Live cell experiments were performed on four different microscopes; 1: Olympus iX81 FluoView 1000 inverted confocal microscope (Olympus, Hamburg, DE), equipped with a PlanApo 60×1.30 oil objective. 2: Olympus SpinSR10 spinning disk confocal super resolution microscope equipped with a Yokogawa CSU-W1 SoRa. Images are acquired with a 60× Plan Apo 1.42 NA objective. 3: Zeiss LSM 880 equipped with and Airyscan detector and image acquisition with a 63×1.40NA oil immersion objective. 4: The FRAP experiments were executed on an Andor Dragonfly equipped with a Mosaic FRAP module. The Andor Dragonfly is built on a Nikon TiE inverted microscope equipped with a 60×1.40 NA oil immersion objective. Bleaching of the EGFP-Rab5 was done with 405 nm laser. Bleaching experiments were done by using 100% laser power, 100 ms bleaching time for the full-size endosome and 20 ms for converged Rab5 domains.
Data obtained from FRAP were normalized and corrected for bleaching (Pelkmans et al., 2001 (link)) and fitted by nonlinear regression to a function that assumes a single diffusion coefficient (Yguerabide et al., 1982 (link)); The values for F(0), F(∞) and t1/2 were calculated usingGraphPad Prism 8 , and immobile fractions (IF) were calculated as described by Lippincott-Schwartz et al. (2001) (link).
Data obtained from FRAP were normalized and corrected for bleaching (Pelkmans et al., 2001 (link)) and fitted by nonlinear regression to a function that assumes a single diffusion coefficient (Yguerabide et al., 1982 (link)); The values for F(0), F(∞) and t1/2 were calculated using