Extended Data Fig. 3: EGF drives clathrin-independent endocytosis and triggers formation of CLICs.

a, The anti-α3 integrin antibody by itself does not stimulate α3 integrin internalization. MDA-MB-231 cells were incubated on ice at sub-saturating concentrations (0.2 µg/ml) with fluorescent anti-α3 integrin antibody (PE in red) and washed. Uptake was measured at 37 °C for 10 min in the presence of increasing concentrations of non-fluorescence tagged anti-α3 integrin antibody (0 to 100 µg/ml). Representative images of 3 independent experiments. Scale bar, 20 µm. b, Quantification of data as in (b). Fluorescence intensity of anti-α3 integrin–PE antibody uptake in the presence of non-fluorescence tagged anti-α3 integrin antibody (≈150 cells per condition). One-way ANOVA Dunnett’s multiple comparison test. ns p > 0.05. c, EGF-induced β1 integrin uptake (quantification in Fig. 4c). MDA-MB-231 cells were serum starved for 30 min at 37 °C and then incubated for 10 min at 37 °C with 1 µg/ml of anti-β1 integrin antibody K20 in the presence or absence of 100 ng/ml EGF. Note that EGF stimulates β1 integrin uptake. SUM intensity confocal images are shown. Scale bar, 10 µm. d, Investigation of caveolar function in EGF-stimulated β1 integrin uptake. Wild-type and caveolin-1 (CAV1) knockout Hela cells were serum starved for 30 min at 37 °C and then incubated for 10 min at 37 °C with 1 µg/ml of anti-β1 integrin antibody K20 in the presence or absence of EGF. Note that EGF stimulated β1 integrin uptake persists in the absence of caveolin-1 expression. One-way ANOVA Dunnett’s multiple comparison test on all data points. e, TNF-induced β1 integrin uptake (quantification in Fig. 4c). MDA-MB-231 cells were serum starved for 30 min at 37 °C and then incubated for 10 min at 37 °C with 1 µg/ml of anti-β1 integrin antibody K20 in the presence or absence of 100 ng/ml TNF. Note that TNF stimulates β1 integrin uptake. SUM intensity confocal images are shown. Scale bar, 10 µm. f, Monitoring pH at the cell surface next to α3 integrin. MDA-MB-231 cells were continuously incubated with AcidiFluor-coupled anti-α3 integrin antibody at 37 °C and imaged in TIRF modalities. pH 4 buffer was added at the indicated timepoint. Note the acidification-induced increase in fluorescence. g,h, Lattice light-sheet microscopy imaging. Mean intensity (g) and maximum intensity (h) of β1 integrin endocytic tracks are shown from 3 independent experiments as in Fig. 5c–e. 1,802 tracks from 16 CTRL cells, and 2,157 tracks from 18 EGF-treated cells. Unpaired t-test. Note that mean and maximum intensities decreased after EGF stimulation. i, Monitoring pH responsiveness of Tf-AcidiFluor Orange. MDA-MB-231 cells were incubated with 10 µg/ml Tf-AcidiFluor Orange for 10 min before fixation and acid stripping. Images were acquired in pH 7.4 imaging buffer before switching to pH 5 imaging buffer. j, Monitoring functionality of Tf-AcidiFluor Orange. MDA-MB-231 cells were either directly imaged (autofluorescence) or pulsed with Tf-AcidiFluor Orange (10 µg/ml) and unlabelled Tf at 1 mg/ml, or with Tf-AcidiFluor Orange (10 µg/ml) in the absence of competing unlabelled Tf. k–m, Representative electron micrographs. (k) CLICs containing the indicated HRP-coupled cargo proteins. Scale bars, 200 nm. (l) Vesicular structure containing HRP-coupled anti-α3 integrin antibody (ASC-1). Scale bar, 200 nm. (m) Macropinocytic structure containing HRP-coupled anti-β1 integrin antibody (K20). Scale bar, 1 µm. Representative images of quantifications in Fig. 5b. n, Workflow for the classification of AP2-positive and AP2-negative uptake events, as imaged by LLSM. For all quantifications in this figure, means ± SEM are shown. In (b), means from separate experiments are indicated by solid dots, and measurements of individual cells have different coloured symbols for each experiment. ns p > 0.05, **** p < 0.0001. Source numerical data are provided.

Source data