Fig 3. Rab6 to Rab11 transition on surface of maturing secretory compartments in Drosophila secondary cells coincides with exosome and DCG biogenesis (related to S3 Movie).

Panel shows ex vivo images of a single SC taken at six discrete timepoints with time since start of imaging shown above in minutes. Rows within panel display cellular organisation at each timepoint through DIC imaging (A-F), fluorescent CFP-Rab6 signal (A’-F’), fluorescent YFP-Rab11 signal (A”-F”), a combined fluorescence image (A”‘-F”‘) and a combined DIC and fluorescence image (A”“-F”“). Three coloured arrows (blue, yellow and white) each indicate the position of one maturing secretory compartment across time. (A-A”“) The compartments marked by either a blue or yellow arrow begin with CFP-Rab6 and YFP-Rab11 co-labelling and have DCGs already present. The compartment marked by the white arrow is significantly larger, is labelled strongly with CFP-Rab6, but has no YFP-Rab11 on its surface and no DCG. (B-B”“, C-C”“) The blue and yellow arrowed compartments lose CFP-Rab6 labelling over time and become more heavily marked by YFP-Rab11. The compartment marked with a white arrow significantly contracts in size and is only weakly labelled by CFP-Rab6 by the end of the time course. In contrast, YFP-Rab11 begins to accumulate on the compartment. Simultaneously, this compartment begins forming internal structures, with ILVs appearing first (B’) and then a DCG (C). ILVs are marked by both CFP-Rab6 and YFP-Rab11 and at least partly surround the DCG (C). (D-D”“, E-E”“, F-F”“). The two more mature highlighted compartments (marked with blue and yellow arrows) lose CFP-Rab6 identity and are strongly labelled by YFP-Rab11 by the end of the time course. The compartment labelled with a white arrow still retains some CFP-Rab6, but YFP-Rab11 continues to increase in levels. Approximate outline of SC is marked by dashed circles. Scale bars: 10 μm. This Rab transition and concurrent DCG formation was observed four times with different accessory glands. Genotype for images: w¹¹¹⁸; TI{TI}Rab6^CFP/+; TI{TI}Rab11^EYFP/+.