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通路，包括能量稳态、钙处理 和脂肪的产生。在许多细胞类型中，会产生和维持不同的线粒体群体 在亚细胞隔间内驱动对不同区域的生理挑战的独特反应 手机。而许多调节线粒体形状并因此发挥功能的分子参与者， 已经确定，完全了解它们在建立这些亚群中的功能和相互作用 细胞内线粒体的数量仍然很难定义。对亚细胞线粒体认识的缺失 形状和功能在很大程度上是由于可视化和操纵这些动态细胞器的能力有限 高时空分辨率的真实生理环境。我们的方法旨在 通过利用新开发的技术来解决这些知识差距，使基因标记和 操控，跨越多种细胞类型，线粒体形态的高空间和时间成像， 体内的动力学和功能。在第一个项目中，实验室成员将针对这四种已知的哺乳动物 线粒体的主要效应蛋白--动力蛋白样蛋白1的受体(MFF、FIS1、MIEF1/2) 分裂，以测试它们在大脑皮层不同线粒体亚群的产生和维持中的作用 活体内的神经元和骨骼肌细胞。通过使用功能丧失实验，CRISPR/Cas标记 靶向基序分析与高分辨率成像相结合，我们将绘制分子机制图 跨多个线粒体亚群调控亚细胞线粒体分裂动力学。在项目中 第二，实验室成员将实施稀疏、明亮的皮质神经元标记方法和 骨骼肌细胞线粒体与三磷酸腺苷、钙、pH和反应性的荧光指示剂 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 亚群决定了体内线粒体和细胞的功能。通过操纵不同的子群和 在体内可视化多种细胞类型对线粒体和细胞功能的影响，我们将提供一个 了解线粒体的普遍作用和细胞特异性作用的独特综合方法 在细胞内发现的亚群。

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.