Electron tomograms from IL-2Rbeta labelled Hep2beta cells

Hep2β cells were incubated on ice with DMEM containing 10% FCS and 0.2% BSA for 20 min. To label IL-2Rβ, an antibody recognizing the extracellular part of the receptor was added for 20 min at 4°C and washed and cells were incubated with protein A coupled to 10-nm gold beads at 4°C (dilution 1/25), washed and further incubated for 7 or 12 min at 37°C to allow endocytosis. Cells were then fixed in 2% GA in 0.1 M NaCac buffer, pH 7.4. Cells were postfixed with 1% reduced osmium tetroxide in 0.1 M NaCac. Cells were gradually dehydrated in EtOH and Ac, infiltrated into epoxy resin and polymerized for 48 h at 60°C.

For electron tomographic analysis, two consecutive 150-nm-thick sections were cut from trimmed Epon block. Images were acquired with Tecnai FEG 20 operating at 200 kV equipped with 4k × 4k Ultrascan 4000 CCD camera using SerialEM software (http://bio3d.colorado.edu/SerialEM/). Tilt series were collected over a tilt range of ± 62° with one-degree interval at nominal magnification of 14,500× providing a 2× binned pixel size of 1.53 nm. Gold particles of 10 nm in size were placed on top/below the sections to serve as fiducial markers; however, all gold particles were erased from the images to reduce the artefacts cast by these markers in the reconstructions. The dual-axis tomograms were reconstructed using IMOD software package (http://bio3d.colorado.edu/imod) prior segmentation, modelling and visualization with Amira software.

Basquin C, Trichet M, Vihinen H, Malardé V, Lagache T, Ripoll L, Jokitalo E, Olivo-Marin JC, Gautreau A, Sauvonnet N. Membrane protrusion powers clathrin-independent endocytosis of interleukin-2 receptor. EMBO J. 2015 Aug 13;34(16):2147-61. doi: 10.15252/embj.201490788. Epub 2015 Jun 29. PMID: 26124312; PMCID: PMC4557667.