High resolution measurement of membrane receptor endocytosis
Main Article Content
Keywords
Dendra2, fluorescence microscopy, membrane protein half-life, TIRF
Abstract
We present a new approach to quantify the half-life of membrane proteins on the cell surface, through tagging the protein with the photoconvertible fluorescent protein, Dendra2. Upon exposure to 405 nm light, Dendra2 is photoconverted from green to red emission. Total internal reflection fluorescence microscopy (TIRF) is applied to limit visualization of fluorescence to proteins located on the plasma membrane. Conversion of Dendra2 works as a pulse chase experiment through monitoring only the population of protein that has been photoconverted. As the protein is endocytosed the red emission decreases due to the protein leaving the TIRF field of view. This method is not impacted by the insertion of new protein into the plasma membrane as newly synthesized protein only exhibits green emission. We used this approach to determine the half-life of ENaC on the plasma membrane illustrating the high temporal resolution capability of this technique compared to current methods.
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References
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25. Staub O, Abriel H, Plant P, Ishikawa T, Kanelis V, Saleki R, et al. Regulation of the epithelial Na+ channel by Nedd4 and ubiquitination. Kidney international. 2000;57(3):809-15.
26. Rotin D, Kanelis V, Schild L. Trafficking and cell surface stability of ENaC. American Journal of Physiology-Renal Physiology. 2001;281(3):F391-F9.
27. Konstas A-A, Koch J-P, Korbmacher C. cAMP-dependent activation of CFTR inhibits the epithelial sodium channel (ENaC) without affecting its surface expression. Pflügers Archiv European Journal of Physiology. 2003;445(4):513-21.
28. Berdiev BK, Qadri YJ, Benos DJ. Assessment of the CFTR and ENaC association. Molecular BioSystems. 2009;5(2):123-7.
29. Lippincott‐Schwartz J, Patterson GH. Fluorescent proteins for photoactivation experiments. Methods in cell biology. 2008;85:45-61.
30. Zhang L, Gurskaya NG, Merzlyak EM, Staroverov DB, Mudrik NN, Samarkina ON, et al. Method for real-time monitoring of protein degradation at the single cell level. BioTechniques. 2007;42(4):450.
31. Yang K-Q, Xiao Y, Tian T, Gao L-G, Zhou X-L. Molecular genetics of Liddle's syndrome. Clinica Chimica Acta. 2014;436:202-6.
2. Sheng M, Pak DT. Ligand-gated ion channel interactions with cytoskeletal and signaling proteins. Annual review of physiology. 2000;62(1):755-78.
3. Hynes NE, Lane HA. ERBB receptors and cancer: the complexity of targeted inhibitors. Nature Reviews Cancer. 2005;5(5):341-54.
4. Laird DW. Life cycle of connexins in health and disease. Biochem J. 2006;394(Pt 3):527-43. Epub 2006/02/24. doi: 10.1042/BJ20051922. PubMed PMID: 16492141; PubMed Central PMCID: PMCPMC1383703.
5. Colley BS, Biju K, Visegrady A, Campbell S, Fadool DA. Neurotrophin B receptor kinase increases Kv subfamily member 1.3 (Kv1. 3) ion channel half-life and surface expression. Neuroscience. 2007;144(2):531-46.
6. Zhou P. Determining protein half-lives. Signal Transduction Protocols. 2004:67-77.
7. De La Rosa DA, Li H, Canessa CM. Effects of aldosterone on biosynthesis, traffic, and functional expression of epithelial sodium channels in A6 cells. The Journal of general physiology. 2002;119(5):427-42.
8. Sungkaworn T, Jobin M-L, Burnecki K, Weron A, Lohse MJ, Calebiro D. Single-molecule imaging reveals receptor–G protein interactions at cell surface hot spots. Nature. 2017;550:543. doi: 10.1038/nature24264
9. Yu L, Helms MN, Yue Q, Eaton DC. Single-channel analysis of functional epithelial sodium channel (ENaC) stability at the apical membrane of A6 distal kidney cells. Am J Physiol Renal Physiol. 2008;295(5):F1519-27. Epub 2008/09/12. doi: 10.1152/ajprenal.00605.2007. PubMed PMID: 18784262; PubMed Central PMCID: PMCPMC2584908.
10. Kleyman TR, Zuckerman JB, Middleton P, McNulty KA, Hu B, Su X, et al. Cell surface expression and turnover of the α-subunit of the epithelial sodium channel. American Journal of Physiology-Renal Physiology. 2001;281(2):F213-F21.
11. Mohan S, Bruns JR, Weixel KM, Edinger RS, Bruns JB, Kleyman TR, et al. Differential current decay profiles of epithelial sodium channel subunit combinations in polarized renal epithelial cells. Journal of Biological Chemistry. 2004;279(31):32071-8.
12. Staub O, Gautschi I, Ishikawa T, Breitschopf K, Ciechanover A, Schild L, et al. Regulation of stability and function of the epithelial Na+ channel (ENaC) by ubiquitination. The EMBO journal. 1997;16(21):6325-36.
13. Lu C, Pribanic S, Debonneville A, Jiang C, Rotin D. The PY motif of ENaC, mutated in Liddle syndrome, regulates channel internalization, sorting and mobilization from subapical pool. Traffic. 2007;8(9):1246-64. Epub 2007/07/04. doi: 10.1111/j.1600-0854.2007.00602.x. PubMed PMID: 17605762.
14. Gurskaya NG, Verkhusha VV, Shcheglov AS, Staroverov DB, Chepurnykh TV, Fradkov AF, et al. Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light. Nature biotechnology. 2006;24(4):461.
15. Chudakov DM, Lukyanov S, Lukyanov KA. Using photoactivatable fluorescent protein Dendra2 to track protein movement. Biotechniques. 2007;42(5):553.
16. Chudakov DM, Lukyanov S, Lukyanov KA. Tracking intracellular protein movements using photoswitchable fluorescent proteins PS-CFP2 and Dendra2. Nature protocols. 2007;2(8):2024-32.
17. Adam V, Nienhaus K, Bourgeois D, Nienhaus GU. Structural basis of enhanced photoconversion yield in green fluorescent protein-like protein Dendra2. Biochemistry. 2009;48(22):4905-15.
18. Zhang L, Gurskaya NG, Merzlyak EM, Staroverov DB, Mudrik NN, Samarkina ON, et al. Method for real-time monitoring of protein degradation at the single cell level. Biotechniques. 2007;42(4):446.
19. Heidary DK, Fox A, Richards CI, Glazer EC. A High-Throughput Screening Assay Using a Photoconvertable Protein for Identifying Inhibitors of Transcription, Translation, or Proteasomal Degradation. SLAS DISCOVERY: Advancing Life Sciences R&D. 2017;22(4):399-407.
20. Yildiz A, Vale RD. Total internal reflection fluorescence microscopy. Cold Spring Harbor Protocols. 2015;2015(9):pdb. top086348.
21. Macro L, Jaiswal JK, Simon SM. Dynamics of clathrin-mediated endocytosis and its requirement for organelle biogenesis in Dictyostelium. J Cell Sci. 2012;125(23):5721-32.
22. Merrifield CJ, Feldman ME, Wan L, Almers W. Imaging actin and dynamin recruitment during invagination of single clathrin-coated pits. Nature cell biology. 2002;4(9):691.
23. Gaidarov I, Santini F, Warren RA, Keen JH. Spatial control of coated-pit dynamics in living cells. Nature cell biology. 1999;1(1):1.
24. Shimkets RA, Warnock DG, Bositis CM, Nelson-Williams C, Hansson JH, Schambelan M, et al. Liddle's syndrome: heritable human hypertension caused by mutations in the β subunit of the epithelial sodium channel. Cell. 1994;79(3):407-14.
25. Staub O, Abriel H, Plant P, Ishikawa T, Kanelis V, Saleki R, et al. Regulation of the epithelial Na+ channel by Nedd4 and ubiquitination. Kidney international. 2000;57(3):809-15.
26. Rotin D, Kanelis V, Schild L. Trafficking and cell surface stability of ENaC. American Journal of Physiology-Renal Physiology. 2001;281(3):F391-F9.
27. Konstas A-A, Koch J-P, Korbmacher C. cAMP-dependent activation of CFTR inhibits the epithelial sodium channel (ENaC) without affecting its surface expression. Pflügers Archiv European Journal of Physiology. 2003;445(4):513-21.
28. Berdiev BK, Qadri YJ, Benos DJ. Assessment of the CFTR and ENaC association. Molecular BioSystems. 2009;5(2):123-7.
29. Lippincott‐Schwartz J, Patterson GH. Fluorescent proteins for photoactivation experiments. Methods in cell biology. 2008;85:45-61.
30. Zhang L, Gurskaya NG, Merzlyak EM, Staroverov DB, Mudrik NN, Samarkina ON, et al. Method for real-time monitoring of protein degradation at the single cell level. BioTechniques. 2007;42(4):450.
31. Yang K-Q, Xiao Y, Tian T, Gao L-G, Zhou X-L. Molecular genetics of Liddle's syndrome. Clinica Chimica Acta. 2014;436:202-6.
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Published
Dec 12, 2018
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Zhang Z, Heidary DK, Richards CI. High resolution measurement of membrane receptor endocytosis. J Biol Methods [Internet]. 2018 Dec. 12 [cited 2024 Apr. 20];5(4):e105. Available from: https://jbmethods.org/index.php/jbm/article/view/266
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Author Biography
Christopher I. Richards, University Of Kentucky
Associate Professor Chemistry
