Fig. 7. Rab5 regulates the `kiss and run' fusion between early/late endosomes. To determine whether rab5 modulates `kiss and run' interactions between endosomes, cells were incubated with mixtures of dextrans (FDx10 and TRDx70 or FDx70 and TRDx70) for 30 minutes, then washed and further incubated for the indicated times. The distribution of dextrans was observed by confocal fluorescence microscopy. Immediately after internalization, the different-sized dextrans (FDx10, green signal and TRDx70, red signal) were co-localizing in the same organelles, labeled in yellow, in control and rab5(Q79L)-expressing cells. After a 30 minute chase, distinctly labeled endosomes were observed in control cells allowed to internalize the mixture of FDx10 and TRDx70. At the same time point in mutant cells, the different-sized dextrans were still co-localizing in the endocytic compartments. After a 120 minute chase, the segregation of different-sized dextrans was almost complete in control cells, in contrast with the mutants, where yellow endosomes showing the co-localization of the FDx10 and TRDx70 could still be observed. After a longer chase period (240 minutes), the size-dependent segregation was still observed in control cells, while the separation of the different-sized dextrans started to become apparent in mutant cells. The inset represents a control cell after a pulse-chase of 30/240 minutes of FDx70 and TRDx70, which clearly demonstrates that the segregation of dextrans depends on the size of the molecules, and not on the properties of the different fluorophores, since similar-sized dextrans co-localize in the same vesicles. Bar, 10 µm.