Ligand-Independent Traffic of Notch Buffers Activated Armadillo in Drosophila (original) (raw)
Figure 6
Endocytosis and traffic of CeN reflects Notch.
Expression of endogenous Notch in a wing disc in which UAS_-CeN_ is expressed under the control of _dpp_-Gal4. (A) Discs were fixed and permeabilized before staining with an antibody against the extracellular domain of Notch (red channel) and CD8 (blue channel). Optical sections through the apical (A) and the basal (A1) side of the cell. In apical levels, eGFP (green channel) and CD8 localize mostly in the membranes at the level of the adherens junctions (see also Figure 4A). Note that both in apical and basal levels the CD8 and eGFP vesicles colocalize. (B–D) Notch and CD8 tracked over time, by pulsing CeN expressing live wing discs with an antibody against the extracellular domain of Notch (red channel) and CD8 (blue channel), and chasing for 0 (B), 30 (C), and 60 min (D). (B–D, apical; B1–D1, basal sections). After 0 min of chasing, the endogenous Notch and CD8 localize in the apical membrane of the cells (B), and there are no vesicles in basal levels (B1). After 30 min of chase, the endogenous Notch has been cleared completely from the apical membranes and localizes in vesicles mostly in apical levels; at this time point, CD8 also goes to vesicles in the apical level, although some remain in the membrane (C–C1). After 60 min of chase, the endogenous Notch localizes in vesicles in apical and basal levels; at this time point, CD8 also goes to vesicles in both levels (D–D1). Circles mark some of the NECD, CeN, and CD8 colocalizing vesicles. In all cases the apical and basal images were taken in equivalent levels in the dorsal region of the wing pouch. The apical sections were taken at the level of the adherens junctions and the basal 7 µm underneath. See Figure S10 for an extended version of this figure. Scale bar, 10 µm.