Abstract
Polarized epithelial cells have two distinct plasma membrane domains, i.e., an apical membrane domain and a basolateral membrane domain, that are the result of polarized trafficking of proteins and lipids. Several members of the Rab-type small GTPases, which are general regulators of membrane trafficking, have been reported to be involved in the regulation of polarized trafficking in epithelial cells, but their precise role in polarized trafficking is poorly understood. In a recent study we used Madin-Darby canine kidney (MDCK) II cells as a model of polarized cells and concluded from the results that Rab27A and its effector synaptotagmin-like protein 2-a (Slp2-a) regulate apical transport of Rab27-bearing vesicles in polarized epithelial cells. Both Rab27A and Slp2-a are uniformly localized at the plasma membrane in subconfluent, non-polarized MDCK II cells, but their expression increases as the cells become polarized, and they are specifically localized at the apical membrane in polarized MDCK II cells (i.e., two-dimensional cell culture). Slp2-a is also localized at the apical membrane of tubular MDCK II cysts (i.e., three-dimensional cell culture) and promotes the formation of a single apical domain in the cysts by regulating polarized trafficking of Rab27-bearing vesicles. In this chapter we describe the assay procedures for analyzing the expression and localization of Rab27A and Slp2-a in non-polarized and polarized renal epithelial cells.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Rodriguez-Boulan E, Kreitzer G, Müsch A (2005) Organization of vesicular trafficking in epithelia. Nat Rev Mol Cell Biol 6:233–247
RodrÃguez-Fraticelli AE, Gálvez-Santisteban M, MartÃn-Belmonte F (2011) Divide and polarize: recent advances in the molecular mechanism regulating epithelial tubulogenesis. Curr Opin Cell Biol 23:638–646
Fukuda M (2008) Regulation of secretory vesicle traffic by Rab small GTPases. Cell Mol Life Sci 65:2801–2813
Hutagalung AH, Novick PJ (2011) Role of Rab GTPases in membrane traffic and cell physiology. Physiol Rev 91:119–149
Stenmark H (2009) Rab GTPases as coordinators of vesicle traffic. Nat Rev Mol Cell Biol 10:513–525
Yasuda T, Saegusa C, Kamakura S, Sumimoto H, Fukuda M (2012) Rab27 effector Slp2-a transports the apical signaling molecule podocalyxin to the apical surface of MDCK II cells and regulates claudin-2 expression. Mol Biol Cell 23:3229–3239
Gálvez-Santisteban M, Rodriguez-Fraticelli AE, Bryant DM, Vergarajauregui S, Yasuda T, Bañón-RodrÃguez I, Bernascone I, Datta A, Spivak N, Young K, Slim CL, Brakeman PR, Fukuda M, Mostov KE, MartÃn-Belmonte F (2012) Synaptotagmin-like proteins control the formation of a single apical membrane domain in epithelial cells. Nat Cell Biol 14:838–849
Fukuda M, Mikoshiba K (2001) Synaptotagmin-like protein 1–3: a novel family of C-terminal-type tandem C2 proteins. Biochem Biophys Res Commun 281:1226–1233
Fukuda M, Saegusa C, Mikoshiba K (2001) Novel splicing isoforms of synaptotagmin-like proteins 2 and 3: identification of the Slp homology domain. Biochem Biophys Res Commun 283:513–519
Kuroda TS, Fukuda M, Ariga H, Mikoshiba K (2002) The Slp homology domain of synaptotagmin-like proteins 1–4 and Slac2 functions as a novel Rab27A binding domain. J Biol Chem 277:9212–9218
Fukuda M (2006) Rab27 and its effectors in secretory granule exocytosis: a novel docking machinery composed of a Rab27 · effector complex. Biochem Soc Trans 34:691–695
Fukuda M (2013) Rab27 effectors, pleiotropic regulators in secretory pathways. Traffic 14:949–963
Kuroda TS, Fukuda M (2004) Rab27A-binding protein Slp2-a is required for peripheral melanosome distribution and elongated cell shape in melanocytes. Nat Cell Biol 6:1195–1203
Fukuda M (2005) Versatile role of Rab27 in membrane trafficking: focus on the Rab27 effector families. J Biochem 137:9–16
Saegusa C, Tanaka T, Tani S, Itohara S, Mikoshiba K, Fukuda M (2006) Decreased basal mucus secretion by Slp2-a-deficient gastric surface mucous cells. Genes Cells 11:623–631
Yu M, Kasai K, Nagashima K, Torii S, Yokota-Hashimoto H, Okamoto K, Takeuchi T, Gomi H, Izumi T (2007) Exophilin4/Slp2-a targets glucagon granules to the plasma membrane through unique Ca2+-inhibitory phospholipid-binding activity of the C2A domain. Mol Biol Cell 18:688–696
Holt O, Kanno E, Bossi G, Booth S, Daniele T, Santoro A, Arico M, Saegusa C, Fukuda M, Griffiths GM (2008) Slp1 and Slp2-a localize to the plasma membrane of CTL and contribute to secretion from the immunological synapse. Traffic 9:446–457
Ménasché G, Ménager MM, Lefebvre JM, Deutsch E, Athman R, Lambert N, Mahlaoui N, Court M, Garin J, Fischer A, de Saint Basile G (2008) A newly identified isoform of Slp2a associates with Rab27a in cytotoxic T cells and participates to cytotoxic granule secretion. Blood 112:5052–5062
Yasuda T, Fukuda M (2014) Slp2-a controls renal epithelial cell size through regulation of Rap—ezrin signaling independently of Rab27. J Cell Sci 127:557–570
Kuroda TS, Itoh T, Fukuda M (2005) Functional analysis of Slac2-a/melaonophilin as a linker protein between Rab27A and myosin Va in melanosome transport. Methods Enzymol 403:419–431
Fukuda M, Kanno E (2005) Analysis of the role of Rab27 effector Slp4-a/granuphilin-a in dense-core vesicle exocytosis. Methods Enzymol 403:445–457
Imai A, Yoshie S, Nashida T, Shimomura H, Fukuda M (2004) The small GTPase Rab27B regulates amylase release from rat parotid acinar cells. J Cell Sci 117:1945–1953
Dukes JD, Whitley P, Chalmers AD (2011) The MDCK variety pack: choosing the right strain. BMC Cell Biol 12:1–4
MartÃn-Belmonte F, Yu W, RodrÃguez-Fraticelli AE, Ewald A, Werb Z, Alonso MA, Mostov K (2008) Cell-polarity dynamics controls the mechanism of lumen formation in epithelial morphogenesis. Curr Biol 18:507–513
Acknowledgments
We thank Megumi Aizawa for technical assistance and members of the Fukuda Laboratory for valuable discussions. This work was supported in part by JSPS KAKENHI Grant Number 242766 (to T.Y.) and by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, and Technology (MEXT) of Japan (to M.F.).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer Science+Business Media New York
About this protocol
Cite this protocol
Yasuda, T., S. Mrozowska, P., Fukuda, M. (2015). Functional Analysis of Rab27A and Its Effector Slp2-a in Renal Epithelial Cells. In: Li, G. (eds) Rab GTPases. Methods in Molecular Biology, vol 1298. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2569-8_11
Download citation
DOI: https://doi.org/10.1007/978-1-4939-2569-8_11
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2568-1
Online ISBN: 978-1-4939-2569-8
eBook Packages: Springer Protocols