Figure 3. pH-dependent fluorescence of tagged Notch in endosomes. (A) pH-dependent emission profiles of GFP and Cherry (adapted from Doherty et al., 2010). a.u., arbitrary unit. (B and B′) intracellular NiGFP (GFP) remained associated with Cherry-Rab5 endosomes in living pupae. See also Video 1. (C–G) NiCherry (Cherry) did not colocalize with GFP-Rab5 (GFP in C; see also Video 3) nor YFP-Rab11 (YFP in D) but partly overlapped with GFP-Lamp1 (GFP in E) and YFP-Rab7 (YFP in F and G; see also Video 4). Snapshots of a video showing the tethering and fusion of NiCherry-positive endosomes with YFP-Rab7 endosomes (G; t is in minutes and seconds). (H and I) Live imaging of NiGFP4Cherry5 in living pupae in control (H) and Rbcn-3ARNAi pupae. The silencing of Rbcn-3A led to the detection of GFP (green) and Cherry (red) fluorescence in enlarged Notch-containing endosomes in living notum cells. (J–L) Imaging of NiGFP4Cherry5 of living third instar larvae showing the pouch of wing imaginal discs in control (K) and chloroquine-fed (L) N55e11 rescued larvae. In control larvae, GFP showed the apical cortex of wing pouch cells and Cherry revealed intracellular dots, particularly along the anterior wing margin, a region of high Delta-Notch signaling. Upon chloroquine treatment, the GFP and Cherry signals of NiGFP4Cherry5 colocalized in dots (L), and the GFP/Cherry signal intensity ratio was significantly increased (J). Error bars represent the standard error of the mean (n = 5). (M and M′) Apical distribution of NiGFP4Cherry5 (GFP [green] and Cherry [red]) was largely unaffected by the loss of lgd activity (M; mutant cells were marked by the loss of a nuclear RFP marker) in living genetically mosaic pupae. In contrast, enlarged high GFP and low Cherry endosomes were specifically detected basally in lgd mutant cells (M′). The number and size of the high Cherry endosomes was not detectably changed upon the loss of lgd activity (M′; this observation was confirmed using GFP as a clone marker; not depicted). The white lines indicate the borders of the mutant clone. Bars: (B–F, H, and M′) 5 µm; (G) 1 µm; (K) 25 µm.
Figure 3.

pH-dependent fluorescence of tagged Notch in endosomes. (A) pH-dependent emission profiles of GFP and Cherry (adapted from Doherty et al., 2010). a.u., arbitrary unit. (B and B′) intracellular NiGFP (GFP) remained associated with Cherry-Rab5 endosomes in living pupae. See also Video 1. (C–G) NiCherry (Cherry) did not colocalize with GFP-Rab5 (GFP in C; see also Video 3) nor YFP-Rab11 (YFP in D) but partly overlapped with GFP-Lamp1 (GFP in E) and YFP-Rab7 (YFP in F and G; see also Video 4). Snapshots of a video showing the tethering and fusion of NiCherry-positive endosomes with YFP-Rab7 endosomes (G; t is in minutes and seconds). (H and I) Live imaging of NiGFP4Cherry5 in living pupae in control (H) and Rbcn-3ARNAi pupae. The silencing of Rbcn-3A led to the detection of GFP (green) and Cherry (red) fluorescence in enlarged Notch-containing endosomes in living notum cells. (J–L) Imaging of NiGFP4Cherry5 of living third instar larvae showing the pouch of wing imaginal discs in control (K) and chloroquine-fed (L) N55e11 rescued larvae. In control larvae, GFP showed the apical cortex of wing pouch cells and Cherry revealed intracellular dots, particularly along the anterior wing margin, a region of high Delta-Notch signaling. Upon chloroquine treatment, the GFP and Cherry signals of NiGFP4Cherry5 colocalized in dots (L), and the GFP/Cherry signal intensity ratio was significantly increased (J). Error bars represent the standard error of the mean (n = 5). (M and M′) Apical distribution of NiGFP4Cherry5 (GFP [green] and Cherry [red]) was largely unaffected by the loss of lgd activity (M; mutant cells were marked by the loss of a nuclear RFP marker) in living genetically mosaic pupae. In contrast, enlarged high GFP and low Cherry endosomes were specifically detected basally in lgd mutant cells (M′). The number and size of the high Cherry endosomes was not detectably changed upon the loss of lgd activity (M′; this observation was confirmed using GFP as a clone marker; not depicted). The white lines indicate the borders of the mutant clone. Bars: (B–F, H, and M′) 5 µm; (G) 1 µm; (K) 25 µm.

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