Extended Data Fig. 1: Identification and characterization of AP2-negative clathrin-associated recycling carriers.
From: Clathrin-associated carriers enable recycling through a kiss-and-run mechanism
a, Top: Genomic PCR analysis showing biallelic integration of TagRFP into the genomic locus of AP2S1 to generate the clonal gene-edited SUM159 cell line AP2-TagRFP+/+ (top left), and then integration of HaloTag into the CLTA genomic locus (top right) to generate the clonal double-edited cell line AP2-TagRFP+/+ CLTA-Halo+/+. Bottom: Western blot analysis of cell lysates probed with antibodies for AP2σ2 (bottom left), CLTA (bottom right), and α-actinin (loading control). b, Top: Genomic PCR analysis showing biallelic integration of miRFP670nano (670nano) into the genomic locus of CLTA of AP2-TagRFP+/+ cells to generate the clonal double-edited cell line AP2-TagRFP+/+ CLTA-670nano+/+. Bottom: Western blot analysis of cell lysates probed with antibodies for CLTA and α-actinin. c, AP2-TagRFP+/+ CLTA-Halo+/+ cells were transiently transfected with the coincidence-detecting PI(4,5)P2 sensor EGFP-PH(PLCδ1)-Aux1 (EGFP-sensor), labelled with the JFX650-HaloTag ligand, and then imaged in 3D using spinning-disk confocal microscopy (15 imaging planes spaced at 0.35 μm). The distributions of AP2-TagRFP, CLTA-Halo, and EGFP-sensor at the bottom surface and the middle plane of the cell are shown. The AP2-negative clathrin-positive carriers that recruited EGFP-sensor are highlighted by arrows. d, AP2-TagRFP+/+ CLTA-Halo+/+ cells stably expressing EGFP-sensor were treated with sgRNA targeting AP2S1 to knock out the expression of AP2σ2 (AP2σ2-KO). The wild-type and AP2σ2-KO cells were incubated with 5 μg/mL Alexa Fluor 647-conjugated transferrin for 5 min at 37°C, acid washed, and then fixed. After nuclei were stained with DAPI, cells were imaged in 3D using spinning-disk confocal microscopy (30 imaging planes spaced at 0.35 μm). The representative images (maximum z-projection of stacks) show the markedly reduced internalization of transferrin in the AP2σ2-KO cells. e, AP2-TagRFP+/+ CLTA-Halo+/+ cells stably expressing EGFP-sensor with AP2σ2-KO were labelled with the JFX650-HaloTag ligand, and then imaged at the bottom surface every 1.5 s by TIRF microscopy. The AP2-negative clathrin-positive carrier recruited the EGFP-sensor is highlighted by arrows. f, AP2-TagRFP+/+ CLTA-Halo+/+ cells stably expressing EGFP-sensor with or without AP2σ2-KO were imaged by TIRF microscopy. The frequency of sensor-positive fusion events from control and AP2σ2-KO cells is shown (mean ± 95% CI; n = 16 and 17 cells from one representative experiment). P value was determined by two-tailed unpaired Student’s t-test. g, AP2-670nano+/+ cells stably expressing EGFP-sensor were transiently transfected with LYN11-FRB-ECFP and mCherry-FKBP-5-ptaseOCRL or mCherry-FKBP-5-ptaseOCRL(D523G), and then imaged at 2-s intervals by TIRF microscopy. Rapamycin was added (set as 0 s) during continuous imaging to trigger acute depletion of PI(4,5)P2 by recruiting mCherry-FKBP-5-ptaseOCRL (inserts) from the cytosol to the plasma membrane. The acute recruitment of the catalytically inactive mCherry-FKBP-5-ptaseOCRL(D523G), which is unable to hydrolyse PI(4,5)P2, did not affect the recruitment of the EGFP-sensor. h,i CLTA-670nano+/+ cells stably expressing α-TagRFP-AP2 (internally TagRFP-tagged α subunit of AP2) were transfected with EGFP-PH(PLCδ1)-Aux1 (h) or EGFP-Tubbyc-Aux1 (i), and then imaged at 1.5-s intervals by TIRF microscopy. The T-projection and kymographs of a representative time series are shown. j,k, CLTA-670nano+/+ cells stably expressing α-TagRFP-AP2 were transfected with the PI(4,5)P2-binding-defective mutant EGFP-PH(PLCδ1)-mt-Aux1 (j) or EGFP-Tubbyc-mt-Aux1 (k), and then imaged at 1.5-s intervals by TIRF microscopy. The T-projection of a representative time series is shown. l, AP2-TagRFP+/+ CLTA-Halo+/+ cells were transiently transfected with mNeonGreen-PH(PLCδ1) (top) or mNeonGreen-Tubbyc (bottom), and then imaged at the bottom surface at 2-s intervals by spinning-disk confocal microscopy. Montages showing weak recruitment of mNeonGreen-PH(PLCδ1) or mNeonGreen-Tubbyc to the AP2-negative clathrin-associated carriers. m, U2OS cells stably expressing AP2-670nano were transiently transfected with EGFP-sensor and CLTA-mScarlet-I, and then imaged every 1 s by TIRF microscopy. The AP2-negative clathrin-positive carrier that recruited the EGFP-sensor is highlighted by arrows. n, COS7 cells stably expressing EGFP-sensor and AP2-670nano were transiently transfected with CLTA-mScarlet-I, and then imaged every 1 s by TIRF microscopy. The AP2-negative clathrin-positive carrier that recruited the EGFP-sensor is highlighted by arrows. Experiments were repeated three times (a-c and g-n) or twice (d-f) with similar results. Scale bars, 10 μm (overview) and 1 μm (magnification) in c, e, h-k, m, and n; 10 μm in d and g; 1 μm in kymographs (e, h, i, m, and n) and montages (l). Source numerical data and unprocessed blots are available in source data.
