RT-SQuARED-M method to study the precise role of mitochondria in the oxidative damage caused by environmental toxins

 描述（由申请人提供）：细胞内信号传导的复杂性表现在各种信号传导途径之间的串扰以及它们受普遍存在的分析物（如ATP、ROS和细胞代谢物）的调节中，其中线粒体处于中心阶段。因此，监测线粒体的实时活动以充分了解其对细胞信号传导的影响至关重要。在这里，该团队提出研究线粒体裂变/融合事件，统称为mtDynamics，是否以及如何通过真实的时间调节线粒体ROS的产生来调节环境致癌过程中的信号传导事件。为了研究线粒体的这种精确作用，我们提出开发一种基于定量显微镜的线粒体氧化还原环境和动力学的真实的时间同时定量评估（RT-SQuARED-M）的分析方法。在R21阶段，将进一步开发和验证RT-SQuARED-M方法的性能。在达到设定的R21里程碑后，将测试R33阶段更广泛的科学假设。开创性研究和新的初步数据表明，抑制mtFission，这允许不受反对的mtFusion和受抑制的线粒体自噬，导致a）增加细胞增殖，B）增强热点的mtROS产生，和c）线粒体上关键细胞周期调节因子的非典型募集。基于这些数据，假设由氧化损伤诱导的环境多烃引起的致癌性是由于mtDynamics驱动的局部mtROS调节不同的线粒体位点进行信号传导和线粒体自噬而引发的。利用RT-SQuARED-M方法，我们预期发现：1）在DRP 1驱动的mtFission位点上较低的mtROS定义了线粒体吞噬热点，而在MFN 1/2驱动的mtFusion位点上较高的mtROS定义了信号传导热点; 2）多烃类致癌物B[a]P和TCDD降低Drp 1驱动的mtFission，抑制线粒体吞噬清除，并允许异常的基于mtROS的生长因子-线粒体中的细胞周期信号传导以启动肺和皮肤癌发生。发现这种可以被环境致癌物调节的精确线粒体信号传导将是线粒体在环境毒性中作用的范式转变。这项研究如果成功，将揭示线粒体动力学参与肺和皮肤癌变背后的作用机制，并强调关键的线粒体分裂/融合蛋白（Drp 1和/或Mfn 1/2）作为预防肺和皮肤癌变的潜在靶点。拟议的研究团队拥有成功开发和应用创新RT-SQuARED-M方法所需的所有方法和程序的专业知识，以了解肺部和皮肤的致癌作用。

 DESCRIPTION (provided by applicant): The complexity of intracellular signaling is demonstrated in the crosstalk between various signaling pathways and in their regulation by the ubiquitous analytes, such as ATP, ROS and cell metabolites, with mitochondria at the center stage. Thus, it is critical to monitor real-time activities of mitochondria to fully understand ther impact on cell signaling. Here, this team proposes to investigate if and how mitochondrial fission/fusion events, collectively called mtDynamics, modulate signaling events during environmental carcinogenesis by regulating mitochondrial ROS production in real time. To investigate such a precise role of mitochondria, we propose to develop a quantitative microscopy-based analytical method of Real Time Simultaneous Quantitative Assessment of the Redox Environment and Dynamics of Mitochondria (RT- SQuARED-M). In the R21 phase the performance of the RT-SQuARED-M method will be further developed and validated. After meeting the set R21 milestone, a broader scientific hypothesis in the R33 phase will be tested. The pioneering studies and new preliminary data demonstrate that repression of mtFission, which allows for unopposed mtFusion and repressed mitophagy, leads to a) increased cell proliferation, b) enhanced mtROS production from hotspots, and c) non-canonical recruitment of crucial cell cycle regulators on mitochondria. Based on these data, it is hypothesized that carcinogenicity caused by oxidative damage-inducing environmental polyhydrocarbons is initiated as mtDynamics-driven local mtROS modulates distinct mitochondrial sites for signaling and mitophagy. Using the RT-SQuARED-M method, we anticipate discovering 1) lower mtROS at the DRP1-driven mtFission sites define the mitophagic hotspots, while the higher mtROS at the MFN1/2-driven mtFusion sites define the signaling hotspots; 2) polyhydrocarbon carcinogens B[a]P and TCDD lower Drp1 driven-mtFission to repress mitophagic clearance and allow aberrant mtROS-based growth factor-cell cycle signaling in mitochondria to initiate lung and skin carcinogenesis. Discovery of such precise mitochondrial signaling that could be modulated by environmental carcinogens would be a paradigm shift in the role of mitochondria in environmental toxicity. This study, if successful, will reveal the mechanism of action behind the involvement of mitochondrial dynamics in lung and skin carcinogenesis and highlight key mitochondrial fission/fusion proteins (Drp1 and/or Mfn1/2) as potential targets for preventing initiation of lung and skin carcinogenesis. The proposed research team has expertise in all the methods and procedures required for successful development and application of the innovative RT- SQuARED-M method in understanding carcinogenesis of the lung and skin.