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Establish a simple pre-embedding correlative light and electron microscopy to evaluate the structural impacts of the APEX2 reporter in PK15 cells.

Establish a simple pre-embedding correlative light and electron microscopy to evaluate the structural impacts of the APEX2 reporter in PK15 cells.

Session

Stream 6 (Frontiers): Correlative Imaging of Organelle Organization and Architecture

Authors

Mr. Heng-Wei Lee (2), Dr. Ivan-Chen Cheng (2), Dr. Yi-Fan Jiang (1)

Affiliations

1. Graduate Institute of Molecular and Comparative Pathobiology, School of Veterinary Medicine, National Taiwan University
2. School of Veterinary Medicine, National Taiwan University

Keywords

Reporter genes, correlative light and electron microscopy, APEX2, endoplasmic reticulum, serial sections, electron tomography

Abstract text

The reporter genes have been widely used in cell culture systems to indicate the distribution of specific genes or organelles under microscopes. Recently, the fixative-resistant peroxidase, APEX2, has been applied in biological samples to visualize the gene expression and cellular structures under the electron microscope (1). However, as an artificial product in the cells, the cellular impact of the constructs remains to be estimated. In this report, the methods of pre-embedding correlative light and electron microscopy (CLEM) method was simplified with the common equipment in a biological lab (2). The genes of interest with either green fluorescent protein (GFP) or APEX2 were expressed in the porcine kidney (PK) 15 cells for structural comparisons. To recognize the positions of the GFP-positive area, the toners were transferred from an Aclar film onto the glass coverslips using the heating blocks of the dry bath at 130℃. After the observations of fluorescent signals, the cells and toners were fixed and processed for TEM observations. To check the cellular structures at the target area, serial sectioning and electron tomography were performed for 3D structural analysis. In the area with ER-targeted GFP, the rough endoplasmic reticulum (rER) loops, nuclear envelope, and mitochondria-associated membranes were observed in PK15 cells. The results were further confirmed by 3D structural analysis in APEX2-expressing cells. Although the signals of APEX2 were also observed on ER-associated structures, obvious regular and interconnected ER clusters were observed, suggesting that the APEX2 constructs may have additional impacts on ER membranes. However, more observations and studies are still necessary to confirm both the structural and functional changes of the cells expressing different reporter genes. Also, investigations are still necessary to reveal the mechanism for ER membrane organization in the cells.  

References

1. Lam, S.S., Martell, J.D., Kamer, K.J., Deerinck, T.J., Ellisman, M.H., Mootha, V.K., and Ting, A.Y. (2015). Directed evolution of APEX2 for electron microscopy and proximity labeling. Nat Methods 12, 51-54.
2. Padman, B.S., Bach, M., and Ramm, G. (2014). An improved procedure for subcellular spatial alignment during live-cell CLEM. PLoS One 9, e95967.