Axiovert 200

GFP::DNAI1 was first observed directly with a DMI4000 Leica microscope using a mercury bulb for excitation to verify correct protein expression and localisation. For FRAP analysis of cells expressing GDP::DNAI1, a Zeiss inverted microscope (Axiovert 200) equipped with an oil immersion objective (magnification x63 with a 1.4 numerical aperture) and a spinning disk confocal head (CSU22, Yokogawa) was used (Buisson et al., 2013) . Images were acquired using Volocity software with an EMCCD camera (C-9100, Hamamatsu) operating in streaming mode. A sample was taken directly from the culture grown at 6 to 8 x 10 6 cells/mL and trapped between slide and coverslip. The samples were kept at 27°C using a fast response mini-stage temperature controller. Time-lapse sequences were acquired to analyse GFP signal recovery after photobleaching. Movies were taken using a time lapse of 3 minutes. Exposure time was 0.8 second per frame (binning was 1x1 pixels). In the case of cells expressing mNeonGreen::DHC-ODAB, the same settings were used except that the microscope was equipped with a 100x objective (1.4 numerical aperture). In this case, 8 cells were identified per series and their position recorded before photobleaching. Sequences of 20 seconds were filmed for each with an exposure time of 0.1 second per frame. Time lapse varied between 5 and 13 minutes.