In Situ Characterization of Bak Clusters Responsible for Cell Death Using Single Molecule Localization Microscopy.
In Situ Characterization of Bak Clusters Responsible for Cell Death Using Single Molecule Localization Microscopy.
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DOI：
10.1038/srep27505
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发表时间：
2016-06-13
影响因子：
4.6
通讯作者：
Ozawa T
中科院分区：
文献类型：
--
作者：
Nasu Y;Benke A;Arakawa S;Yoshida GJ;Kawamura G;Manley S;Shimizu S;Ozawa T
细胞凋亡在多细胞生物的发育和组织稳态中起着关键作用。巴克蛋白在线粒体外膜上的聚集是通过引起促凋亡蛋白从线粒体释放到胞质溶胶中来诱导细胞凋亡的原因。然而，蛋白质簇如何渗透线粒体膜仍不清楚，因为阐明簇的特征，如大小和蛋白质密度已受到阻碍的衍射有限的分辨率的光学显微镜。在这里，我们描述了一种方法来定量表征巴克集群在原位基于单分子定位。我们发现，巴克蛋白在细胞凋亡过程中在线粒体上形成纳米级的密集簇。基于每个巴克蛋白的定位的定量分析揭示，尽管簇大小是高度异质性的，但巴克蛋白的密度在簇之间是均匀的。我们的方法提供了前所未有的信息的大小和蛋白质密度的巴克集群可能至关重要的透化，并适用于不同的集群形成的分析。
Apoptosis plays a pivotal role in development and tissue homeostasis in multicellular organisms. Clustering of Bak proteins on the mitochondrial outer membrane is responsible for the induction of apoptosis by evoking a release of pro-apoptotic proteins from mitochondria into cytosol. However, how the protein cluster permeabilizes the mitochondrial membrane remains unclear because elucidation of the cluster characteristics such as size and protein density has been hampered by the diffraction-limited resolution of light microscopy. Here, we describe an approach to quantitatively characterize Bak clusters in situ based on single molecule localization. We showed that Bak proteins form densely packed clusters at the nanoscale on mitochondria during apoptosis. Quantitative analysis based on the localization of each Bak protein revealed that the density of Bak protein is uniform among clusters although the cluster size is highly heterogeneous. Our approach provides unprecedented information on the size and protein density of Bak clusters possibly critical for the permeabilization and is applicable for the analysis of different cluster formations.