Abstract
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, Fis1, 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 hFis1 spread widely around the mitochondrial surface while Mfn2 only exists at the contact sites.
Highlights
Protein localization correlates with its particular function in cells or tissues
We developed the APEX2–focus ion beam scanning electron microscopy (FIB-scanning electron microscopy (SEM)) approach to determine the target protein localization within 3D cellular ultrastructural context by combining two state-of-the-art techniques, APEX2 and FIB-SEM
Through investigation of the staining patterns in the 3D volume data, we found mitochondrial fission factor (Mff) and hFis1 distributed widely around the surface of mitochondrial outer membrane, and they were consistent with MiD51/ 49 except that the staining signals of MiD51/49 were reduced at the periphery of mitochondrial cluster
Summary
Protein localization correlates with its particular function in cells or tissues. Mapping protein localization information onto their cellular ultrastructural context is of great importance for cell biology study and can be achieved via electron microscopy (EM). EM contrast of immune-localization comes from the antibody-conjugated gold particles (De Mey et al 1981) or quantum dots (Giepmans et al 2005). This approach is significantly limited due to the limited efficiency of immunolabeling, the spatial hindrance of large antibodies, and the fact that the well-preserved ultrastructure and the antigen immuno-activity are always mutually exclusive
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