Rab GTPases pp 233-243 | Cite as

Assay of Rab17 and Its Guanine Nucleotide Exchange Factor Rabex-5 in the Dendrites of Hippocampal Neurons

  • Yasunori Mori
  • Mitsunori FukudaEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1298)


Neurons are functionally and morphologically compartmentalized into axons and dendrites, and the localization of specific proteins within these compartments is critical to the proper formation of neuronal networks, which includes neurite morphogenesis and synapse formation. The small GTPase Rab17 is specifically localized in dendrites and is not found in axons, and it regulates the dendrite morphogenesis and postsynaptic development of mouse hippocampal neurons. However, the spatiotemporal regulation of Rab17 is poorly understood. We recently identified Rabex-5, originally described as a Rab5-guanine nucleotide exchange factor (GEF), as a physiological Rab17-GEF that promotes translocation of Rab17 from the cell body to the dendrites of developing hippocampal neurons. Knockdown of Rab17 in mouse hippocampal neurons resulted in reductions in dendrite growth, branch numbers, filopodium density, and active synapse numbers. Knockdown of Rab17-GEF Rabex-5 in hippocampal neurons resulted in decreased targeting of Rab17 to the dendrites, which led to a reduction in dendrite growth. In this chapter we describe the assay procedures for analyzing Rab17 and Rabex-5 in cultured mouse hippocampal neurons, and we particularly focus on the measurement of total dendrite (or axon) length and total dendrite (or axon) branch numbers, filopodium density, number of active synapses, and dendritic Rab17 signals.

Key words

Rab17 Rabex-5 Dendrite Postsynapse Hippocampal neuron 



We thank Megumi Aizawa for technical assistance and members of the Fukuda Laboratory for valuable discussions. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, and Technology (MEXT) of Japan (to M.F. and Y.M.) and by a grant from the Daiichi-Sankyo Foundation of Life Science (to M.F.).


  1. 1.
    Yoshihara Y, De Roo M, Muller D (2009) Dendritic spine formation and stabilization. Curr Opin Neurobiol 19:146–153CrossRefPubMedGoogle Scholar
  2. 2.
    Fukuda M (2008) Regulation of secretory vesicle traffic by Rab small GTPases. Cell Mol Life Sci 65:2801–2813CrossRefPubMedGoogle Scholar
  3. 3.
    Stenmark H (2009) Rab GTPases as coordinators of vesicle traffic. Nat Rev Mol Cell Biol 10:513–525CrossRefPubMedGoogle Scholar
  4. 4.
    Barr F, Lambright DG (2010) Rab GEFs and GAPs. Curr Opin Cell Biol 22:461–470CrossRefPubMedCentralPubMedGoogle Scholar
  5. 5.
    Touchot N, Chardin P, Tavitian A (1987) Four additional members of the ras gene superfamily isolated by an oligonucleotide strategy: molecular cloning of YPT-related cDNAs from a rat brain library. Proc Natl Acad Sci U S A 84:8210–8214CrossRefPubMedCentralPubMedGoogle Scholar
  6. 6.
    Mori Y, Fukuda M (2013) Rabex-5 determines the neurite localization of its downstream Rab proteins in hippocampal neurons. Commun Integr Biol 6:e25433CrossRefPubMedCentralPubMedGoogle Scholar
  7. 7.
    Mori Y, Matsui T, Furutani Y, Yoshihara Y, Fukuda M (2012) Small GTPase Rab17 regulates dendritic morphogenesis and postsynaptic development of hippocampal neurons. J Biol Chem 287:8963–8973CrossRefPubMedCentralPubMedGoogle Scholar
  8. 8.
    Horiuchi H, Lippé R, McBride HM, Rubino M, Woodman P, Stenmark H, Rybin V, Wilm M, Ashman K, Mann M, Zerial M (1997) A novel Rab5 GDP/GTP exchange factor complexed to Rabaptin-5 links nucleotide exchange to effector recruitment and function. Cell 90:1149–1159CrossRefPubMedGoogle Scholar
  9. 9.
    Yoshimura S, Gerondopoulos A, Linford A, Rigden DJ, Barr FA (2010) Family-wide characterization of the DENN domain Rab GDP-GTP exchange factors. J Cell Biol 191:367–381CrossRefPubMedCentralPubMedGoogle Scholar
  10. 10.
    Mori Y, Matsui T, Fukuda M (2013) Rabex-5 protein regulates dendritic localization of small GTPase Rab17 and neurite morphogenesis in hippocampal neurons. J Biol Chem 288:9835–9847CrossRefPubMedCentralPubMedGoogle Scholar
  11. 11.
    Meijering E, Jacob M, Sarria J-CF, Steiner P, Hirling H, Unser M (2004) Design and validation of a tool for neurite tracing and analysis in fluorescence microscopy images. Cytometry A 58:167–176CrossRefPubMedGoogle Scholar
  12. 12.
    Fukuda M, Kowalchyk JA, Zhang X, Martin TFJ, Mikoshiba K (2002) Synaptotagmin IX regulates Ca2+-dependent secretion in PC12 cells. J Biol Chem 277:4601–4604CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Laboratory of Membrane Trafficking Mechanisms, Department of Developmental Biology and Neurosciences, Graduate School of Life SciencesTohoku UniversitySendaiJapan

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