Fig. 4

Trafficking of N-HA and N^K2R-HA through the secretory and endosomal pathway. A–L A pulse-chase assay to analyse the subcellular localisation of N-HA and N^K2R-HA. The pulse of N-HA and N^K2R-HA expression was limited to 2 h. Following to the pulse, the larvae were shifted back to restrictive 18 °C for different chase intervals. The localisation of the constructs was revealed by HA antibody staining. A–F Analysis of N-HA and N^K2R-HA localisation compared to endogenous N (YFP-N, gene trap). Each box shows a Z-projection (upper panel) and the focal plane (lower panels). The schematic cell and the arrow in the Z-projection shows the focal plane used in each box. Co-localisation of the transgenic constructs and YFP-N is highlighted by small arrows in the corresponding lower panels. A, B After a pulse of 2 h, N-HA and N^K2R-HA were localised in dot-like structures adjacent to the nucleus that in rare cases co-localised with YFP-N. C, D After a subsequent chase of 2 h, N-HA and N^K2R-HA were both localised throughout the apical plasma membrane, while YFP-N was mainly localised to the subapical membrane region, indicated by the honeycomb pattern. E, F After a 4 h chase, N-HA and N^K2R-HA were strongly localised to the nucleus, throughout the apical plasma membrane and in vesicles that co-localise with YFP-N. G, H Localisation of N-HA and N^K2R-HA to the ER. The ER was indicated by Calnexin antibody staining. After the 2-h pulse, the N-HA and N^K2R-HA-positive dot-like structures adjacent to the nucleus are positive for the ER marker Calnexin (arrows), indicating the early phase of synthesis of the constructs in the ER. I, J After 1 h chase, the variants co-localised with the Golgi marker Golgin in the cytosol apical to the nucleus (arrows), indicating that both constructs were in the secretory pathway on their way to the apical plasma membrane via maturation in the Golgi. K, L The N-HA and N^K2R-HA-positive vesicles seen after the 4-h chase were positive for Rab5-CFP and Rab7-YFP (arrows and magnification), indicating that both receptors entered the endosomal pathway. M–Q Endocytosis of N-HA and N^K2R-HA from the apical plasma membrane. To analyse the endocytosis of N-HA and N^K2R-HA from the apical plasma membrane, the expression of the constructs was increased to a 14.5-h pulse followed by a chase for 28 h. All boxes show a Z-projection (upper panel) and a magnification of the apical focal plane (insert). Areas of magnification are highlighted by the white boxes. M Endogenous N was predominantly localised in the apical plasma membrane (honeycomb pattern, insert) and in vesicles throughout the whole A/B axis (z-section in upper panel). The arrow in the z-sections highlights the apical side of the epithelium. N After 14.5-h pulse of expression, N-HA localised at the apical plasma membrane (insert), similar to endogenous N. O After a subsequent 28-h chase, N-HA levels in the apical plasma membrane were reduced (insert), while it accumulated in intracellular vesicles (z-section in upper panel). P Like endogenous N and N-HA, N^K2R-HA was localised to the apical plasma membrane after 14.5-h pulse of expression (insert). In contrast to N-HA, N^K2R-HA accumulated also in vesicles at the basal region of the cell (z-section in upper panel). Q Similar to N-HA, the levels of N^K2R-HA in the apical membrane were reduced after a 28-h chase (insert), while the accumulation in basal vesicles continued (z-section in upper panel)