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Clathrin-associated carriers enable recycling through a kiss-and-run mechanism

Nature Cell Biology (2024)Cite this article

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Abstract

Endocytosis and recycling control the uptake and retrieval of various materials, including membrane proteins and lipids, in all eukaryotic cells. These processes are crucial for cell growth, organization, function and environmental communication. However, the mechanisms underlying efficient, fast endocytic recycling remain poorly understood. Here, by utilizing a biosensor and imaging-based screening, we uncover a recycling mechanism that couples endocytosis and fast recycling, which we name the clathrin-associated fast endosomal recycling pathway (CARP). Clathrin-associated tubulovesicular carriers containing clathrin, AP1, Arf1, Rab1 and Rab11, while lacking the multimeric retrieval complexes, are generated at subdomains of early endosomes and then transported along actin to cell surfaces. Unexpectedly, the clathrin-associated recycling carriers undergo partial fusion with the plasma membrane. Subsequently, they are released from the membrane by dynamin and re-enter cells. Multiple receptors utilize and modulate CARP for fast recycling following endocytosis. Thus, CARP represents a previously unrecognized endocytic recycling mechanism with kiss-and-run membrane fusion.

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Fig. 1: Identification of intracellular clathrin-associated carriers that transiently visit the plasma membrane.
Fig. 2: Fusion of the intracellular clathrin-associated carriers with the plasma membrane.
Fig. 3: Imaging-based screening of proteins associated with AP2-negative carriers that recruited the EGFP-sensor.
Fig. 4: Regulation of CARP by Rab11 and Rab1.
Fig. 5: Regulation of CARP by Arf1.
Fig. 6: CARP carriers are released from the plasma membrane by dynamin.
Fig. 7: CARP carriers are derived from early endosomes.
Fig. 8: CARP carriers mediate the recycling of receptors back to the plasma membrane.

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Data availability

The data that support the findings of the current study are included in Extended Data Figs. 1–10, Supplementary Tables 1–6 and Supplementary Videos 1–15. MS data have been deposited in iProX (IPX0009213000). The uncropped western blots and PCR gels have been provided as source data. All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

Code availability

The download links for published codes are provided in the Methods. Other custom codes are available at https://github.com/Clathrin2019/CARP.

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Acknowledgements

We thank T. Kirchhausen for critical reading and discussions and TALEN constructs used for genome-editing of AP2σ2, CLTA and dynamin2. We thank L. Lavis for the generous gifts of the HaloTag and SNAP-tag ligands. We thank J. Brugge for generously providing SUM159 cells. We express our gratitude to B. Song, J. Bonifacino and Z. Chen for generously providing plasmid constructs. We thank Y. Luo, W. Shen, W. Dong and G. Zhong from ZEISS China and ZEISS Microscopy Customer Centre Beijing for their technical support in collecting and analysing imaging data on Lattice Lightsheet 7. We thank I. Hanson for editing. This work was supported by grants from the National Natural Science Foundation of China (92354305, 32321004 and 91957106 to K.H.; 92154001 to A.S.), the Ministry of Science and Technology of the People’s Republic of China (National Key R&D Program of China 2022YFA1304500 and 2021YFA0804802 to K.H.), the Youth Innovation Promotion Association CAS (2023105 to N.L.) and the State Key Laboratory of Molecular Developmental Biology of China.

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Author notes

  1. These authors contributed equally: Jiachao Xu, Yu Liang, Nan Li, Song Dang.

Authors and Affiliations

  1. State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China

    Jiachao Xu, Yu Liang, Nan Li, Song Dang, Xinyi Zhang, Yaran Yang, Yongtao Du & Kangmin He

  2. University of Chinese Academy of Sciences, Beijing, China

    Yu Liang, Yuting Guo, Xiaoyu Yang, Xinyi Zhang, Yongtao Du, Dong Li & Kangmin He

  3. National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China

    Amin Jiang, Yuting Guo, Xiaoyu Yang & Dong Li

  4. National Center for Protein Sciences and Core Facilities of Life Sciences at Peking University, College of Life Sciences, Peking University, Beijing, China

    Yiqun Liu

  5. Department of Microbiology and Infectious Disease Center, NHC Key Laboratory of Medical Immunology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China

    Yi Yuan & Xiaoyun Liu

  6. Department of Biochemistry and Molecular Biology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

    Anbing Shi

  7. Cell Architecture Research Center, Huazhong University of Science and Technology, Wuhan, China

    Anbing Shi

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  1. Jiachao Xu
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Contributions

J.X., N.L. and Y.L. performed imaging experiments. Y.L. and J.X. performed imaging analysis. S.D. generated constructs for imaging-based screening and genome-editing. N.L., S.D., Y.L. and J.X. generated and characterized the genome-edited cell lines. D.L., Y.L., A.J., Y.G. and X.Y. performed the experiments and analysis with SIM and lattice light-sheet microscopy. Y.Q.L., J.X. and Y.L. conducted the FIB-SEM experiment and analysis. Y.D., Y.R.Y. and A.S. generated or prepared materials. X.L., Y.Y. and X.Z. performed cell surface proteomics. Y.L., J.X. and K.H. composed the figures. K.H. conceived and supervised the work and wrote the manuscript.

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Correspondence to Kangmin He.

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Extended data

Extended Data Fig. 1 Identification and characterization of AP2-negative clathrin-associated recycling carriers.

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.

Source data

Extended Data Fig. 2 Live-cell imaging as well as correlative fluorescence and FIB-SEM imaging of the fusion of clathrin-associated recycling carriers with the plasma membrane.

a, Top: Genomic PCR analysis showing biallelic integration of mScarlet-I into the genomic locus of AP1S1 of AP2-670nano+/+ cells to generate the clonal double-edited cell line AP2-670nano+/+ AP1σ1-mScarlet-I+/+. Bottom: Western blot analysis of cell lysates probed with antibodies for RFP and α-actinin. b, AP2-670nano+/+ cells stably expressing EGFP-sensor were transiently transfected with AP1μ1A-mScarlet-I, and then imaged by TIRF microscopy at the bottom surface every 1 s for 180 s. Left: T-projection and kymographs of a representative time series. Middle: Montage of the event indicated by the yellow arrow in the kymograph. Right: Intensity cohorts of AP1μ1A (red), sensor (green), and AP2 (blue) of the AP2-negative sensor-positive fusion events, and the fraction of fusion events recruited AP1μ1A (n = 12 cells). c, AP2-TagRFP+/+ CLTA-670nano+/+ cells stably expressing EGFP-sensor were treated with two different sgRNAs to knock out the expression of AP1μ1A (KO1 and KO2). Knockout was validated by western blot analysis using antibodies against the indicated proteins. d-f, Top: Genomic PCR analysis showing the integration of mScarlet3 into the genomic locus of EXOC7, EXOC1, or EXOC2 to generate a pool of gene-edited cells expressing AP2-670nano+/+ Exo70-mScarlet3en (d), AP2-670nano+/+ Sec3-mScarlet3en (e), or AP2-670nano+/+ Sec5-mScarlet3en (f), respectively. Bottom: Knock-in was validated by western blot analysis using antibodies against GAPDH and Exo70, Sec3, or Sec5. g, Genome-edited AP2-670nano+/+ and Sec5-mScarlet3 (pool) cells stably expressing EGFP-sensor were imaged and displayed as in (b). Intensity cohorts from 11 cells. h,i, AP2-670nano+/+ cells stably expressing EGFP-sensor were transiently transfected with Sec8-mScarlet-I or Sec15-mScarlet-I, and then imaged and displayed as in (b). Intensity cohorts from 12 (h) and 12 (i) cells. j, AP2-TagRFP+/+ CLTA-670nano+/+ cells stably expressing EGFP-sensor were treated with control siRNA or two different siRNAs targeting Exo70 (KD1 and KD2). Elimination of Exo70 expression was confirmed by western blot analysis using antibodies against Exo70 and GAPDH. k, AP2-TagRFP+/+ CLTA-670nano+/+ cells stably expressing EGFP-sensor were treated with control siRNA or two different siRNAs targeting VAMP2 (KD1 and KD2). Elimination of VAMP2 expression was confirmed by western blot analysis using antibodies against VAMP2 and GAPDH. l, FRAP was conducted on CLTA-TagRFP+/+ AP2-Halo+/+ cells stably expressing EGFP-sensor using a confocal laser scanning microscope. Left: The single frame of the cells before photobleaching. The squared region was enlarged and shown on the right. Right: The white circular region on the cell was photobleached and then the cells were imaged again at 3-s intervals for 2 min. Montage of the squared region showing recovery of the fluorescence of CLTA-TagRFP in the CARP carrier (red arrow) and two CCPs (cyan arrows). m, The relative fluorescent intensity traces of CLTA-TagRFP from CARP carriers (n = 28 from 16 cells) and CCPs (n = 28 from 18 cells). n, The workflow of correlative spinning-disk confocal fluorescence imaging with FIB-SEM. AP2-TagRFP+/+ CLTA-Halo+/+ cells stably expressing EGFP-sensor were cultured on a glass-bottom dish with position markers (I), stained with the JF646-HaloTag ligand, fixed, and then imaged by spinning-disk confocal microscopy (II). The samples were post-fixed, stained with 1% aqueous uranyl acetate, dehydrated through a series of graded ethyl alcohols, and then embedded in resin (III). The cell samples were then imaged volumetrically in 3D using FIB-SEM (IV). The collected FIB-SEM slices were automatically aligned to reconstruct the 3D image (V). The fluorescence image and the FIB-SEM image were aligned (VI) and the AP2-negative sensor-positive clathrin-containing structures identified by fluorescence imaging were segmented and reconstructed (VII). See Methods for details. Experiments were repeated three times with similar results. Cells in b-m were from one experiment. Data are shown as mean ± s.e.m. in cohorts in b and g-i; mean ± 95% CI in the fraction of events in b; mean ± s.d. in m. Scale bars, 10 μm (overview) and 1 μm (magnification) in b, g-i, and l; 1 μm in kymographs (b and g-i) and montages (b, g-i, and l). Source numerical data and unprocessed blots are available in source data.

Source data

Extended Data Fig. 3 CARP carriers are distinct from other known clathrin-associated structures.

a, Diagram illustrating the trafficking of thermosensitive VSVG (VSVGts) following a temperature shift. VSVGts-mScarlet-I is retained in the ER at 40 °C. Upon a temperature shift from 40 °C to 32 °C, VSVGts-mScarlet-I moves out of the ER, translocates to the Golgi complex, and then redistributes to the plasma membrane via post-Golgi vesicles. b, AP2-670nano+/+ cells stably expressing EGFP-sensor were transiently transfected with VSVGts-mScarlet-I, and then cultured at 40 °C for 12 h. The cells were then shifted to and imaged at 32 °C by TIRF microscopy. Left: Kymographs from representative time series (every 1.5 s for 5 min) collected at 15 or 45 min after shifting to 32 °C. Middle: Tracks of identified fusion events with or without VSVGts association at 15, 30, or 45 min at 32 °C. Right: The fraction of fusion events recruited VSVGts at different times after being shifted to 32 °C (n = 26, 28, and 28 cells). c, Diagram illustrating the trafficking of newly synthesized TfR1 following its synchronous release from the ER using the RUSH system. TfR1 is fused with the streptavidin-binding peptide (SBP) and mCherry (TfR1-SBP-mCherry). Streptavidin fused with the ER retention signal KDEL functions as the ER hook, which retains the newly synthesized TfR1-SBP-mCherry in the ER. Upon the addition of biotin, TfR1-SBP-mCherry is synchronously released from the ER, trafficked to the Golgi, and subsequently delivered to the plasma membrane via post-Golgi vesicles. d, AP2-670nano+/+ cells stably expressing EGFP-sensor were transiently transfected with Str-KDEL-TfR1-SBP-mCherry, and then treated with biotin (40 μM) to trigger the release of TfR1 from ER and imaged by TIRF microscopy. Left: Kymographs from the representative time series (every 1.5 s for 5 min) collected at 15 or 45 min after biotin addition. Middle: Tracks of identified fusion events with or without TfR1 association at 15, 30, or 45 min after the addition of biotin. Right: The fraction of fusion events recruited TfR1 at different times after the addition of biotin (n = 13, 15, and 14 cells). e, AP2-670nano+/+ cells stably expressing EGFP-sensor were transiently transfected with NPY-mRuby and imaged by TIRF microscopy. From left to right: the T-projection and kymographs from a time series, the montage of the NPY exocytosis event indicated by the arrow in the kymograph, and the fraction of sensor-positive fusion events recruited NPY (n = 16 cells). f,g, Genome-edited AP2-670nano+/+ and mScarlet-I-Eps15 (pool) cells (f), or AP2-670nano+/+ and mScarlet-I-Eps15R (pool) cells (g) stably expressing the EGFP-sensor were imaged by TIRF microscopy at the bottom surface every 1 s for 180 s. The T-projections and kymographs of a time series are shown. Experiments were repeated three times with similar results. Cells were from three experiments (b and d) or one experiment (e). Data are shown as mean ± 95% CI in b, d, and e. P values were determined by one-way ANOVA with Dunnett’s multiple comparisons test in b and d. Scale bars, 1 μm in kymographs (b and d-g) and montage (e); 10 μm and 1 μm (magnification) in T-projections (e-g). Source numerical data are available in source data.

Source data

Extended Data Fig. 4 Imaging-based screening and validation of screening results.

a, The workflow of live-cell TIRF imaging-based screening of proteins related to AP2-negative sensor-positive fusion events. AP2-670nano+/+ cells stably expressing EGFP-sensor were transiently transfected with constructs expressing different proteins tagged with mScarlet-I, and then imaged by TIRF microscopy at the bottom surface every 1 s for 180 s. Each time series was first subjected to multi-channel detection and tracking with the EGFP-sensor channel as the 'master' channel to identify the sensor-positive AP2-negative tracks. Each time series was then subjected to multi-channel detection and tracking with the mScarlet-I channel as the 'master' channel. The AP2-negative sensor-positive tracks with significant overlap with the mScarlet-I tracks were classified as positive for the mScarlet-I-tagged protein. The fraction of AP2-negative sensor-positive tracks with positive signals from the mScarlet-I channel was calculated for each protein. The fluorescence intensity distribution heatmap reflecting the relative timing of recruitment of each protein to the EGFP-sensor was plotted. b, For each indicated mScarlet-1-tagged protein, the kymograph from a representative time series is shown. AP2-negative sensor-positive fusion events that recruited the indicated proteins are highlighted by arrows. c,d, For each indicated mScarlet-1-tagged protein, the kymograph from a representative time series is shown. AP2-negative sensor-positive fusion events lacking the recruitment of the indicated proteins are highlighted by arrows (c). The fraction of AP2-negative sensor-positive fusion events exhibiting significant signals from the indicated mScarlet-I-tagged protein is shown in (d) (mean ± 95% CI; n = 8-12 cells). The 95th percentile of the calculated fraction of related events from 133 cells (pooled from the twelve proteins) is 0.18. In this study, the mScarlet-I-tagged protein was considered to be associated with CARP carriers if the averaged fraction of related events exceeds 0.20 (dotted line). e, AP2-670nano+/+ cells stably expressing the EGFP-sensor were genome-edited again to express HIP1R-mScarlet-I+/+, mScarlet-I-SNX9+/+, mScarlet-I-EHD1 (pool), mScarlet-I-Rab7a (pool), LAMP1-mScarlet-I (pool), or flotillin-1-mScarlet-I (pool), and then imaged by TIRF microscopy at the bottom surface every 1 s for 180 s. From left to right: the T-projection and kymographs of a time series; the fraction of sensor-positive events recruited the indicated proteins (mean ± 95% CI); and the intensity cohorts (mean ± s.e.m.; n = 10, 16, 14, 12, 12, and 12 cells). Experiments were repeated three times with similar results. Cells in d and e were from one experiment. Scale bars, 1 μm in kymographs (b, c, and e); 10 μm and 1 μm (magnification) in T-projections (e). Source numerical data are available in source data.

Source data

Extended Data Fig. 5 Dynamic association of proteins with the AP2-positive or negative carriers at the plasma membrane.

a, AP2-670nano+/+ cells stably expressing EGFP-sensor were transiently transfected with various proteins tagged with mScarlet-I and then imaged by TIRF microscopy as described in Fig. 3a. The EGFP-sensor and AP2-670nano dual-positive tracks were analysed for the dynamic association of mScarlet-I-tagged proteins. The fraction of events containing significant signals from the mScarlet-I-tagged protein was plotted for each protein (mean ± 95% CI; n = 7-15 cells from one representative experiment, with the exact numbers of cells shown in the source data). b, Genome-edited cells expressing AP2-670nano+/+ and CLTA-mEGFP (pool) were transiently transfected with the indicated mScarlet-I-tagged proteins, and then imaged by TIRF microscopy at the bottom surface every 1 s for 180 s. Representative montages and kymographs show the dynamic association of Sec3, Rab11a, Arf1, AP1, and dynamin2 (Dyn2) with the AP2-negative clathrin-associated carriers during their approach or stalling at the plasma membrane. mScarlet-I-tagged flotillin-1, Rab7a, Sec31A, and Sec23A are not recruited to the AP2-negative clathrin-associated carriers. Experiments were repeated twice (a) or three times (b) with similar results. Scale bars, 1 μm. Source numerical data are available in source data.

Source data

Extended Data Fig. 6 Regulation of CARP by Rab11 and Rab1 but not Rab4.

a, Left: Genomic PCR analysis showing biallelic integration of mScarlet-I into the genomic locus of RAB11A to generate the clonal double-edited cell line AP2-670nano+/+ mScarlet-I-Rab11a+/+. Right: Western blot analysis of cell lysates probed with antibodies for Rab11a and α-actinin. b, Left: Genomic PCR analysis showing biallelic integration of mScarlet-I into the genomic locus of RAB11B to generate the clonal double-edited cell line AP2-670nano+/+ mScarlet-I-Rab11b+/+. Right: Western blot analysis of cell lysates probed with antibodies for Rab11b and α-actinin. c, AP2-670nano+/+ cells stably expressing EGFP-sensor were transiently transfected with mScarlet-I-SH3BP5L (left) or mScarlet-I-Rab11FIP2 (right), and then imaged by TIRF microscopy. Montage and kymograph showing EGFP-sensor recruitment to the SH3BP5L- or Rab11FIP2-positive carrier. d, AP2-TagRFP+/+ CLTA-670nano+/+ cells stably expressing EGFP-sensor were treated with either control siRNA or siRNA targeting Rab11a, Rab11b, or both Rab11a and Rab11b (Rab11a/Rab11b-KD1). Elimination of Rab11a and/or Rab11b expression was confirmed by western blot analysis using antibodies against Rab11a, Rab11b, and α-actinin. e, AP2-670nano+/+ mScarlet-I-Rab11b+/+ cells stably expressing EGFP-sensor were subjected to knockout of Rab11b, and then treated with control siRNA (Rab11b-KO) or two different siRNAs targeting Rab11a. Knockout of Rab11b and knockdown of Rab11a was confirmed by western blot analysis using antibodies against Rab11a, Rab11b, and α-actinin. f, Left: Western blot analysis of SUM159 cells without or with EGFP-Rab4b transfection, probed with antibodies for Rab4b and α-actinin. The expression of endogenous Rab4b in SUM159 cells is negligible. Right: AP2-TagRFP+/+ CLTA-670nano+/+ cells stably expressing EGFP-sensor were treated with either control siRNA or siRNA targeting Rab4a, Rab4b, or both Rab4a and Rab4b. Elimination of Rab4a expression was confirmed by western blot analysis using antibodies against Rab4a and α-actinin. g, AP2-TagRFP+/+ CLTA-670nano+/+ cells stably expressing EGFP-sensor were treated with either control siRNA or siRNA targeting Rab4a, Rab4b, or both Rab4a and Rab4b, and then imaged by TIRF microscopy. The frequency of AP2-negative sensor-positive fusion events from siRNA-treated cells is shown (mean ± 95% CI; n = 14, 14, 14, and 15 cells). h, Left: Genomic PCR analysis showing single allelic integration of mScarlet-I into the genomic locus of RAB1A to generate the clonal double-edited cell line AP2-670nano+/+ mScarlet-I-Rab1a+/-. Right: Western blot analysis of cell lysates probed with antibodies for RFP and α-actinin. i, Left: Genomic PCR analysis showing biallelic integration of mScarlet-I into the genomic locus of RAB1B to generate the clonal double-edited cell line AP2-670nano+/+ mScarlet-I-Rab1b+/+. Right: Western blot analysis of cell lysates probed with antibodies for RFP and α-actinin. j, AP2-TagRFP+/+ CLTA-670nano+/+ cells stably expressing EGFP-sensor were treated with either control siRNA or siRNA targeting Rab1a, Rab1b, or both Rab1a and Rab1b (Rab1a/Rab1b-KD1). The knockdown efficiency was measured by real-time quantitative PCR (qPCR; mean ± s.d.; n = 5 independent experiments). The siRNA-treated cells were imaged at 1-s intervals by TIRF microscopy, and the imaging results are shown in Fig. 4m. k, AP2-TagRFP+/+ CLTA-670nano+/+ cells stably expressing EGFP-sensor were treated with either control siRNA or a second set of siRNAs targeting Rab1a, Rab1b, or both Rab1a and Rab1b (Rab1a/Rab1b-KD2). Left: Knockdown efficiency measured by qPCR (mean ± s.d.; n = 3 independent experiments). Right: siRNA-treated cells were imaged by TIRF microscopy. Plots showing the frequency of fusion events (mean ± 95% CI; n = 19, 18, 17, and 18 cells). l, mScarlet-I-Rab1b+/+ or mScarlet-I-Rab1a+/- cells were transiently transfected with the indicated proteins and then imaged by spinning-disk confocal microscopy. Intracellular distribution and colocalization of Rab1b or Rab1a with the indicated proteins are shown. m, EGFP-Rab11a+/+ cells were treated with control siRNA or siRNA targeting Rab1a, Rab1b, or both Rab1a and Rab1b, and then imaged by TIRF microscopy. The first frame and T-projection of EGFP-Rab11a from a time series in each treatment are shown. n, AP2-670nano+/+ mScarlet-I-Rab11a+/+cells stably expressing EGFP-sensor were treated with control siRNA or siRNA targeting both Rab1a and Rab1b, and then imaged in 3D using spinning-disk confocal microscopy (15 imaging planes spaced at 0.35 μm). Simultaneous knockdown of Rab1a and Rab1b expression caused the loss of mScarlet-I-Rab11a-positive vesicles around the bottom surface of the cell, while only having a minor effect on the mScarlet-I-Rab11a-positive vesicles near the perinuclear region. Experiments were repeated three (a-i and k-n) or five times (j) with similar results. Cells in g and k (right) were from one experiment. P values were determined by one-way ANOVA with Dunnett’s multiple comparisons test in g and k. Scale bars, 1 μm in c; 10 μm and 1 μm (magnification) in l; 10 μm in m and n. Source numerical data and unprocessed blots are available in source data.

Source data

Extended Data Fig. 7 Dynamics and functions of Arf1 in CARP carrier formation.

a, Left: Genomic PCR analysis showing biallelic integration of mScarlet-I into the genomic locus of ARF1 to generate the clonal double-edited cell line AP2-670nano+/+ Arf1-mScarlet-I+/+. Right: Western blot analysis of cell lysates probed with antibodies for Arf1 and α-actinin. b, Left: Genomic PCR analysis showing biallelic integration of mScarlet-I into the genomic locus of ARF3 to generate the clonal double-edited cell line AP2-670nano+/+ Arf3-mScarlet-I+/+. Right: Western blot analysis of cell lysates probed with antibodies for RFP and α-actinin. c, AP2-TagRFP+/+ CLTA-670nano+/+ cells stably expressing EGFP-sensor were treated with control siRNA or siRNA targeting Arf1, Arf3, Arf4, both Arf1 and Arf3 (Arf1/Arf3-KD), or both Arf1 and Arf4 (Arf1/Arf4-KD). The knockdown efficiency was measured by qPCR (mean ± s.d.; n = 6 independent experiments). d, Arf1 expression was knocked out (Arf1-KO) in AP2-670nano+/+ Arf1-mScarlet-I+/+ cells stably expressing EGFP-sensor. The cells were transiently transfected with mScarlet-I-tagged wild-type Arf1, constitutively active Arf1(Q71L), or dominant negative Arf1(T31N), and then imaged at 1-s intervals by TIRF microscopy. Plots showing the frequency of AP2-negative sensor-positive fusion events (mean ± 95% CI; n = 19, 14, 19, 20, and 20 cells). e, AP2-TagRFP+/+ CLTA-670nano+/+ cells stably expressing EGFP-sensor were treated with control siRNA or siRNA targeting either BIG1, BIG2, or GBF1. The knockdown efficiency was measured by qPCR (mean ± s.d.; n = 3 independent experiments). The siRNA-treated cells were imaged at 1-s intervals by TIRF microscopy, and the imaging results are shown in Fig. 5e. f, AP2-TagRFP+/+ CLTA-670nano+/+ cells stably expressing EGFP-sensor were treated with control siRNA or a second set of siRNAs targeting either BIG1, BIG2, or GBF1. Left: Knockdown efficiency measured by qPCR (mean ± s.d.; n = 3 independent experiments). Right: siRNA-treated cells were imaged at 1-s intervals by TIRF microscopy. Plots showing the frequency of fusion events (mean ± 95% CI; n = 12, 12, 12, and 12 cells). g,h, Genome-edited Arf1-mEGFP cells (pool) were transiently transfected with mScarlet-I-Rab11b (g) or mScarlet-I-Rab1b (h), and then imaged by spinning-disk confocal microscopy at the plane near the bottom surface. i, CLTA-670nano+/+ cells stably expressing EGFP-sensor were transiently transfected with Arf1-mScarlet-I, and then imaged at 0.3-s intervals by TIRF microscopy. Montage showing the dynamic association of clathrin at the rear part of a highly motile Arf1-labelled tubule before its fusion and the recruitment of the sensor at the plasma membrane (arrows). j, Genome-edited Arf1-mEGFP cells (pool) were transiently transfected with CLTA-mScarlet-I, and then imaged by SIM. Examples illustrate the distribution of clathrin at subregions of tubular Arf1 carriers. k, AP2-670nano+/+ cells stably expressing EGFP-sensor and genome-edited for mScarlet-I-Rab1a+/-, mScarlet-I-Rab1b+/+, mScarlet-I-Rab11a+/+, mScarlet-I-Rab11b+/+ or AP1σ1-mScarlet-I+/+ were treated with control siRNA or siRNA targeting Arf1, then imaged by spinning-disk confocal microscopy. Experiments were repeated six (c) or three times (a-b and d-k) with similar results. Cells in d and f (right) were from one experiment. P values were determined by one-way ANOVA with Dunnett’s multiple comparisons test. Scale bars, 10 μm and 1 μm (magnification) in g and h; 1 μm in i; 0.5 μm in j; 10 μm in k. Source numerical data and unprocessed blots are available in source data.

Source data

Extended Data Fig. 8 Dynamin catalysers the scission of CARP carriers from the plasma membrane.

a, Cells stably expressing EGFP-sensor were transiently transfected with dynamin2-670nano (Dyn2-670nano) and mScarlet-I-Sec8, and then imaged at 1-s intervals by TIRF microscopy. Montage and kymographs showing the recruitment of dynamin2 during the late stage of sensor recruitment in a Sec8-positive fusion event (arrows). b, Left: Genomic PCR analysis showing the integration of mScarlet-I into the genomic locus of DNM2 to generate a pool of gene-edited cells expressing AP2-670nano+/+ Dyn2-mScarlet-Ien. Right: Western blot analysis of cell lysates probed with antibodies for RFP and GAPDH. c, U2OS cells stably expressing EGFP-sensor were transiently transfected with Dyn2-mScarlet-I and AP2-670nano, and then imaged at 1-s intervals by TIRF microscopy. Representative montage, kymographs, and relative fluorescence intensity traces with estimated uncertainties (s.d.) show the recruitment of dynamin2 during the late stage of sensor recruitment to an AP2-negative carrier (arrows). d, COS7 cells stably expressing EGFP-sensor and AP2-670nano were transiently transfected with Dyn2-mScarlet-I, and then imaged and displayed as in (c). e, AP2-670nano+/+ cells stably expressing EGFP-sensor were transiently transfected with endophilin-A2-mScarlet-I (EndoA2-mScarlet-I), and then imaged and displayed as in (c). f, AP2-TagRFP+/+ CLTA-670nano+/+ cells stably expressing EGFP-sensor were treated with control siRNA or two different siRNAs targeting dynamin2 (KD1 and KD2). The depletion efficiency of dynamin2 mRNA by siRNA was measured by qPCR (mean ± s.d.; n = 3 independent experiments). g, Left: Genomic PCR analysis showing biallelic integration of mScarlet-I into the genomic locus of ARPC3 to generate the clonal double-edited cell line AP2-670nano+/+ ARPC3-mScarlet-I+/+. Right: Western blot analysis of cell lysates probed with antibodies for RFP and α-actinin. h, Left: Genomic PCR analysis showing biallelic integration of mScarlet-I into the genomic locus of DBNL to generate the clonal double-edited cell line AP2-670nano+/+ mScarlet-I-mAbp1+/+. Right: Western blot analysis of cell lysates probed with antibodies for RFP and GAPDH. i, AP2-670nano+/+ mScarlet-I-mAbp1+/+ cells stably expressing EGFP-sensor were imaged at 1-s intervals by TIRF microscopy. Left: T-projections of a time series. Middle: The montage and kymographs show that mAbp1 is recruited during the late stage of sensor recruitment to an AP2-negative carrier and remains associated with the carrier after the loss of sensor signal (arrows). Right: The fraction of fusion events recruited mAbp1 (mean ± 95% CI; n = 10 cells from one experiment). Experiments were repeated three times with similar results. Scale bars, 1 μm in kymographs and montages (a, c-e, and i); 10 μm and 1 μm (magnification) in T-projection (i). Source numerical data and unprocessed blots are available in source data.

Source data

Extended Data Fig. 9 CARP carriers are derived from early endosomes independent of the endosomal retrieval complexes.

a, AP2-670nano+/+ cells stably expressing EGFP-sensor were transiently transfected with mScarlet-I-myosin Vb, and then imaged by TIRF microscopy. Montage, kymographs, and relative fluorescence intensity traces with estimated uncertainties (s.d.) showing dynamic recruitment of EGFP-sensor to a myosin Vb-containing carrier (arrows). b, AP2-TagRFP+/+ CLTA-670nano+/+ cells stably expressing EGFP-sensor were treated with either control siRNA or siRNA targeting Rab5a, Rab5b, Rab5c, or Rab5a/5b/5c. The knockdown efficiency was measured by qPCR (mean ± s.d.; n = 4 independent experiments). c, AP2-TagRFP+/+ CLTA-670nano+/+ cells stably expressing EGFP-sensor were treated with either control siRNA or siRNA targeting VPS35, VPS29, C16orf62, SNX3, SNX17, SNX27, COMMD1, or CCDC22. Left: Knockdown efficiency measured by qPCR (mean ± s.d.; n = 3 independent experiments). Middle: Due to the lack of appropriate qPCR primers for CCDC22, the efficiency of CCDC22 knockdown was assessed using western blot with antibodies against CCDC22 and α-actinin. Right: siRNA-treated cells were imaged by TIRF microscopy. The frequency of AP2-negative sensor-positive fusion events from the siRNA-treated cells is shown (mean ± 95% CI; n = 15, 14, 14, 14, 14, 14, 15, 14, and 14 cells). d, AP2-TagRFP+/+ CLTA-670nano+/+ cells stably expressing EGFP-sensor were treated with either control siRNA or siRNA targeting SNX1, SNX2, both SNX1 and SNX2 (SNX1/2), SNX5, SNX6, or both SNX5 and SNX6 (SNX5/6). Left: Knockdown efficiency measured by qPCR (mean ± s.d.; n = 3 independent experiments). Right: siRNA-treated cells were imaged by TIRF microscopy. The frequency of fusion events from the siRNA-treated cells is shown (mean ± 95% CI; n = 13, 13, 13, 13, 13, 13, 13 cells). e, AP2-mScarlet+/+ cells were subjected to knockout of VPS29, VPS35, SXN17, SNX27, or CCDC22. Knockout was validated by western blot analysis. f, AP2-mScarlet+/+ cells were subjected to knockout of SNX1, SNX2, SNX1/2, SNX5, SNX6, or SNX5/6. Knockout was validated by western blot analysis. g, Cells stably expressing EGFP-sensor were genome-edited to express Halo-EEA1 (pool). The cells were stained with the JFX650-HaloTag ligand, pretreated with DMSO (control) or nocodazole for 1 h, and then imaged by spinning-disk confocal microscopy near the bottom surface every 1.5 s for 300 s. The first frame and T-projection of Halo-EEA1 from representative time series are shown for the DMSO- or nocodazole-treated cells. The EEA1-positive endosomes in the time series were detected and tracked as shown in Fig. 7f. h, Cells stably expressing EGFP-sensor and genome-edited to express Halo-EEA1 (pool) were transiently transfected with AP1σ1-mScarlet-I, and then imaged by spinning-disk confocal microscopy near the bottom surface. The montage shows the recruitment of EGFP-sensor to the AP1-positive structure budded from the EEA1-positive endosome close to the plasma membrane (arrows). i, Cells expressing EGFP-sensor and Halo-EEA1 (pool) were transiently transfected with mScarlet-I-Rab11b, stained with the JFX650-HaloTag ligand and then treated with nocodazole for 1 h. The cells were 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 Rab11b-positive structure budded from the EEA1-positive endosome close to the plasma membrane (arrows). j,k, Genome-edited cells expressing Arf1-mEGFP and Halo-EEA1 (pool) were transiently transfected with mScarlet-I-Rab1b (j) or mScarlet-I-Rab11b (k), stained with the JFX650-HaloTag ligand, and then imaged near the middle plane by SIM. Left: Sub-organelle distribution of Arf1 with Rab1b or Rab11b on an EEA1-positive early endosome. Middle: Intensity profiles of the proteins (between the arrows) on the early endosome. Right: Polar plots of the relative spatial distribution of Rab1b or Rab11b with respect to Arf1 on individual early endosomes. Experiments were repeated three (a and c-k) or four times (b) with similar results. Cells in c (right) and d (right) were from one experiment. P values were determined by one-way ANOVA with Dunnett’s multiple comparisons test. Scale bars, 1 μm in a, h, and i; 10 μm in g; 0.5 μm in j and k. Source numerical data and unprocessed blots are available in source data.

Source data

Extended Data Fig. 10 Receptors can utilize and modulate CARP for recycling.

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.

Source data

Supplementary information

Reporting Summary

Supplementary Table

Supplementary Table 1. Plasmids used in this study. Supplementary Table 2. siRNA sequences used in this study. Supplementary Table 3. Primers used for qPCR. Supplementary Table 4. gRNA sequences used for generating knock-in cell lines. Supplementary Table 5. gRNA sequences used for generating knockout cell lines. Supplementary Table 6. Antibodies used in this study.

Supplementary Video 1

Gene-edited AP2-TagRFP+/+ CLTA-670nano+/+ cells stably expressing the EGFP-sensor imaged at the bottom surfaces every 1.5 s by TIRF microscopy.

Supplementary Video 2

Gene-edited CLTA-670nano+/+ cells stably expressing α-TagRFP-AP2 and transiently expressing EGFP-Tubbyc-Aux1 imaged at the bottom surfaces every 1.5 s by TIRF microscopy.

Supplementary Video 3

Gene-edited AP2-TagRFP+/+ CLTA-Halo+/+ cells stably expressing the EGFP-sensor imaged every 3.9 s by lattice light-sheet microscopy.

Supplementary Video 4

Gene-edited CLTA-TagRFP+/+ AP2-Halo+/+ cells stably expressing the EGFP-sensor imaged at the bottom surfaces every 2 s by GI-SIM.

Supplementary Video 5

Gene-edited AP1σ1-mScarlet-I+/+ AP2-670nano+/+ cells stably expressing the EGFP-sensor imaged at the bottom surfaces every 1 s by TIRF microscopy.

Supplementary Video 6

Gene-edited AP2-670nano+/+ Exo70-mScarlet3en cells stably expressing the EGFP-sensor imaged at the bottom surfaces every 1 s by TIRF microscopy.

Supplementary Video 7

Gene-edited AP2-670nano+/+ Sec3-mScarlet3en cells stably expressing the EGFP-sensor imaged at the bottom surfaces every 1 s by TIRF microscopy.

Supplementary Video 8

Gene-edited AP2-670nano+/+ mScarlet-I-Rab11a+/+ cells stably expressing the EGFP-sensor imaged at the bottom surfaces every 1 s by TIRF microscopy.

Supplementary Video 9

Gene-edited AP2-670nano+/+ mScarlet-I-Rab11b+/+ cells stably expressing the EGFP-sensor imaged at the bottom surfaces every 1 s by TIRF microscopy.

Supplementary Video 10

Gene-edited AP2-670nano+/+ mScarlet-I-Rab1a+/- cells stably expressing the EGFP-sensor imaged at the bottom surfaces every 1 s by TIRF microscopy.

Supplementary Video 11

Gene-edited AP2-670nano+/+ mScarlet-I-Rab1b+/+ cells stably expressing the EGFP-sensor imaged at the bottom surfaces every 1 s by TIRF microscopy.

Supplementary Video 12

Gene-edited AP2-670nano+/+ Arf1-mScarlet-I+/+ cells stably expressing the EGFP-sensor imaged at the bottom surfaces every 1 s by TIRF microscopy.

Supplementary Video 13

Gene-edited AP2-670nano+/+ Dyn2-mScarlet-Ien cells stably expressing the EGFP-sensor imaged at the bottom surfaces every 1 s by TIRF microscopy.

Supplementary Video 14

Gene-edited Halo-EEA1en cells stably expressing the EGFP-sensor and transiently expressing Arf1-mScarlet-I imaged at the bottom surfaces every 1.5 s by spinning-disk confocal microscopy.

Supplementary Video 15

Gene-edited AP2-670nano+/+ cells stably expressing SNAP-β2AR and the EGFP-sensor were stimulated by ISO for 20 min and imaged at the bottom surfaces every 2 s by TIRF microscopy.

Source data

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Xu, J., Liang, Y., Li, N. et al. Clathrin-associated carriers enable recycling through a kiss-and-run mechanism. Nat Cell Biol (2024). https://doi.org/10.1038/s41556-024-01499-4

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