Abstract
In mammalian cells, secretory proteins are transported to their destination compartment via the secretory pathway. Cargos start their journey in the endoplasmic reticulum and then reach the Golgi complex where they are processed and sorted to be delivered to their target intracellular compartment. To analyze and visualize this flow of proteins, one needs to synchronize their transport. We recently developed the retention using selective hooks (RUSH) system enabling simultaneous and synchronous release of secretory cargos from the endoplasmic reticulum in a population of cells. Here, we describe how to subclone the gene coding for a cargo of interest into a RUSH plasmid and to monitor its synchronized transport along the secretory pathway in fixed samples and in living cells.
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References
Boncompain G, Perez F (2013) The many routes of Golgi-dependent trafficking. Histochem Cell Biol 140:251–260
Glick BS, Nakano A (2009) Membrane traffic within the Golgi apparatus. Annu Rev Cell Dev Biol 25:113–132
Matlin KS, Simons K (1983) Reduced temperature prevents transfer of a membrane glycoprotein to the cell surface but does not prevent terminal glycosylation. Cell 34:233–243
Saraste J, Kuismanen E (1984) Pre- and post-Golgi vacuoles operate in the transport of Semliki Forest virus membrane glycoproteins to the cell surface. Cell 38:535–549
Lippincott-Schwartz J, Yuan LC, Bonifacino JS, Klausner RD (1989) Rapid redistribution of Golgi proteins into the ER in cells treated with brefeldin A: evidence for membrane cycling from Golgi to ER. Cell 56:801–813
Kreis TE, Lodish HF (1986) Oligomerization is essential for transport of vesicular stomatitis viral glycoprotein to the cell surface. Cell 46:929–937
Lafay F (1974) Envelope proteins of vesicular stomatitis virus: effect of temperature-sensitive mutations in complementation groups III and V. J Virol 14:1220–1228
Boncompain G, Divoux S, Gareil N et al (2012) Synchronization of secretory protein traffic in populations of cells. Nat Methods 9:493–498
Boncompain G, Perez F (2012) Synchronizing protein transport in the secretory pathway. Curr Protoc Cell Biol 15:Unit15.19
Jordan M, Schallhorn A, Wurm FM (1996) Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation. Nucleic Acids Res 24:596–601
Boncompain G, Perez F (2013) Fluorescence based analysis of trafficking in mammalian cells. Methods Cell Biol 118:179–194
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Boncompain, G., Perez, F. (2014). Synchronization of Secretory Cargos Trafficking in Populations of Cells. In: Ivanov, A. (eds) Exocytosis and Endocytosis. Methods in Molecular Biology, vol 1174. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0944-5_14
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DOI: https://doi.org/10.1007/978-1-4939-0944-5_14
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Publisher Name: Humana Press, New York, NY
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Online ISBN: 978-1-4939-0944-5
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