Analysis of Fis1 in Mitophagy in Mammals

 描述(申请人提供)：由于线粒体在细胞新陈代谢和其他功能中起着至关重要的作用，因此保持细胞内线粒体群体的质量至关重要。有丝分裂是线粒体通过自噬而降解，被认为是剔除功能失调或受损的线粒体的主要途径。研究最多的丝裂原吞噬途径涉及PINK1和Parkin，但关于这一途径的生理重要性仍然存在疑问。小鼠的PINK1和Parkin基因敲除只显示出基本线粒体功能的微小缺陷，与广泛存在的有丝分裂缺陷不一致。这一观察结果表明，有丝分裂存在替代或多余的途径。我们的初步数据表明，Fis1，一种先前被认为参与线粒体分裂的蛋白质，是有丝分裂的主要参与者，包括不依赖于PINK1/Parkin的有丝分裂。此外，缺乏FIS1的小鼠存在严重的生理缺陷。因此，对Fis1突变小鼠的研究可能揭示吞噬细胞的生理功能。该研究项目的长期目标是了解FIS1依赖的有丝分裂吞噬的分子机制以及有丝分裂吞噬在小鼠发育中的作用。在目标1中，我们开发了一种功能分析来剖析哺乳动物线粒体单亲遗传的分子机制。利用这个系统，我们将识别参与消除小鼠早期胚胎中父系线粒体的分子。在目标2中，我们开发了一种细胞系统，在该系统中，氧化磷酸化增加刺激有丝分裂。我们将利用这个细胞系统来理解有丝分裂吞噬的分子机制，包括FIS1的作用，泛素化的作用，以及自噬适配器p62的招募。在目标3中，我们利用Fis1基因敲除小鼠模型来了解有丝分裂吞噬的生理功能。我们将研究Fis1在男性生殖细胞成熟中的作用，在男性生殖细胞成熟过程中，Fis1的缺失会导致线粒体和过氧化体密度增加。综上所述，这些目标将有助于深入理解FIS1在体内的作用以及有丝分裂在哺乳动物生理中的作用。

 DESCRIPTION (provided by applicant): Because mitochondria have vital roles in cell metabolism and other functions, it is critical to maintain the quality of the mitochondrial population within cells. Mitophagy, the degradation of mitochondria via autophagy, is thought to be the major pathway for culling dysfunctional or damaged mitochondria. The best- studied mitophagy pathway involves Pink1 and Parkin, but questions remain about the physiological importance of this pathway. Mouse knockouts of Pink1 and Parkin show only subtle defects in basal mitochondrial function, inconsistent with a widespread defect in mitophagy. This observation suggests that there are alternative or redundant pathways for mitophagy. Our preliminary data indicate that Fis1, a protein previously thought to be involved in mitochondrial fission, is a major player in mitophagy, including mitophagy that is Pink1/Parkin-independent. Moreover, mice lacking Fis1 have severe physiological defects. Therefore, the study of Fis1 mutant mice may reveal the physiological functions of mitophagy. The long-term goals of this research project are to understand the molecular mechanism of Fis1-dependent mitophagy and the role of mitophagy in mouse development. In Aim 1, we develop a functional assay to dissect the molecular mechanism underlying uniparental inheritance of mitochondria in mammals. Using this system, we will identify molecules involved in elimination of paternal mitochondria in the early mouse embryo. In Aim 2, we develop a cellular system in which increased oxidative phosphorylation stimulates mitophagy. We will use this cellular system to understand the molecular mechanism of mitophagy, including the role of Fis1, the role of ubiquitination, and the recruitment of the autophagy adaptor p62. In Aim 3, we utilize a Fis1 knockout mouse model to understand the physiological functions of mitophagy. We will study the role of Fis1 in male germ cell maturation, where loss of Fis1 results in increased mitochondrial and peroxisomal density. Taken together, these Aims will lead to a deep understanding of the in vivo roles of Fis1 and the role of mitophagy in mammalian physiology.