For live imaging cells were seeded on sterilized glass bottom culture dishes with a 10 mm microwell (MatTek Corporation, Ashland, MA) at 10400 cells/cm² for vesicle trafficking. Imaging was accomplished at 37 °C 20-29 h post-transfection under normoxic conditions. RPMI without phenol red (Gibco 1640, Invitrogen) containing 10 % FBS and 1 mg/l Hoechst 33342 for DNA staining (Invitrogen) was used as recording medium. In 2D-time series, 500 frames with a resolution of 512x512, 1000 Hz were taken every 393 – 786 ms with either a Leica TCS SP5 (Leica Microsystems, Wetzlar, Germany) or Zeiss LSM510META (Zeiss, Goettingen, Germany) confocal microscope for vesicle trafficking. Hoechst 33342, GFP and Cherry were excited using the 405 nm, 488 nm and 543 nm laser respectively.

Lysosomes were stained for live imaging using lysotracker®red DND-99 (Invitrogen) at a final concentration of 66.7 nM according to the manual provided by the company. Early endosomes were visualized using the CellLightTMReagent*BacMam 2.0* system for Rab5a-GFP overexpression (Invitrogen) as suggested in the manual. 30 particles per cell (PPC) were used for the transfection.

For statistical tests, the averaged vesicle speed of in total 5 samples was used. 5-48 vesicles/vacuoles of 1-5 cells were measured per sample. Using the chi-square test, a normal distribution of the calculated averages was accepted with p < 0.05. Vesicle vs. vacuole speed and displacement was compared using the one-tailed student’s t-test.