Abstract
Endocytosis mediates the entry of surface and extracellular cargoes into the cell. In this chapter, we describe assays to quantitively measure the endocytosis of both soluble and transmembrane cargo proteins, taking advantage of cleavable fluorescent dyes labeling cargo proteins or antibodies recognizing cargo proteins. After removing surface-bound fluorescent dye, internalized cargoes are measured using confocal imaging and flow cytometry. We also describe strategies to determine the role of clathrin-mediated endocytosis (CME) in the internalization of a cargo by using a small molecule inhibitor of CME and knockout (KO) of the AAGAB gene, which encodes an essential regulator of CME.
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References
McMahon HT, Boucrot E (2011) Molecular mechanism and physiological functions of clathrin-mediated endocytosis. Nat Rev Mol Cell Biol 12(8):517–533. https://doi.org/10.1038/nrm3151
Haucke V (2006) Cargo takes control of endocytosis. Cell 127(1):35–37. https://doi.org/10.1016/j.cell.2006.09.012
Traub LM, Bonifacino JS (2013) Cargo recognition in clathrin-mediated endocytosis. Cold Spring Harb Perspect Biol 5(11):a016790. https://doi.org/10.1101/cshperspect.a016790
Mettlen M, Chen PH, Srinivasan S, Danuser G, Schmid SL (2018) Regulation of Clathrin-mediated endocytosis. Annu Rev Biochem 87:871–896. https://doi.org/10.1146/annurev-biochem-062917-012644
Kaksonen M, Roux A (2018) Mechanisms of clathrin-mediated endocytosis. Nat Rev Mol Cell Biol 19(5):313–326. https://doi.org/10.1038/nrm.2017.132
Wood LA, Larocque G, Clarke NI, Sarkar S, Royle SJ (2017) New tools for “hot-wiring” clathrin-mediated endocytosis with temporal and spatial precision. Int J Cell Biol 216(12):4351–4365. https://doi.org/10.1083/jcb.201702188
Kelly BT, McCoy AJ, Späte K, Miller SE, Evans PR, Höning S, Owen DJ (2008) A structural explanation for the binding of endocytic dileucine motifs by the AP2 complex. Nature 456(7224):976–979. https://doi.org/10.1038/nature07422
Gulbranson DR, Crisman L, Lee M, Ouyang Y, Menasche BL, Demmitt BA, Wan C, Nomura T, Ye Y, Yu H, Shen J (2019) AAGAB controls AP2 adaptor assembly in Clathrin-mediated endocytosis. Dev Cell 50(4):436–446 e435. https://doi.org/10.1016/j.devcel.2019.06.013
Kamei N, Yamamoto S, Hashimoto H, Nishii M, Miyaura M, Tomada K, Nakase I, Takeda-Morishita M (2019) Optimization of the method for analyzing endocytosis of fluorescently tagged molecules: impact of incubation in the cell culture medium and cell surface wash with glycine-hydrochloric acid buffer. J Control Release 310:127–140. https://doi.org/10.1016/j.jconrel.2019.08.020
Bitsikas V, Corrêa IR Jr, Nichols BJ (2014) Clathrin-independent pathways do not contribute significantly to endocytic flux. eLife 3:e03970. https://doi.org/10.7554/eLife.03970
Tobys D, Kowalski LM, Cziudaj E, Muller S, Zentis P, Pach E, Zigrino P, Blaeske T, Honing S (2020) Inhibition of clathrin-mediated endocytosis by knockdown of AP-2 leads to alterations in the plasma membrane proteome. Traffic 22(1-2):6–22. https://doi.org/10.1111/tra.12770
Mayle KM, Le AM, Kamei DT (2012) The intracellular trafficking pathway of transferrin. Biochim Biophys Acta 1820:264–281. https://doi.org/10.1016/j.bbagen.2011.09.009
Ishikawa Y, Tacheva-Grigorova SK, Maeda M, Aguet F, Lee S-U, Ericsson M, Kirchhausen T, Maeda T (2013) Cellular mechanisms for transferrin receptor endocytosis vital for terminal erythroid differentiation. Blood 122(21):3434. https://doi.org/10.1182/blood.V122.21.3434.3434
Harding C, Heuser J, Stahl P (1983) Receptor-mediated endocytosis of transferrin and recycling of the transferrin receptor in rat reticulocytes. Int J Cell Biol 97(2):329–339. https://doi.org/10.1083/jcb.97.2.329
Bryant NJ, Govers R, James DE (2002) Regulated transport of the glucose transporter GLUT4. Nat Rev Mol Cell Biol 3(4):267–277
Elkin SR, Oswald NW, Reed DK, Mettlen M, MacMillan JB, Schmid SL (2016) Ikarugamycin: a natural product inhibitor of Clathrin-mediated endocytosis. Traffic 17(10):1139–1149. https://doi.org/10.1111/tra.12425
Gulbranson DR, Davis EM, Demmitt BA, Ouyang Y, Ye Y, Yu H, Shen J (2017) RABIF/MSS4 is a Rab-stabilizing holdase chaperone required for GLUT4 exocytosis. Proc Natl Acad Sci U S A 114(39):E8224–E8233. https://doi.org/10.1073/pnas.1712176114
Acknowledgments
We thank Yan Ouyang, Harrison Puscher, and Ximing Dou for technical assistance, and Dr. Stefan Höning for advice. This work was supported by National Institutes of Health (NIH) grants DK124431 (J.S.), GM126960 (J.S.), AG061829 (J.S.), an American Diabetes Association Basic Science Award (J.S.).
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Wang, S., Wan, C., Squiers, G.T., Shen, J. (2022). Endocytosis Assays Using Cleavable Fluorescent Dyes. In: Shen, J. (eds) Membrane Trafficking. Methods in Molecular Biology, vol 2473. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2209-4_14
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DOI: https://doi.org/10.1007/978-1-0716-2209-4_14
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