Institute of Biological Chemistry, Academia Sinica, College of Life Sciences, National Taiwan University, Taipei, Taiwan.
Autophagy. 2011 Oct;7(10):1230-8. doi: 10.4161/auto.7.10.16626.
Mitophagy, the selective removal of mitochondria through the autophagic pathway, is involved in cellular mitochondria quality control. Dysfunctional mitochondria can be selectively eliminated through Parkin-mediated mitophagy. Parkin is a ubiquitin E3 ligase that selectively translocates onto impaired mitochondria to initiate mitophagy, and mutations in Parkin have been identified in autosomal recessive forms of Parkinson disease. Here with the use of a genetically encoded, mitochondria-matrix targeting photosensitizer, we established a robust strategy that allows for spatiotemporally controlled initiation of Parkin-mediated mitophagy in single cells with light. The method can specifically target varying numbers of mitochondria into the Parkin-mediated mitophagy pathway for clearance. Combined with live cell imaging, we demonstrated that mitochondria can be cleared by Parkin-mediated mitophagy without juxtanuclear mito-aggresome formation. Autophagy proceeded with the asynchronous appearance of small LC3B-coated structures on Parkin-labeled mitochondria subsections in a nucleation-expansion manner. Our method allows for quantitative measurement on the Parkin-mediated mitophagy process, and can be multiplexed in imaging for higher throughput studies.
线粒体自噬是通过自噬途径选择性去除线粒体的过程，参与细胞中线粒体的质量控制。功能失调的线粒体可以通过 Parkin 介导的线粒体自噬选择性消除。Parkin 是一种泛素 E3 连接酶，它可以选择性地转移到受损的线粒体上，启动线粒体自噬，Parkin 的突变已被确定存在于常染色体隐性遗传形式的帕金森病中。在这里，我们使用一种遗传编码的、靶向线粒体基质的光敏感剂，建立了一种强大的策略，可以用光在单细胞中时空控制地启动 Parkin 介导的线粒体自噬。该方法可以特异性地将不同数量的线粒体靶向到 Parkin 介导的线粒体自噬途径中进行清除。结合活细胞成像，我们证明了线粒体可以通过 Parkin 介导的线粒体自噬而不形成核周线粒体聚集物进行清除。自噬过程中，LC3B 小包裹结构以成核-扩展的方式异步出现在被 Parkin 标记的线粒体分段上。我们的方法允许对 Parkin 介导的线粒体自噬过程进行定量测量，并可以在成像中进行多重化，以进行更高通量的研究。
