APEX2-enhanced electron microscopy distinguishes sigma-1 receptor localization in the nucleoplasmic reticulum
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Timur A. Mavlyutov1,*, Huan Yang1,*, Miles L. Epstein2, Arnold E. Ruoho2, Jay Yang3 and Lian-Wang Guo1,4,5,6
1Department of Surgery, Wisconsin Institute for Medical Research, University of Wisconsin, Madison, WI 53705, USA
2Department of Neuroscience, University of Wisconsin, Madison, WI 53706, USA
3Department of Anesthesiology, Wisconsin Institute for Medical Research, University of Wisconsin, Madison, WI 53705, USA
4McPherson Eye Research Institute, University of Wisconsin, Madison, WI 53705, USA
5Department of Surgery, Davis Heart and Lung Research Institute, The Ohio State University, Columbus, OH 43210, USA
6Department of Physiology and Cell Biology, Davis Heart and Lung Research Institute, The Ohio State University, Columbus, OH 43210, USA
*These authors contributed equally to this work
Lian-Wang Guo, email: Lianwang.Guo@osumc.edu
Keywords: the sigma-1 receptor, APEX2-enhanced electron microscopy, nucleoplasmic reticulum, nuclear envelope, serial sectioning
Abbreviations: Sig1R-sigma-1 receptor, ER-endoplasmic reticulum, NR-nucleoplasmic reticulum, NE-nuclear envelope, EM-electron microscopy
Received: March 02, 2017 Accepted: May 04, 2017 Published: May 16, 2017
The sigma-1 receptor (Sig1R) is an endoplasmic reticulum chaperonin that is attracting tremendous interest as a potential anti-neurodegenerative target. While this membrane protein is known to reside in the inner nuclear envelope (NE) and influences transcription, apparent Sig1R presence in the nucleoplasm is often observed, seemingly contradicting its NE localization. We addressed this confounding issue by applying an antibody-free approach of electron microscopy (EM) to define Sig1R nuclear localization. We expressed APEX2 peroxidase fused to Sig1R-GFP in a Sig1R-null NSC34 neuronal cell line generated with CRISPR-Cas9. APEX2-catalyzed gold/silver precipitation markedly improved EM clarity and confirmed an apparent intra-nuclear presence of Sig1R. However, serial sectioning combined with APEX2-enhanced EM revealed that Sig1R actually resided in the nucleoplasmic reticulum (NR), a specialized nuclear compartment formed via NE invagination into the nucleoplasm. NR cross-sections also indicated Sig1R in ring-shaped NR membranes. Thus, this study distinguishes Sig1R in the NR which could otherwise appear localized in the nucleoplasm if detected with low-resolution methods. Our finding is important for uncovering potential Sig1R regulations in the nucleus.
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