Nasu Yusuke, Benke Alexander, Arakawa Satoko, Yoshida Go J, Kawamura Genki, Manley Suliana, Shimizu Shigeomi, Ozawa Takeaki
Department of Chemistry, School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Laboratory of Experimental Biophysics, École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.
Sci Rep. 2016 Jun 13;6:27505. doi: 10.1038/srep27505.
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.
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。Bak蛋白在线粒体外膜上的聚集通过引发促凋亡蛋白从线粒体释放到细胞质中，从而诱导细胞凋亡。然而，蛋白质簇如何使线粒体膜通透仍不清楚，因为光学显微镜的衍射极限分辨率阻碍了对簇特征（如大小和蛋白质密度）的阐明。在这里，我们描述了一种基于单分子定位对Bak簇进行原位定量表征的方法。我们发现，在细胞凋亡过程中，Bak蛋白在线粒体上形成纳米级的密集簇。基于每个Bak蛋白定位的定量分析表明，尽管簇大小高度异质，但Bak蛋白密度在簇之间是均匀的。我们的方法提供了关于Bak簇的大小和蛋白质密度的前所未有的信息，这可能对通透化至关重要，并且适用于分析不同的簇形成。
