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

体内调节线粒体亚群动态和功能的分子和细胞机制

基本信息

批准号：
10027150
负责人：
Tommy L Lewis
金额：
$ 43.7万
依托单位：
OKLAHOMA MEDICAL RESEARCH FOUNDATION
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-08-01 至 2025-05-31
项目状态：
未结题

项目摘要

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.

项目摘要线粒体调节许多关键细胞途径，包括能量稳态，钙处理和脂质产生。在多种细胞类型中，创建和维护不同的线粒体种群在亚细胞隔室中，在不同地区的生理挑战中采取了独特的反应细胞。虽然许多调节线粒体形状并因此功能的分子玩家具有已确定，完全了解其功能以及建立这些亚群时的互动细胞内的线粒体仍然难以定义。理解亚细胞线粒体的赤字形状和功能在很大程度上是由于有限的可视化和操纵能力，这些动态细胞器在高空间和时间分辨率下的真正生理环境。我们的方法旨在通过利用新开发的技术实现遗传标签和操纵多种细胞类型，具有高空间和时间成像的线粒体形态，体内动力和功能。在项目第一中，实验室成员将针对四个已知的哺乳动物 Dynamin样蛋白ONE（DRP1）的受体（MFF，FIS1，MIEF1/2），线粒体的主要效应子裂变，以测试其在皮质中不同线粒体亚群的创建和维持中的作用神经元和体内骨骼肌细胞。通过使用功能实验的损失，CRISPR/CAS标记和靶向摩托图分析，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细胞亚细胞裂变动力学。在项目中两个，实验室成员将实施稀疏，明亮的皮质神经元和明亮标签的方法骨骼肌细胞线粒体，带有荧光记者三磷酸腺苷，钙，pH和反应性氧气，并将其与活小鼠中的2光片成像搭配，以揭示这些线粒体如何亚群在体内为线粒体和细胞功能提供了信息。通过操纵不同的亚群和可视化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整合方法，以理解线粒体的通用和细胞特异性作用细胞中发现的亚群。