subcellular compartments and develop Fe3+ and Fe2+ heme specific sensors; and (b) deploy these sensors in mammalian cell lines and a C. elegans model of environmental toxicity. Altogether, these studies will result in the first sensors for quantitative imaging of labile heme relevant to its role in cell signaling and establish heme a a vital mitochondria-derived signaling molecule that initiates the adaptation to heavy metal toxicity in both cell and animal models.

描述（由申请人提供）：由于线粒体是能量代谢和中间代谢物形成的中心枢纽，因此它们必须能够将其功能变化的信号传递给细胞核，以便细胞能够相应地重塑代谢。当线粒体功能受到包括重金属在内的环境毒物损害时，这一点尤其重要。然而，鉴于其重要性，人们对这些细胞器间信号的性质和动力学及其转导机制知之甚少。血红素调节的转录因子控制着从能量代谢到细胞增殖的抗氧化应激反应的多种细胞过程，以及线粒体血红素稳态对重金属的敏锐敏感性，我们认为线粒体来源的血红素发出细胞对重金属反应的信号然而，目前尚不存在可用于研究基于血红素的信号传输的化学和遗传工具，重点是生成和应用基因编码的比率荧光血红素传感器。在细胞和动物模型中研究线粒体衍生的器官内和器官间血红素信号对重金属毒性的反应。在 R21 阶段，目的是开发并应用第一个比率血红素传感器，用于酵母中的定量血红素成像。最后，目标是（a）表征血红素与血红素传感器结合的热力学，并将其应用于酵母线粒体基质、膜间空间、细胞质和细胞核中。成功地对铅毒性反应的血红素信号进行成像；(b) 在铅毒性的哺乳动物细胞模型中以特定方式应用传感器，这些目标的成功完成将为开始扩大研究范围提供动力。开发可用于细胞区室之间血红素同步成像且具有氧化态特异性的血红素传感器，并将其应用于铅毒性动物模型。在此阶段的重点是（a）使颜色味觉多样化。这用于亚细胞区室之间同步荧光成像的血红素传感器，并开发 Fe3+ 和 Fe2+ 血红素特异性传感器；(b) 将这些传感器部署到哺乳动物细胞系和秀丽隐杆线虫环境毒性模型中。总而言之，这些研究将产生第一个传感器。用于对与其在细胞信号传导中的作用相关的不稳定血红素进行成像，并确定血红素是一种重要的线粒体衍生信号分子，可在细胞和动物模型中定量启动对重金属毒性的适应。