For transmission EM, cells grown on glass coverslips were fixed, osmicated, dehydrated in a graded ethanol series and acetone, and infiltrated gradually with epon (TAAB) as described by Puhka et al. (2007) (link). 60-nm-thick sections were cut parallel to the coverslip and poststained with uranyl acetate and lead citrate. Specimens were observed using a Tecnai 12 (FEI) microscope equipped with an Orius SC 1000B bottom-mounted charge-coupled device camera (Gatan) at acceleration voltage of 80 kV.
The negative-staining samples were prepared by mixing 2 µM protein with 500 µM unilamellar vesicles (with a diameter of 400 nm) in 20 mM Hepes buffer, pH 7.5, and 150 mM NaCl at RT for 5 min. The mixture was applied to the glow-discharged Pioloform (Agar Scientific)- and carbon-coated copper grids and stained with 3% uranyl acetate. At each step, excess solution was removed by filter paper. The membrane morphologies were examined with a JEM-1400 (Jeol) with a Orius SC 1000B bottom-mounted charge-coupled device camera (Gatan). The lipid composition of MIM used for EM was POPC:POPE:POPS:PI(4,5)P2:cardiolipin = 40:34:3:5:18. The final concentrations of FAM92A1 and lipid were 2 and 500 µM, respectively.
The negative-staining samples were prepared by mixing 2 µM protein with 500 µM unilamellar vesicles (with a diameter of 400 nm) in 20 mM Hepes buffer, pH 7.5, and 150 mM NaCl at RT for 5 min. The mixture was applied to the glow-discharged Pioloform (Agar Scientific)- and carbon-coated copper grids and stained with 3% uranyl acetate. At each step, excess solution was removed by filter paper. The membrane morphologies were examined with a JEM-1400 (Jeol) with a Orius SC 1000B bottom-mounted charge-coupled device camera (Gatan). The lipid composition of MIM used for EM was POPC:POPE:POPS:PI(4,5)P2:cardiolipin = 40:34:3:5:18. The final concentrations of FAM92A1 and lipid were 2 and 500 µM, respectively.
