Metamorph 6

Two-dimensional cultures (Supplementary Movie 4 and Supplementary Fig. 4a): For time-lapse phase-contrast microscopy, transient or stably transfected MDCK cells were plated on 35-mm glass dishes (MatTek) and put in an open chamber (Life Imaging) equilibrated in 5% CO₂ and maintained at 37 °C. Time-lapse sequences were recorded every 5 min for 48 h (Supplementary Fig. 2a) or every second (Supplementary Movie 4) using a Nikon Eclipse Ti inverted microscope with a × 20 0.45 NA Plan FluorELWD objective (Supplementary Fig. 4a) or a × 100 1.4 NA PL-APO objective (Supplementary Movie 4) controlled by Metamorph 6.1 software (Universal Imaging). This microscope was equipped with a cooled CCD camera (HQ2; Roper Scientific). In Supplementary Fig. 4a, cytokinetic abscission time was quantified in mCherry-tubulin-transfected cells by time-lapse microscopy starting from furrow ingression until the cut of the intracellular bridge. In all, 100 cells per condition were analysed. Supplementary Movie 4 was deconvolved using Huygens Professional software SVI (2 iterations, signal/noise 10).

For US28-pHluorin TIRF imaging, HeLa cells on poly-l-lysine-coated round 18 mm coverslips in a 12 wells plate were transfected with 500 ng HA-US28-pHluorin plasmid using Lipofectamine 2000 (Invitrogen). 24 hours after transfection, coverslips were placed in an imaging chamber, perfused with Tyrode’s solution and imaged on a microscope (Zeiss, Axiovert 200M) equipped with an EMCCD camera (Cascade, Roper Scientific). For TIRF imaging, a laser beam from an air-cooled argon ion laser was coupled into a 100 × 1.45 N.A. TIRF objective via a TIRF condenser (TILL Photonics). Images were acquired at 2 Hz with MetaMorph 6.2 software (Universal Imaging). Imaging experiments were performed at room temperature (21°C–24°C).