Determining the target protein localization in 3D using the combination of FIB-SEM and APEX2  

作　　者：Yang Shi Li Wang Jianguo Zhang Yujia Zhai Fei Sun 

机构地区：[1]National Key Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China [2]Center for Biological Imaging, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China [3]University of Chinese Academy of Sciences, Beijing 100049, China [4]Sino-Danish Center for Education and Research, Beijing 100190, China

出　　处：《Biophysics Reports》2017年第4期92-99,共8页生物物理学报（英文版）

摘　　要：Determining the cellular localization of proteins of interest at nanometer resolution is necessary for elucidating their functions. Besides super-resolution fluorescence microscopy, conventional electron microscopy （EM） combined with immunolabeling or clonable EM tags provides a unique approach to correlate protein localization information and cellular ultrastructural information. However, there are still rare cases of such correlation in three-dimensional （3D） spaces. Here, we developed an approach by combining the focus ion beam scanning electron microscopy （FIB-SEM） and a promising clonable EM tag APEX2 （an enhanced ascorbate peroxidase 2） to determine the target protein localization within 3D cellular ultrastructural context. We further utilized this approach to study the 3D localization of mitochondrial dynamics-related proteins （MID49/51, Mff, Fisl, and Mfn2） in the cells where the target proteins were overexpressed. We found that all the target proteins were located at the surface of the mitochondrial outer membrane accompanying with mitochondrial clusters. Mid49/51, Mff, and hFisl spread widely around the mitochondrial surface while Mfn2 only exists at the contact sites.Determining the cellular localization of proteins of interest at nanometer resolution is necessary for elucidating their functions. Besides super-resolution fluorescence microscopy, conventional electron microscopy （EM） combined with immunolabeling or clonable EM tags provides a unique approach to correlate protein localization information and cellular ultrastructural information. However, there are still rare cases of such correlation in three-dimensional （3D） spaces. Here, we developed an approach by combining the focus ion beam scanning electron microscopy （FIB-SEM） and a promising clonable EM tag APEX2 （an enhanced ascorbate peroxidase 2） to determine the target protein localization within 3D cellular ultrastructural context. We further utilized this approach to study the 3D localization of mitochondrial dynamics-related proteins （MID49/51, Mff, Fisl, and Mfn2） in the cells where the target proteins were overexpressed. We found that all the target proteins were located at the surface of the mitochondrial outer membrane accompanying with mitochondrial clusters. Mid49/51, Mff, and hFisl spread widely around the mitochondrial surface while Mfn2 only exists at the contact sites.

关 键 词：Enhanced ascorbate peroxidase Focus ion beam scanning electron microscopy Mitochondrial dynamics Protein location Three-dimensional space 

分 类 号：Q24[生物学—细胞生物学]