Extended Data Fig. 10: Receptors can utilize and modulate CARP for recycling.
From: Clathrin-associated carriers enable recycling through a kiss-and-run mechanism
a, AP2-670nano+/+ cells stably expressing mRuby3-sensor were transiently transfected with TfR1-pHluorin, and then imaged at 0.15-s intervals by TIRF microscopy. The montage shows the burst and subsequent lateral dispersal of TfR1-pHluorin fluorescence, followed by the recruitment of EGFP-sensor at the fusion site (arrows). b, AP2-670nano+/+ cells stably expressing EGFP-sensor and SNAP-β2AR were stained with membrane-impermeable JF549i-SNAP-tag ligand, treated with ISO for 3 min, and then imaged by TIRF microscopy every 2 s for 300 s. From left to right: T-projection and kymographs from a time series; montage showing sensor recruitment to a β2AR exocytosis event (yellow arrow in the kymograph); and tracks of AP2-negative sensor-positive fusion events with or without β2AR recruitment at different periods after ISO stimulation. c, Cells stably expressing EGFP-sensor were transiently transfected with SNAP-β2AR and Halo-EEA1, stained with JFX650-HaloTag ligand, treated with nocodazole for 1 h, stained with JF549i-SNAP-tag ligand, treated with ISO, and then imaged by spinning-disk confocal microscopy near the bottom surface every 1.5 s for 300 s. The montage shows the recruitment of EGFP-sensor to the β2AR-loaded tubular structure budded from the EEA1-positive endosome (arrows). Sensor recruitment is accompanied by the disappearance of the tubular structure. d, AP2-TagRFP+/+ CLTA-670nano+/+ cells stably expressing EGFP-sensor were treated with control siRNA or two different siRNAs targeting EGFR (KD1 and KD2). Left: Reduction in the expression of EGFR by siRNA was confirmed by western blot analysis using antibodies against EGFR and GAPDH. Right: siRNA-treated cells were treated with EGF at 120 s during continuous imaging at 4-s intervals by TIRF microscopy. The plots show the relative frequency of AP2-negative sensor-positive fusion events at different time points (mean ± s.e.m.; n = 48, 39, and 48 cells from five independent experiments). e, AP2-TagRFP+/+ CLTA-670nano+/+ cells stably expressing the EGFP-sensor were cultured with 0, 5, or 10% of FBS for 8-10 h, or cultured in EBSS for 2 h, then imaged at 1-s intervals by TIRF microscopy. Left: Images showing the spatial frequency distribution of AP2-negative events from cells cultured with 0 or 10% FBS. Right: Plots showing the frequency and lifetime of AP2-negative events from cells with different treatments (mean ± 95% CI; n = 16 cells each from one experiment). P values were determined by one-way ANOVA with Dunnett’s multiple comparisons test. f, Cells were pretreated with DMSO or BFA for 30 min and then incubated with Alexa Fluor 568-conjugated transferrin for 10 min on ice. Cells were then transferred to 37°C for 5 or 45 min in the medium containing DMSO or BFA, acid washed, fixed, and imaged using spinning-disk confocal microscopy. g, Cells stably expressing SNAP-β2AR were pretreated with DMSO or BFA for 30 min, stained with JF549i-SNAP-tag ligand, and then treated with ISO for 10 min. The cells were then fixed (0 min) or incubated further in the medium containing the β-blocker propranolol with DMSO or BFA for 20 min, and then fixed and imaged using spinning-disk confocal microscopy. h, Cells were treated with control siRNA or siRNA targeting both Rab1a and Rab1b, and then incubated with Alexa Fluor 568-conjugated transferrin for 10 min on ice. Cells were transferred to 37°C for 5 or 45 min, acid-washed, fixed, and imaged using spinning-disk confocal microscopy. i, Cells stably expressing SNAP-β2AR were treated with control siRNA or siRNA targeting both Rab1a and Rab1b, stained with JF549i-SNAP-tag ligand, and then treated with ISO for 10 min. The cells were then fixed (0 min) or further incubated in the medium containing the β-blocker propranolol for 20 min, and then fixed and imaged using spinning-disk confocal microscopy. Experiments were repeated three times (a-c and e), five times (d), or twice (f-i) with similar results. Scale bars, 1 μm in a-c; 10 μm in f-i. Source numerical data and unprocessed blots are available in source data.
