DOI: 10.23729/70f2c4b1-50cf-4c9c-a252-ee072ecf5203
title: Electron tomograms from LDs in A431 WT, SKO cells, seipin NE-trap and seipin Degron cells

description: For electron tomogprahy cells were grown on glass coverslips and fixed with 3% GA in 0.1 M Na-cacodylate buffer, pH 7.4, supplemented with 1 mM CaCl2 and 0.1% malachite green. Malachite green was used to enhance preservation of lipids (Hayat, 1993). Specimens were osmicated with reduced osmium, stained with uranyl acetate en bloc, dehydrated and embedded in Epon resin as described in Puhka et al, 2007. Dual axis tilt series were recorded from three consecutive semi-thick (250 nm) sections using Tecnai FEG 20 microscope operating at 200 kV. The sections were tilted at 1-degree intervals using a high tilt specimen holder between ±62 degrees. Images were acquired with SerialEM software using a 4k × 4k Ultrascan 4000 CCD camera at nominal magnification 7,800×, providing a 2×binned pixel size of 2.84 nm. The alignment of the tilt series as well as reconstructions were done with IMOD software package using 10-nm colloidal gold particles as fiducial markers. For manual segmentation and visualization, tomograms were denoised using nonlinear anisotrophic diffusion filter, K=1 for 4 iterations. Tomographic reconstructions were segmented, modeled and visualized using MIB software and modeling, visualization and surface area measurements of LD and ER-LD contact profiles were performed in Amira.

For CLEM cells were grown on fibronectin coated dishes with gridded glass cover slips. The cells were fixed and imaged by Zeiss LSM880 and Airyscan microscopy (to image end-seipin-GFPx7 and BFP-KDEL signal. After LM, cells were post-fixed with 1% reduced osmium tetroxide in 0.1 M sodium cacodylate buffer, pH 7.4 for 1 h at RT, en bloc stained with 1% uranyl acetate in 0.3 M sucrose for 1 h at 4°C, and flat embedded. The target cells were identified according to the finder grid pattern transferred to the block surface, and a pyramid covering the area of interest was trimmed. Serial 230-nm-thick sections parallel to the cover slip were cut with a 35° diamond knife on ultramicrotome and collected on Pioloform-coated single slot copper grids. The target areas for 3D-imaging were defined by correlating the ER profiles in TEM micrographs and BFP-KDEL signal in fluorescence microscopy images using Corel PHOTO-PAINT. One to three consecutive semi-thick sections having the overlapping signal/features/profiles were subjected to ET. The acquisition of dual axis tilt series and reconstructions were done as described (Salo et al., 2016), except using nominal magnification of 11.500x. Segmentation and visualization were done with MIB and Amira, respectively. For data in Figures S2A and S2B, A431 cells and primary human fibroblasts were fixed and imaged as described (Salo et al., 2016). For visualization, tomograms were denoised using nonlinear anisotrophic diffusion filter, K=1.2 for 11 iterations. Measurements of membrane-LD contact site parameters were done using ImageJ FIJI and analysis of contact site areas using MIB.

Cells of S166D-seipin mutant, grown on fibronectin-coated coverslips were fixed for immunoelectron tomography. Briefly, after PLP fixation and permeabilization with 0.01% saponin, the cells were labeled with anti-GFP antibody for 1 h and nano-gold-conjugated anti-rabbit FAB fragments for 1 h, post-fixed with 1% glutaraldehyde, and quenched with 50 mM glycine. Nano-gold particles were then intensified for 2 min using the HQ SILVER Enhancement Kit followed by gold toning in subsequent incubations in 2% sodium acetate, 0.05% HAuCl4, and 0.3% sodium thiosulfate prior to osmication, dehydration, and flat embedding. A 230-nm-thick section cut parallel to the cover slip was subjected to electron tomography at a nominal magnification of 19,000×, providing a 2× binned pixel size of 1.2 nm, using a Tecnai FEG 20 microscope operated at 200 kV. The alignment of the tilt series as well as reconstruction (SIRT technique using 14 iterations) were done with the IMOD software package (version 4.10.32). Segmentation and visualization were done using MIB (version 2.601) and Amira (version 5.3.2), respectively. The immunolabeling is not shown, as the labeling was clearly denser at the outskirts and edges of the S166D-induced ER sacs, suggesting that steric hindrance may prevent diffusion of the antibodies to these structures.

2016_S5-S3_Ctrl includes TEM images from BSCL2 patient and control lymphoblasts (Combot et al., 2022).