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：
8927908
负责人：
Amit Ram Reddi
金额：
$ 19.77万
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 2017-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8927908
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 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 public health relevance 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（由适用提供）：由于线粒体是能量代谢和中间代谢产物形成的中心枢纽，因此它们必须能够传输信号，以传达其功能与核的变化，以便细胞可以重塑代谢作用。当包括重金属在内的环境有毒物质损害线粒体功能时，线粒体 - 核信号传导尤为重要。但是，这些轨道间信号的性质和动力学及其被转导的机制的理解很少。 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 exclusive 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.但是，不存在用于研究基于血红素信号的传播的化学和遗传工具。在当前的赠款应用中，重点是在细胞和动物模型中生成和应用一般编码的比率荧光传感器，以研究对重金属毒性的线粒体衍生的内部内部和管间血红素信号的翻译。在R21阶段，意图是开发出来的，并应用了第一个比率的血红素传感器，以在重金属毒性的酵母和哺乳动物细胞模型中进行定量血红素成像。最后，目标是（a）表征血红素与血红素传感器的热力学，并在酵母线粒体基质，膜间空间，细胞质和细胞核中特异性地将其特异性地应用于定量图像血红素信号，以响应铅毒性；（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提供动力，以开始R33阶段，其中将扩展研究以开发血红素传感器，这些传感器可用于在细胞隔室之间简单地对血红素进行简单成像，并且是氧化态特异性的，并将其应用于PB毒性的动物模型。在此阶段，重点是（a）多样化血红素传感器的色泽，以在亚细胞隔室之间进行简单的荧光成像，并开发Fe3+和Fe2+血红素特异性传感器；（b）将这些传感器部署在哺乳动物细胞系和秀丽隐杆线虫的环境毒性模型中。这些研究总之将导致第一传感器为了定量成像不稳定的血红素与其在细胞信号传导中的作用相关，并建立血红素是一个重要的线粒体衍生的信号传导分子，从而使细胞和动物模型中的重金属毒性适应了重金属毒性。