Fig. 5
NK2R-HA accumulates at the limiting membrane of abnormal endosomes. A–B2 Endosomal localisation of N-HA and NK2R-HA after 14.5 h expression. Endosomes were indicated by tub-Rab7-YFP. The arrows point some of the analysed endosomes. A N-HA localised on Rab7-positive endosomes. B1 Similar to N-HA, apical NK2R-HA localised on Rab7-positive endosomes. B2 In contrast to N-HA, basally located NK2R-HA-positive vesicles were enlarged and were hardly positive for Rab7. C, D Analysis of basal N-HA and NK2R-HA vesicles with structured illumination microscopy (SIM). N-HA and NK2R-HA were expressed for 24 h. C N-HA clearly co-localised with endogenous YFP-N (arrows) in the basal endosomes. D In contrast, NK2R-HA was restricted to the limiting membrane of the enlarged vesicles. Note, that the endogenous YFP-N was clearly localised in the vesical lumen (the area framed by the NK2R-HA signal), indicating that the ESCRT-mediated ILV formation occurred, but NK2R-HA was not incorporated into the ILVs (arrows). E–J Uptake-assay of N-HA and NK2R-HA in S2 cells. N-HA and NK2R-HA were expressed for 24 h under control of pMT-GAL4. After expression, the cells were shifted to 4 °C and incubated with NECD antibody, which exclusively detects the N extracellular domain on the cell surface. Afterwards, the cells were shifted back to 25 °C to re-initiate endocytosis and fixed and stained with secondary antibody after 0’, 45’ and 90’ of re-initiated endocytosis. Endosomes were revealed by anti Rab5 antibody staining. Co-localisation of N-HA and NK2R-HA with Rab5-positive endosomes is highlighted by the arrows. The arrowheads show N-HA or NK2R-HA vesicles that do not co-localise with Rab5. E, F At t = 0, N-HA and NK2R-HA were predominantly localised at the plasma membrane. G, H At t = 45 min, N-HA and NK2R-HA were endocytosed and located in Rab5-positive endosomes. I, J At t = 90 min, N-HA and NK2R-HA were nearly completely removed from the plasma membrane, indicated by the loss of membrane staining. J In contrast to N-HA, NK2R-HA was also localised at the limiting membrane of enlarged endosomes that were negative for Rab5, indicating that abnormal NK2R-HA-positive endosomes had formed