ZFIN Image: Ariotti et al., 2015, Fig. 7

Image

Figure Caption

Fig. 7

Inducible Control of APEX-GBP Expression In Vivo

(A) EM of muscle tissue from a double transgenic non-heat shocked GFP-CAAX zebrafish crossed with the APEX-GBP zebrafish under control of the hsp70I promoter. No electron density can be observed at the surface of muscle cells. Scale bar represents 1 µm.

(B) EM of a double transgenic heat-shocked GFP-CAAX zebrafish expressing APEX-GBP (hsp70I promoter). Arrows highlight the electron density generated by the osmiophillic DAB reaction product accumulating at the site of the APEX-GBP tag and the GFP-CAAX protein at the PM of muscle cells. Arrowheads of higher magnification panels demonstrate sensitive detection of the transverse tubule network (observed by fluorescence microscopy in Figure 7B). CA, contractile apparatus; TT, transverse tubules; PM, plasma membrane; Nuc, nucleus. Scale bar represents 1 µm.

(C) Backscatter detection of zebrafish muscle tissue demonstrates the electron density generated by the DAB reaction can be observed in the 3view scanning electron microscope. Scale bar represents 1 µm.

(D) Aligned image stack and segmentation of the cell surface of a muscle cell from a GFP-CAAX APEX-GBP (hsp70I promoter) zebrafish line demonstrating the discernable PM in three-dimensions (white, PM; blue, nucleus). Refer to Movie S3 for three-dimensional aligned image stack.

Acknowledgments

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Reprinted from Developmental Cell, 35(4), Ariotti, N., Hall, T.E., Rae, J., Ferguson, C., McMahon, K.A., Martel, N., Webb, R.E., Webb, R.I., Teasdale, R.D., Parton, R.G., Modular Detection of GFP-Labeled Proteins for Rapid Screening by Electron Microscopy in Cells and Organisms, 513-25, Copyright (2015) with permission from Elsevier. Full text @ Dev. Cell