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VAP–SCRN1 interaction regulates dynamic endoplasmic reticulum remodeling and presynaptic function 133 4 Expansion microscopy sample preparation Expansion microscopy was performed according to proExM protocol (Tillberg et al, 2016). Briefly, immunostained cells on 18-mm glass coverslips were incubated overnight in PBS with 0.1 mg/ml Acryloyl-X (Thermo Fisher, A20770) and 0.002% of 0.1 μm yellow–green Fluorospheres (Thermo Fisher, F8803). These bright fluorescent microspheres adhered to cell surfaces, thereby this cell boundary marker simplified the localization of cells in the expanded samples. Cells were washed three times with PBS and transferred to a gelation chamber (13 mm diameter, 120 μl volume) made of silicone molds (Sigma- Aldrich, GBL664107) on a parafilm-covered glass slide. The chamber was prefilled with monomer solution (PBS, 2 M NaCl, 8.625% (w/w) sodium acrylate, 2.5% (w/w) acrylamide, and 0.15% (w/w) N,N′-methylenebisacrylamide) supplemented with 0.4% (w/w) tetramethylethylenediamine (TEMED) accelerator and 0.2% (w/w) ammonium persulfate (APS) initiator. The gelation proceeded for 1 h at 37°C in a humidified incubator. Gels were further immersed into 2 ml of 8 units/ml proteinase-K in digestion buffer (pH 8.0, 50 mM Tris, 1 mM EDTA, 0.5% Triton X-100, 0.08 M guanidine HCl) solution for 4 h at 37°C for digestion. Gels were transferred to 50 ml deionized water for overnight expansion, and water was refreshed once to ensure the expansion reached plateau. Plasma-cleaned #1.5 coverslips for gel imaging were incubated in 0.1% poly-l-lysine to reduce gel’s drift during acquisition. Gels were mounted using custom-printed imaging chambers (https://www. tinkercad.com/things/7qqYCygcbNU) . Expansion factor was calculated as a ratio of a gel’s diameter to the diameter of gelation chamber and was in the range of 4.0–4.1. Confocal microscopy Confocal microscopy of fixed samples on glass coverslips was performed with a LSM 700 confocal laser-scanning microscope (Zeiss) equipped with a Plan-Apochromat 63x NA 1.40 oil DIC, EC Plan-Neofluar 40x NA1.30 Oil DIC, and a Plan-Apochromat 20x NA 0.8 objective. Each confocal image represents a maximum intensity projection of a z-series covering the region of interest. For fluorescence intensity measurements, settings were kept the same for all conditions. Confocal microscopy of fixed samples on expanded gels was performed with a Leica TCS SP8 STED 3X microscope using a HC PL APO 63×/1.20 W CORR CS2 water immersion objective. Images were acquired with lateral pixel size in the range of 70–80 nm and axial of 180 nm using internal HyD detector. If necessary, a drift correction of Z-stack was performed in Huygens Professional version 17.04 (Scientific Volume Imaging, The Netherlands) using cross-correlation between adjacent slices. All images were deconvolved in the same program, using the CMLE algorithm, with SNR:7 and 20 iterations. Movies of 3D reconstructions of z-stacks were performed in Blender version 2.79b (Blender Institute, Amsterdam). Syt uptake assay Hippocampal neurons were pretreated with 50 μM bicuculline (Sigma, 14340) in their original NB medium for 10 min at 37°C in 5% CO2. Next, neurons were directly incubated