LAUSR

Cell culture, transfection and fixation U2OS cells in McCoy's 5A Medium Modified (MCMM) (Life Technologies), HeLa cells and COS7 cells in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with glucose (Life Technologies) and 10% fetal bovine serum (Life Technologies) and penicillin/streptomycin (Hyclone) were grown at 37°C with 5% CO2. For transient expression, cells cultured in 12-well plates (Nunc) at 80% confluence were transfected with 1 μg lifeact-mEos3.2 [1] plasmid (from our lab), mEos3.2-CLC (clathrin light chain) [2] plasmid (gift from Prof. Joerg Bewersdorf) or mEos3.2-Mito [1] plasmid (from our lab) using Lipofectamine 2000 (Life Technologies) following the manufacturer’s protocol. Five hours post transfection, cells were trypsinized and plated on clean coverslips (Fisher Scientific) coated with 10 mg/ml fibronection (Millipore, FC010) to induce spreading for an additional 24 h. Fixations were performed with PBS buffer (pH 7.4) containing 4% paraformaldehyde and 0.2% glutaraldehyde for 15 min at 37°C just before imaging. During live imaging, cells were incubated in DMEM without phenol.