Figure 4.
Abi-dependent CME is required for ligand-induced Notch internalization. (A and B) Single confocal slices of primary hemocytes from HmlΔ-GAL4/+ and UAS-abiRNAi/+; HmlΔ-GAL4/+ (HmlΔ > abiRNAi) late-third instar larvae. Hemocytes were pulsed with Alexa Fluor 555-mBSA (mBSA, a CME marker) and 70 kDa FITC-dextran (Dex70, a macropinocytosis marker) (A) or 10 kDa FITC-dextran (Dex10, a GEEC endocytosis marker) alone (B) for 5 min, chased for 5 min, and stained with DAPI. (C) Quantification of endocytic events in A and B. The ratios of mBSA, Dex70, and Dex10 to DAPI fluorescence intensities are presented as percentages of HmlΔ-GAL4/+. n = 30 hemocytes. (D) Single confocal slices of S2N cells transfected with abi, Chc, or Rabankyrin dsRNA. Transfected S2N cells were pretreated with 0.7 mM CuSO4 and cocultured with S2S cells. Live cocultured cells were incubated with an anti-NECD antibody at 4°C for 30 min to prelabel surface Notch receptors (on S2N cells) and further incubated at 25°C for 30 min to allow endocytosis to resume. After fixation, cocultured cells were sequentially stained for surface (green) and internalized (pseudocolored magenta) Notch receptors under nonpermeant and permeant conditions, respectively. Permeabilized cocultured cells were additionally stained for Myc-Ser (blue). Arrowheads indicate intracellular punctate structures containing internalized Notch. (E) Quantification of the ratio of internal to surface Notch fluorescence intensities. n = 9 cells. (F) Single confocal slices of S2N cells transfected with shi dsRNA, pretreated with 0.7 mM CuSO4 for 24 h, and cocultured with S2S cells for 6 h. Notch receptors on S2N cells were prelabeled with an anti-NECD antibody and allowed to internalize as in D. After fixation and permeabilization, cells were stained for Flag-Chc (green), HA-Abi (pseudocolored magenta), and prelabeled Notch (blue). Arrowheads indicate colocalization of Flag-Chc, HA-Abi, and Notch. Data represent the mean ± SEM. Statistical analyses were performed using Student’s t test (C) or by a one-way ANOVA with the Tukey–Kramer post hoc test (E). Comparisons are with HmlΔ-GAL4/+ in C and mock-transfected isolated (-Ser) S2N cells in E (***P < 0.001). Scale bars: 5 μm (A and B); 10 μm (D and F).

Abi-dependent CME is required for ligand-induced Notch internalization. (A and B) Single confocal slices of primary hemocytes from HmlΔ-GAL4/+ and UAS-abiRNAi/+; HmlΔ-GAL4/+ (HmlΔ > abiRNAi) late-third instar larvae. Hemocytes were pulsed with Alexa Fluor 555-mBSA (mBSA, a CME marker) and 70 kDa FITC-dextran (Dex70, a macropinocytosis marker) (A) or 10 kDa FITC-dextran (Dex10, a GEEC endocytosis marker) alone (B) for 5 min, chased for 5 min, and stained with DAPI. (C) Quantification of endocytic events in A and B. The ratios of mBSA, Dex70, and Dex10 to DAPI fluorescence intensities are presented as percentages of HmlΔ-GAL4/+. n = 30 hemocytes. (D) Single confocal slices of S2N cells transfected with abi, Chc, or Rabankyrin dsRNA. Transfected S2N cells were pretreated with 0.7 mM CuSO4 and cocultured with S2S cells. Live cocultured cells were incubated with an anti-NECD antibody at 4°C for 30 min to prelabel surface Notch receptors (on S2N cells) and further incubated at 25°C for 30 min to allow endocytosis to resume. After fixation, cocultured cells were sequentially stained for surface (green) and internalized (pseudocolored magenta) Notch receptors under nonpermeant and permeant conditions, respectively. Permeabilized cocultured cells were additionally stained for Myc-Ser (blue). Arrowheads indicate intracellular punctate structures containing internalized Notch. (E) Quantification of the ratio of internal to surface Notch fluorescence intensities. n = 9 cells. (F) Single confocal slices of S2N cells transfected with shi dsRNA, pretreated with 0.7 mM CuSO4 for 24 h, and cocultured with S2S cells for 6 h. Notch receptors on S2N cells were prelabeled with an anti-NECD antibody and allowed to internalize as in D. After fixation and permeabilization, cells were stained for Flag-Chc (green), HA-Abi (pseudocolored magenta), and prelabeled Notch (blue). Arrowheads indicate colocalization of Flag-Chc, HA-Abi, and Notch. Data represent the mean ± SEM. Statistical analyses were performed using Student’s t test (C) or by a one-way ANOVA with the Tukey–Kramer post hoc test (E). Comparisons are with HmlΔ-GAL4/+ in C and mock-transfected isolated (-Ser) S2N cells in E (***P < 0.001). Scale bars: 5 μm (A and B); 10 μm (D and F).

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal