Electron tomograms from autophagosomes in NRK52E cells showing connections between phagophore and ER

Data includes ET tilt series and reconstructions from NRK-52E cells showing connections between the phagophore and ER. 18.9.2006
Eeva-Liisa Eskelinen, Päivi Ylä-Anttila

NRK-52E cells were incubated in serum and amino acid free medium for 1 h to induce autophagy. The cells were fixed in 2% GA in 0.1 M sodium cacodylate buffer, pH 7.4, postfixed in reduced osmium tetroxide, and flat embedded in Epon. Serial semithick 250-nm sections were prepared and picked on single slot grids. Colloidal gold particles of 10 nm in diameter were placed below the sections to serve as fiducial markers for alignment of the tomograms. Dual axis tilt series were acquired using SerialEM software running on a Tecnai FEG 20 microscope operating at 200 kV. Images were recorded at one degree intervals with a 1 k x 1 k CCD camera over a tilt range of ±70 degrees, niminl magnification 14,500x, pixel 1.63 nm. IMOD software was used to create 3D reconstructions from the tilt series.

Ylä-Anttila, P., Vihinen, H., Jokitalo, E., & Eskelinen, E. L. (2009). 3D tomography reveals connections between the phagophore and endoplasmic reticulum. Autophagy, 5(8), 1180–1185. https://doi.org/10.4161/auto.5.8.10274
Figs 2-6, videos