Abstract
We hereby describe a method to image cargo trafficking from the cis- to the trans-face of the Golgi apparatus. Briefly, we combine nocodazole treatment that breaks down the Golgi ribbon, temperature blocks that slow down cargo transport, and a drug-controlled aggregation system that controls the size of the cargo and its retention at different stages of the secretory pathway. Using this method, we first position the cargo within the cis-face of the Golgi. When traffic resumes upon temperature block release, kinetics of transport can be assessed by confocal microscopy through colocalization of the cargo with cis- and trans-Golgi markers. This method allows for testing various modes of intra-Golgi transports and can be adapted to investigate other steps of the secretory pathway.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Dancourt J, Barlowe C (2010) Protein sorting receptors in the early secretory pathway. Annu Rev Biochem 79:777–802
Ladinsky MS, Mastronarde DN, McIntosh JR et al (1999) Golgi structure in three dimensions: functional insights from the normal rat kidney cell. J Cell Biol 144:1135–1149
Emr S, Glick BS, Linstedt AD et al (2009) Journeys through the Golgi--taking stock in a new era. J Cell Biol 187:449–453
Glick BS, Luini A (2011) Models for Golgi traffic: a critical assessment. Cold Spring Harb Perspect Biol 3:a005215
Casler JC, Papanikou E, Barrero JJ et al (2019) Maturation-driven transport and AP-1–dependent recycling of a secretory cargo in the Golgi. J Cell Biol 218:1582–1601
Kurokawa K, Osakada H, Kojidani T et al (2019) Visualization of secretory cargo transport within the Golgi apparatus. J Cell Biol 218:1602–1618
Rivera VM, Wang X, Wardwell S et al (2000) Regulation of protein secretion through controlled aggregation in the endoplasmic reticulum. Science 287:826–830
Volchuk A, Amherdt M, Ravazzola M et al (2000) Megavesicles implicated in the rapid transport of intracisternal aggregates across the Golgi stack. Cell 102:335–348
Lavieu G, Zheng H, Rothman JE (2013) Stapled Golgi cisternae remain in place as cargo passes through the stack. Elife 2:e00558
Pellett PA, Dietrich F, Bewersdorf J et al (2013) Inter-Golgi transport mediated by COPI-containing vesicles carrying small cargoes. Elife 2:e01296
Lavieu G, Dunlop MH, Lerich A et al (2014) The Golgi ribbon structure facilitates anterograde transport of large cargoes. Mol Biol Cell 25:3028–3036
Dancourt J, Zheng H, Bottanelli F et al (2016) Small cargoes pass through synthetically glued Golgi stacks. FEBS Lett 590:1675–1686
Rizzo R, Parashuraman S, Mirabelli P et al (2013) The dynamics of engineered resident proteins in the mammalian Golgi complex relies on cisternal maturation. J Cell Biol 201:1027–1036
Wong M, Munro S (2014) The specificity of vesicle traffic to the Golgi is encoded in the golgin coiled-coil proteins. Science 346:1256898
Dunlop MH, Ernst AM, Schroeder LK et al (2017) Land-locked mammalian Golgi reveals cargo transport between stable cisternae. Nat Commun 8:432
Boncompain G, Divoux S, Gareil N et al (2012) Synchronization of secretory protein traffic in populations of cells. Nat Methods 9:493–498
Acknowledgments
This work is supported by ANR-18-IDEX-0001, IdEx Université de Paris, ANR-19-CE18-002001, and ANR-20-CE15-002102.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Dancourt, J., Lavieu, G. (2022). Monitoring Intra-Golgi Transport with Acute Spatiotemporal Control of a Synthetic Cargo. 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_2
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2209-4_2
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2208-7
Online ISBN: 978-1-0716-2209-4
eBook Packages: Springer Protocols