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

 描述(申请人提供)：由于线粒体是能量新陈代谢和中间代谢物形成的中心枢纽，它们必须能够向细胞核传递信号，传达其功能变化，以便细胞能够相应地重塑新陈代谢。当线粒体功能受到包括重金属在内的环境毒物的损害时，线粒体-核信号尤其重要。然而，这些细胞器间信号的性质和动力学以及它们被转导的机制还知之甚少。鉴于血红素调节的转录因子的重要性，它控制着从能量代谢到抗氧化应激反应再到细胞增殖的各种细胞过程，以及线粒体血红素的动态平衡对重金属的敏感度，我们认为线粒体来源的血红素通过线粒体-核逆行调节信号传递细胞对重金属毒性的反应。然而，可用于研究基于血红素的信号传输的化学和遗传工具并不存在。在目前的拨款申请中，重点是在细胞和动物模型中产生和应用一种基因编码的比率荧光血红素传感器，以研究线粒体衍生的器官内和器官间的血红素信号对重金属毒性的反应。在R21阶段，目的是开发和应用第一个比率血红素传感器，用于在重金属毒性的酵母和哺乳动物细胞模型中进行血红素定量成像。最后，我们的目标是(A)表征血红素与血红素传感器结合的热力学，并将它们应用于酵母线粒体基质、膜间隙、胞浆和细胞核中，以定量成像响应铅中毒的血红素信号；以及(B)在铅中毒的哺乳动物细胞模型中以隔室特有的方式应用传感器。这些目标的成功完成将为开始R33阶段提供动力，在该阶段中，研究将扩大到开发可用于细胞间血红素同时成像的血红素传感器，并将其应用于铅中毒的动物模型。在这一阶段，重点是(A)使血红素传感器的颜色味觉多样化，以便在亚细胞间同时进行荧光成像，并开发Fe3+和Fe2+特异性的血红素传感器；以及(B)在哺乳动物细胞系和线虫环境毒性模型中部署这些传感器。总而言之，这些研究将产生第一批传感器 用于定量成像不稳定的血红素与其在细胞信号中的作用，并建立血红素，一个重要的线粒体衍生的信号分子，在细胞和动物模型中启动对重金属毒性的适应。

 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.