Molecular and cellular mechanisms regulating mitochondrial subpopulation dynamics and function in vivo

项目摘要 线粒体调节许多关键的细胞途径，包括能量稳态、钙处理 和脂质产生。在许多细胞类型中，不同的线粒体群体被创建和维持 在亚细胞区室中，驱动对不同区域生理挑战的独特反应 cell.虽然许多调节线粒体形状和功能的分子参与者， 已经确定，完全了解他们的功能和建立这些亚群的相互作用 细胞内线粒体的数量仍然难以确定。对亚细胞线粒体的理解不足 形状和功能在很大程度上是由于有限的能力，可视化，和操纵，这些动态细胞器在一个 高空间和时间分辨率的真实生理环境。我们的方法旨在 利用新开发的基因标签技术，弥补这些知识差距， 操作，跨多种细胞类型，具有线粒体形态的高空间和时间成像， 体内动力学和功能。在项目一中，实验室的成员将针对四种已知的哺乳动物 受体（MFF，FIS 1，MIEF 1/2）的动力蛋白样蛋白1（DRP 1），线粒体的主要效应物， 裂变，以测试它们在皮层细胞中不同线粒体亚群的产生和维持中的作用。 神经元和骨骼肌细胞。通过使用功能丧失实验，CRISPR/Cas标记 和靶向基序分析结合高分辨率成像，我们将绘制分子机制 调节多个线粒体亚群的亚细胞线粒体裂变动力学。在项目 第二，实验室的成员将实施稀疏，明亮标记皮层神经元的方法， 骨骼肌细胞线粒体与腺苷三磷酸，钙，pH和反应性的荧光报告 氧物种，并将其与活体小鼠的双光子成像相结合，以揭示这些线粒体是如何 亚群告知体内线粒体和细胞功能。通过操纵不同的亚群， 可视化对体内多种细胞类型中线粒体和细胞功能的影响，我们将提供一个 独特的综合方法来了解线粒体的普遍和细胞特异性作用 细胞内发现的亚群。

Project Summary Mitochondria regulate a number of critical cellular pathways including energy homeostasis, calcium handling and lipid production. In a number of cell types, distinct populations of mitochondria are created and maintained within subcellular compartments driving unique responses to physiological challenges in different regions of the cell. While many of the molecular players that modulate mitochondrial shape, and therefore function, have been identified, complete understanding of their functions and interactions in establishing these subpopulations of mitochondria within cells remain difficult to define. The deficit in understanding subcellular mitochondrial shape and function is largely due to a limited ability to visualize, and manipulate, these dynamic organelles in a truly physiological environment at high spatial and temporal resolution. Our approaches are designed to address these gaps in knowledge by leveraging newly developed technologies enabling genetic labelling and manipulation, across multiple cell types, with high spatial and temporal imaging of mitochondrial morphology, dynamics and function in vivo. In project one, members of the laboratory will target the four known mammalian receptors (MFF, FIS1, MIEF1/2) of the dynamin-like protein one (DRP1), the main effector of mitochondrial fission, to test their roles in the creation and maintenance of different mitochondrial subpopulations in cortical neurons and skeletal myocytes in vivo. Through the use of loss of function experiments, CRISPR/Cas labeling and targeting-motif analysis coupled with high resolution imaging we will map the molecular mechanisms regulating subcellular mitochondrial fission dynamics across multiple mitochondrial subpopulations. In project two, members of the laboratory will implement methods for sparse, bright labeling of cortical neuron and skeletal myocyte mitochondria with fluorescent reporters for adenosine triphosphate, calcium, pH and reactive oxygen species, and couple it with 2-photon imaging in living mice to reveal how these mitochondrial subpopulations inform mitochondrial and cellular function in vivo. By manipulating different subpopulations and visualizing the effects on mitochondrial and cellular function in multiple cell types in vivo, we will provide a uniquely integrated approach to understanding the universal and cell-specific roles of mitochondrial subpopulations found within cells.