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

 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改变的信号传递给细胞核，以便细胞可以响应地重塑代谢。当线粒体功能受到环境毒物（包括重金属）的损害时，线粒体核信号传导尤为重要。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知之甚少。考虑到血红素调节的转录因子控制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的重要性，以及线粒体血红素稳态对重金属的灵敏度，我们提出线粒体来源的血红素通过细胞核逆行调节来发出对重金属毒性的细胞反应的信号。然而，可用于研究血红素信号传输的化学和遗传工具并不存在。在目前的资助申请中，重点是在细胞和动物模型中产生和应用遗传编码的比率荧光血红素传感器，以研究重金属毒性引起的脑内和器官间血红素信号的转导。在R21阶段，目的是开发和应用第一个比率血红素传感器，用于重金属毒性的酵母和哺乳动物细胞模型中的血红素定量成像。最后，我们的目标是（a）血红素结合血红素传感器的热力学特性，并将它们应用于酵母线粒体基质，膜间空间，胞质溶胶和细胞核中的位点特异性，以定量成像血红素信号响应铅毒性;和（B）应用传感器在特定的方式在哺乳动物细胞模型的铅毒性。这些目标的成功完成将提供动力，开始的R33阶段，其中研究将扩大到开发血红素传感器，可用于同时成像的血红素细胞室之间，是氧化态特异性，并将其应用于铅毒性的动物模型。在该阶段，重点是（a）使血红素传感器的色觉多样化，用于在亚细胞区室之间同时荧光成像，并开发Fe 3+和Fe 2+血红素特异性传感器;以及（B）将这些传感器部署在哺乳动物细胞系中，以及C. elegans环境毒性模型。总之，这些研究将导致第一个传感器 用于与其在细胞信号传导中的作用相关的不稳定血红素的定量成像，并在细胞和动物模型中确立血红素是启动对重金属毒性的适应的重要的神经细胞衍生的信号传导分子。

 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.