Cell-Free Analysis of Mitochondrial Fusion by Fluorescence Microscopy.
Cell-Free Analysis of Mitochondrial Fusion by Fluorescence Microscopy.
复制标题
通过荧光显微镜对线粒体融合的无细胞分析。
DOI：
10.1007/978-1-0716-0676-6_10
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Hoppins S
中科院分区：
文献类型：
--
作者：
Samanas NB;Hoppins S
线粒体外膜和内膜上的动力蛋白相关蛋白介导膜融合。线粒体融合在许多不同的生理环境中受到调节，包括细胞周期进程、分化途径、应激反应和细胞死亡。线粒体融合与线粒体分裂相反，需要线粒体在微管上运动。我们开发了一种无细胞重构线粒体融合试验，以规避影响体内线粒体融合机制活性的途径的复杂性。这允许在限定的条件下并且在不存在其他过程如线粒体分裂或转运的情况下定量线粒体融合。还可以评估蛋白质或小分子对线粒体融合的影响。在这里，我们描述了无细胞线粒体融合试验使用小鼠胚胎成纤维细胞分离的线粒体。
Dynamin-related proteins on both the mitochondrial outer and inner membranes mediate membrane fusion. Mitochondrial fusion is regulated in many different physiological contexts including cell cycle progression, differentiation pathways, stress responses, and cell death. Mitochondrial fusion is opposed by mitochondrial division and requires movement of mitochondria on microtubules. We developed a cell-free reconstituted mitochondrial fusion assay to circumvent the complexity of the pathways impinging on the activity of the mitochondrial fusion machinery in vivo. This allows for quantification of mitochondrial fusion in defined conditions and in the absence of other processes such as mitochondrial division or transport. The impact of proteins or small molecules on mitochondria fusion can also be assessed. Here we describe the cell-free mitochondrial fusion assay using mitochondria isolated from mouse embryonic fibroblasts.