介导线粒体和内质网互作的分子机器动态调控机制

In a eukaryotic cell, mitochondria and endoplasmic reticulum (ER) form interacting networks via mitochondrial-ER contacts (MERCs), which play an important role in concerted regulation of biological processes of a cell through bidirectional trafficking of factors between the two organelles. However, the in vivo molecular mechanism of mitochondria and ER dynamic interaction is not completely known. The applicant was using C. elegans as a model studying skin wound response and repair, and has committed to the field for many years. Recently, we found that skin wounding triggers immediate mitochondrial responses and breaks down the organelle network to fragmentation. Interestingly, the fragmentation of mitochondria after wounding is not dependent on DRP-1 mediated mitochondrial fission. Through genetic screen, we found loss of function of miro-1, which is a mitochondrial Rho GTPase located in the outer mitochondrial membrane, dramatically changed the mitochondrial morphology to thread-like structure in C. elegans epidermis. Furthermore, skin injury does not trigger mitochondrial network breakdown in miro-1 mutant compare to the wild type. Gem1, the yeast homology of MIRO-1, have been shown interacting with ER-mitochondria encounter structure (ERMES), indicating that MIRO-1 may regulate mitochondria and ER tether and respond to skin injury. Thus, based on the preliminary results, in this proposal we will investigate the molecular mechanism by which MIRO-1 regulates mitochondria-ER dynamic interaction in vivo in C. elegans epidermis. Combining CRISPR-Cas9 based genome editing tool, genetics, molecular biotechnology, live imaging, laser injury and single-molecule biomechanical assay to characterize membrane protein-protein interaction, we propose three specific aims to study: 1) investigating the function of MIRO-1 in regulating the establishment and maintenance of mitochondria-ER interaction structure; 2) discovering the key interacting proteins of MIRO-1 in regulating the dynamic mitochondria-ER interaction structure; 3) characterizing the molecular mechanism of biomechanical-biochemical coupling of MIRO-1’s binding to its partners in modulating the dynamic structure of mitochondria-ER interactions before and after injury. Results from these studies will not only elucidate the molecular and biomechanical mechanisms of dynamic mitochondria-ER interaction, but may also provide novel insights for the treatment of mitochondria and ER related human diseases.

真核细胞内线粒体和内质网互作网络调控着细胞许多重要的生物学活动，但是它们在体内互作的动态模式和分子机制知之甚少。申请人以秀丽线虫为模型，着重研究皮肤的创伤应激和修复。最近我们发现皮肤损伤引起细胞内的线粒体快速响应并导致其网状结构瓦解。遗传筛选发现miro-1基因突变导致线粒体线性化排列且不能正常应激损伤。酵母中MIRO-1与线粒体和内质网的结构偶联复合体互作，提示MIRO-1可能参与调控线粒体和内质网的互作结构并响应损伤。本项目将在整体动物水平探索线粒体和内质网的互作，通过多学科技术手段，探索MIRO-1介导线粒体和内质网动态互作结构建立和维持的功能，发现其关键互作蛋白并研究它们之间动态互作的生物力学调控机制，阐明线粒体和内质网互作的分子机理。本项目的开展将为综合理解细胞器互作网络调控机制提供新理论，同时为解决因线粒体和内质网互作网络紊乱造成的疾病提供新思路。

细胞器互作和稳态调控在细胞命运决定、生长发育过程中发挥重要功能。线粒体的网络结构和稳态维持对于其自身的功能和细胞的增殖分裂有重要影响。过去的研究表明，线粒体的动态维持受一系列融合分裂蛋白调控，但是，线粒体如何应激损伤如何维持重建其稳态结构和网络并不清楚。本项目利用CRISPR-Cas9内源性插入、激光损伤、快速活体显微成像、单分子生物力学等技术手段，明确建立和维持线粒体动态互作结构的关键蛋白质机器MIRO-1的功能并阐明其作用的分子生物学及分子力学机理。通过近两年的研究，我们利用线虫表皮为模型，发现损伤快速引起线粒体的片段化。遗传学和显微成像结果表明线粒体片段化加速伤口愈合。遗传筛选并发现MIRO-1是损伤引起的线粒体片段化所必须的。进一步研究发现，损伤引起的钙信号释放通过MIRO-1导致线粒体的片段化，并且这一过程并不依赖于传统的DRP-1依赖的线粒体分裂过程。通过针刺损伤，我们发现运动神经元能够快速的融合实现再生。利用线虫的遗传筛选系统，我们发现了参与神经再生的保守的分子DLK-1；同时我们也发现线粒体的动态变化显著影响神经的再生，在miro-1, drp-1等突变体力，损伤后的神经元发生了显著的退行，为进一步研究神经再生和退行的机制创造了条件。.为进一步研究MIRO-1如何参与调控线粒体形态及其与内质网的互作，通过GFP::MIRO-1的基因敲入和蛋白质谱技术，我们已经找到4个可能与MIRO-1互作并维持线粒体形态的关键基因VDAC-1。初步的研究提示，MIRO-1和VDAC-1可能通过调控线粒体的膜电势来调控线粒体的形态以及对损伤的应激功能。本项目的研究成果为我们进一步理解线粒体的动态调控以及与其它细胞器的动态互作提供了新的研究模式和理论参考。