Mitochondrial Fusion and Division

描述（由申请人提供）：线粒体是一种动态的膜结合细胞器，经历融合和分裂。这些对立事件之间的平衡以协调的方式发生，是细胞器大小、数量和形状的关键决定因素。线粒体融合和分裂由保守的动力蛋白相关gtpase介导，包括用于融合的Mfn（哺乳动物）/Fzo1p（酵母）和用于分裂的Drp1（哺乳动物）/Dnm1p（酵母）。线粒体融合和分裂异常与许多神经退行性疾病有关，如沙克-玛丽-图斯神经病变、显性视神经萎缩、阿尔茨海默病、亨廷顿病和帕金森病。许多这些疾病影响有丝分裂后的神经元，这些神经元沿其长轴突和分支树突含有线粒体。了解这些疾病的发病机制需要更深入地了解介导和协调线粒体融合和分裂的分子机制以及这些事件的生理功能。该研究将揭示线粒体如何融合（Aim 1），线粒体融合和线粒体分裂如何协调（Aim 2），以及线粒体分裂如何控制有丝分裂后神经元中的线粒体分布（Aim 3）。为了研究Aim 1中线粒体融合的分子机制，我们纯化了线粒体融合所需的两种酵母蛋白——Fzo1p GTPase和Fzo1p结合蛋白Ugo1p，并对其进行了生化表征。利用这些蛋白，我们开发了GTP结合、GTP水解和GTP依赖膜融合的检测方法。这些新的分析将使我们能够解剖Fzo1p GTPase和Ugo1p在线粒体融合中的功能。在Aim 2中，我们将确定线粒体融合和分裂是如何协调的。我们已经证明，Drp1的缺失降低了Drp1缺失小鼠胚胎成纤维细胞中Mfn1和Mfn2的水平。我们将确定Drp1水平的变化如何转化为Mfn水平和线粒体融合的调节。在Aim 3中，我们将使用Cre-loxP系统从小鼠有丝分裂后神经元中删除Drp1，从而确定线粒体分裂在神经元中的生理作用。初步数据显示，Drp1缺失导致线粒体分布改变和神经退行性变。我们将确定线粒体分裂如何控制有丝分裂后神经元的细胞器形态和分布。这些研究的成功完成将为线粒体融合、平衡线粒体融合和分裂的协调机制以及线粒体分裂在神经元中的生理作用提供机制上的见解。

DESCRIPTION (provided by applicant): The mitochondrion is a dynamic membrane-bound organelle that undergoes fusion and division. The balance between these opposing events, which occur in a coordinated manner, is a key determinant of organelle size, number, and shape. Mitochondrial fusion and division are mediated by conserved dynamin-related GTPases including Mfn (mammals)/Fzo1p (yeast) for fusion and Drp1 (mammals)/Dnm1p (yeast) for division. Abnormalities in mitochondrial fusion and division are associated with many neurodegenerative diseases such as Charcot-Marie-Tooth neuropathy, dominant optic atrophy, Alzheimer's disease, Huntington's disease, and Parkinson's disease. Many of these diseases affect postmitotic neurons, which contain mitochondria along their long axons and branched dendrites. Understanding the pathogenesis of these diseases requires a deeper knowledge of the molecular mechanisms that mediate and coordinate mitochondrial fusion and division as well as the physiological functions of these events. The proposed research will uncover how mitochondria fuse (Aim 1), how mitochondrial fusion and mitochondrial division are coordinated (Aim 2), and how mitochondrial division controls mitochondrial distribution in postmitotic neurons (Aim 3). To study the molecular mechanisms underlying mitochondrial fusion in Aim 1, we have purified and biochemically characterized two yeast proteins that are required for mitochondrial fusion- Fzo1p GTPase and the Fzo1p- binding protein Ugo1p. Using these proteins, we have developed assays for GTP binding, GTP hydrolysis, and GTP-dependent membrane fusion. These novel assays will allow us to dissect the functions of Fzo1p GTPase and Ugo1p in mitochondrial fusion. In Aim 2, we will determine how mitochondrial fusion and division are coordinated. We have shown that the loss of Drp1 reduces Mfn1 and Mfn2 levels in Drp1-null mouse embryonic fibroblasts. We will determine how changes in Drp1 levels are translated into regulation of Mfn levels and mitochondrial fusion. In Aim 3, we will determine the physiological roles of mitochondrial division in neurons by deleting Drp1 from postmitotic neurons using the Cre-loxP system in mice. Preliminary data show that Drp1 loss induces alterations in mitochondrial distribution and neurodegeneration. We will determine how mitochondrial division controls organelle morphology and distribution in postmitotic neurons. Successful completion of the proposed studies will provide mechanistic insights into mitochondrial fusion, the coordination mechanism that balances mitochondrial fusion and division, and the physiological role of mitochondrial division in neurons.