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

 描述(申请人提供)：细胞内信号的复杂性表现在各种信号通路之间的串扰以及它们被无处不在的分析物，如ATP，ROS和细胞代谢物，以线粒体为中心阶段的调节中。因此，监测线粒体的实时活动以充分了解其对细胞信号的影响是至关重要的。在这里，该团队建议调查线粒体裂变/融合事件，统称为mtDynamic，是否以及如何通过实时调节线粒体ROS的产生来调节环境致癌过程中的信号事件。为了研究线粒体的这种精确作用，我们建议建立一种基于定量显微镜的实时同时定量评估线粒体氧化还原环境和动力学(RT-Squared-M)的分析方法。在R21阶段，将进一步开发和验证RT-平方-M方法的性能。在达到设定的R21里程碑后，将在R33阶段测试更广泛的科学假设。这些开创性的研究和新的初步数据表明，抑制线粒体分裂使线粒体融合和抑制有丝分裂，导致a)增加细胞增殖，b)增加热点mtROS的产生，c)线粒体上关键的细胞周期调节因子的非规范招募。根据这些数据，假设氧化损伤诱导的环境多碳氢化合物的致癌作用是由于mtDynamic驱动的局部mtROS调控不同的线粒体位置进行信号传递和有丝分裂。使用RT-Square-M方法，我们预计会发现1)在Drp1驱动的mtFastings位点上较低的mtROS定义了有丝分裂热点，而在Mfn1/2驱动的mtFusion位点上的较高mtROS定义了信号热点；2)多烃类致癌物质B[a]P和TCDD降低了Drp1驱动的mtF，以抑制有丝分裂清除，并允许线粒体中基于mtROS的生长因子细胞周期信号异常启动肺癌和皮肤癌的发生。这种可以被环境致癌物调节的精确线粒体信号的发现，将是线粒体在环境毒性中作用的一种范式转变。这项研究如果成功，将揭示线粒体动力学在肺癌和皮肤癌发生中的作用机制，并强调关键的线粒体分裂/融合蛋白(Drp1和/或Mfn1/2)是预防肺癌和皮肤癌发生的潜在靶点。拟议的研究小组在成功开发和应用创新的RT-Square-M方法以了解肺癌和皮肤癌的发生所需的所有方法和程序方面拥有专业知识。

 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.