Abstract
Endocytosis and intracellular sorting of transforming growth factor-β (TGF-β) receptors play an important regulatory role in TGF-β signaling. Two major endocytic pathways, clathrin- and caveolae-mediated endocytosis, have been reported to independently mediate the internalization of TGF-β receptors. In this study, we demonstrate that the clathrin- and caveolae-mediated endocytic pathways can converge during TGF-β receptor endocytic trafficking. By tracking the intracellular dynamics of fluorescently-labeled TGF-β type I receptor (TβRI), we found that after mediating TβRI internalization, certain clathrin-coated vesicles and caveolar vesicles are fused underneath the plasma membrane, forming a novel type of caveolin-1 and clathrin double-positive vesicles. Under the regulation of Rab5, the fused vesicles are targeted to early endosomes and thus deliver the internalized TβRI to the caveolin-1 and EEA1 double-positive early endosomes (caveolin-1-positive early endosomes). We further showed that the caveolin-1-positive early endosomes are positive for Smad3/SARA, Rab11 and Smad7/Smurf2, and may act as a multifunctional device for TGF-β signaling and TGF-β receptor recycling and degradation. Therefore, these findings uncover a novel scenario of endocytosis, the direct fusion of clathrin-coated and caveolae vesicles during TGF-β receptor endocytic trafficking, which leads to the formation of the multifunctional sorting device, caveolin-1-positive early endosomes, for TGF-β receptors.
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Acknowledgements
We thank Drs Sanford M Simon, Richard D Minshall, Lois E Greene, Yan Chen, Marino Zerial and Harald Stenmark for their generous gifts of the clathrin-DsRed, caveolin-1-EGFP, EGFP-clathrin, caveolin-ECFP, EGFP-Rab5, ECFP-Rab5, ECFP-Rab5S34N, EGFP-Rab11, ECFP-Rab11 and Rab5Q79L plasmids. We thank Ying Li (Center of Biomedical Analysis, Tsinghua University) for technical assistance in transmission electron microscopy. This work was supported by the National Basic Research Program of China (2013CB933701), the National Natural Science Foundation of China (91413119, 91213305, 81270159 and 31221064) and the Chinese Academy of Sciences.
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Supplementary information
Supplementary information, Figure S1
Myc-TβRI protein expression in HeLa cells. (PDF 87 kb)
Supplementary information, Figure S2
Myc-TβRI labeling showed minor effect on TGF-β/Smad signaling transduction. (PDF 226 kb)
Supplementary information, Figure S3
Distribution of TβRI, caveolin-1 and clathrin at the plasma membrane. (PDF 740 kb)
Supplementary information, Figure S4
Quantification of the triple colocalization of TβRI, caveolin-1 and EEA1. (PDF 307 kb)
Supplementary information, Figure S5
Distribution of TβRI in caveolin-1-positive early endosomes in the mink lung epithelial cells (Mv1Lu). (PDF 166 kb)
Supplementary information, Figure S6
Resolving the ultrastructure of caveolin-1-positive early endosomes by immunoelectron microscopy. (PDF 136 kb)
Supplementary information, Figure S7
Interfering of clathrin- and caveolae-mediated endocytosis. (PDF 180 kb)
Supplementary information, Figure S8
Intracellular distribution of EGFR in caveolin-1-positive early endosomes. (PDF 193 kb)
Supplementary information, Figure S9
The caveolin-1 and clathrin double-positive vesicles were negatively stained with the endosomal SNARE molecules. (PDF 136 kb)
Supplementary information, Figure S10
Caveolin-1 is involved in TβRI recycling. (PDF 426 kb)
Supplementary information, Figure S11
Test for fluorescence bleed-through between different channels by single-labeled specimens. (PDF 111 kb)
Supplementary information, Movie S1
Live-cell TIRFM imaging of caveolae-mediated Myc-TβRI (red) endocytosis. (AVI 463 kb)
Supplementary information, Movie S2
Live-cell TIRFM imaging of a Myc-TβRI spot (red) moving toward a pre-existing caveolae. (AVI 386 kb)
Supplementary information, Movie S3
Live-cell TIRFM imaging of clathrin-mediated Myc-TβRI (red) endocytosis. (AVI 530 kb)
Supplementary information, Movie S4
Live-cell confocal imaging revealed the intracellular movement of a caveolin-GFP (green), Myc-TβRI (red) and Alexa Fluor-633 conjugated tranferrin (blue) triple-positive vesicle. (AVI 496 kb)
Supplementary information, Movie S5
Live-cell confocal imaging revealed the dynamic fusion and separation of a caveolin-1-GFP (green), Myc-TβRI (red) and Alexa Fluor 633-conjugated (blue) triple-positive vesicle. (AVI 361 kb)
Supplementary information, Movie S6
Live-cell spinning-disk confocal imaging of the lateral plasma membrane showed that a caveolin-1-GFP (green) and clathrin-DsRed (red) double-positive vesicle moved into cytoplasm. (AVI 700 kb)
Supplementary information, Movie S7
Live-cell confocal imaging of a Myc-TβRI (blue), caveolin-1-GFP (green) and clathrin-DsRed (red) triple-positive vesicle moving from the lateral plasma membrane into cytoplasm. (AVI 328 kb)
Supplementary information, Movie S8
Live-cell confocal imaging revealed the intracellular movement of the caveolin-1-GFP (green), mRFP-Rab5 (red) and Myc-TβRI (blue) triple-positive vesicle. (AVI 966 kb)
Supplementary information, Movie S9
Live-cell confocal imaging revealed the intracellular movement of the EGFP-Rab11 (green), caveolin-1-mRFP (red) and Myc-TβRI (blue) triple-positive vesicle. (AVI 484 kb)
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He, K., Yan, X., Li, N. et al. Internalization of the TGF-β type I receptor into caveolin-1 and EEA1 double-positive early endosomes. Cell Res 25, 738–752 (2015). https://doi.org/10.1038/cr.2015.60
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DOI: https://doi.org/10.1038/cr.2015.60
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