Rab GTPases pp 319-329 | Cite as

Visualizing Directional Rab7 and TrkA Cotrafficking in Axons by pTIRF Microscopy

  • Kai Zhang
  • Praveen D. Chowdary
  • Bianxiao CuiEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1298)


Rab7 GTPase is known to regulate protein degradation and intracellular signaling via endocytic sorting and is also known to be involved in peripheral neurodegeneration. Mutations in the GTP-binding pocket of Rab7 cause Charcot–Marie–Tooth type 2B (CMT-2B) neuropathy. It has been suggested that the CMT-2B-associated Rab7 mutants may disrupt retrograde survival signaling by degrading the signaling endosomes carrying the nerve growth factor (NGF) and its TrkA receptor. Studying the cotrafficking of Rab7 and retrograde-TrkA endosomes in axons is therefore important to understand how Rab7 mutants affect the NGF signaling in neurons. However, tracking the axonal transport of Rab7 and TrkA with conventional microscopy and assigning the transport directionality in mass neuronal cultures pose some practical challenges. In this chapter, we describe the combination of a single-molecule imaging technique, pseudo-total internal reflection fluorescence (pTIRF) microscopy, with microfluidic neuron cultures that enables the simultaneous tracking of fluorescently labeled Rab7- and TrkA-containing endosomes in axons.

Key words

Pseudo-total internal reflection fluorescence (pTIRF) microscopy Charcot–Marie–Tooth type 2B (CMT-2B) Rab7 Nerve growth factor (NGF) TrkA Endosomes Axonal transport Dorsal root ganglion (DRG) Polydimethylsiloxane (PDMS) Compartmentalized microfluidic chamber 


  1. 1.
    Stenmark H, Olkkonen VM (2001) The Rab GTPase family. Genome Biol 2(5):REVIEWS3007CrossRefPubMedCentralPubMedGoogle Scholar
  2. 2.
    Spinosa MR, Progida C, De Luca A, Colucci AM, Alifano P, Bucci C (2008) Functional characterization of Rab7 mutant proteins associated with Charcot-Marie-Tooth type 2B disease. J Neurosci 28(7):1640–1648CrossRefPubMedGoogle Scholar
  3. 3.
    Cogli L, Piro F, Bucci C (2009) Rab7 and the CMT2B disease. Biochem Soc Trans 37(Pt 5):1027–1031CrossRefPubMedGoogle Scholar
  4. 4.
    Saxena S, Bucci C, Weis J, Kruttgen A (2005) The small GTPase Rab7 controls the endosomal trafficking and neuritogenic signaling of the nerve growth factor receptor TrkA. J Neurosci 25(47):10930–10940CrossRefPubMedGoogle Scholar
  5. 5.
    Deinhardt K, Salinas S, Verastegui C, Watson R, Worth D, Hanrahan S, Bucci C, Schiavo G (2006) Rab5 and Rab7 control endocytic sorting along the axonal retrograde transport pathway. Neuron 52(2):293–305CrossRefPubMedGoogle Scholar
  6. 6.
    Zhang K, Fishel Ben Kenan R, Osakada Y, Xu W, Sinit RS, Chen L, Zhao X, Chen JY, Cui B, Wu C (2013) Defective axonal transport of Rab7 GTPase results in dysregulated trophic signaling. J Neurosci 33(17):7451–7462CrossRefPubMedCentralPubMedGoogle Scholar
  7. 7.
    Ascano M, Richmond A, Borden P, Kuruvilla R (2009) Axonal targeting of Trk receptors via transcytosis regulates sensitivity to neurotrophin responses. J Neurosci 29(37):11674–11685CrossRefPubMedCentralPubMedGoogle Scholar
  8. 8.
    Cui B, Wu C, Chen L, Ramirez A, Bearer EL, Li WP, Mobley WC, Chu S (2007) One at a time, live tracking of NGF axonal transport using quantum dots. Proc Natl Acad Sci U S A 104(34):13666–13671CrossRefPubMedCentralPubMedGoogle Scholar
  9. 9.
    Ure DR, Campenot RB (1997) Retrograde transport and steady-state distribution of 125I-nerve growth factor in rat sympathetic neurons in compartmented cultures. J Neurosci 17(4):1282–1290PubMedGoogle Scholar
  10. 10.
    Taylor AM, Blurton-Jones M, Rhee SW, Cribbs DH, Cotman CW, Jeon NL (2005) A microfluidic culture platform for CNS axonal injury, regeneration and transport. Nat Methods 2(8):599–605CrossRefPubMedCentralPubMedGoogle Scholar
  11. 11.
    Moore SW, Lai Wing Sun K, Xie F, Barker PA, Conti M, Kennedy TE (2008) Soluble adenylyl cyclase is not required for axon guidance to netrin-1. J Neurosci 28(15):3920–3924CrossRefPubMedGoogle Scholar
  12. 12.
    Owen DE, Egerton J (2012) Culture of dissociated sensory neurons from dorsal root ganglia of postnatal and adult rats. Methods Mol Biol 846:179–187CrossRefPubMedGoogle Scholar
  13. 13.
    Darbinyan A, Pozniak P, Darbinian N, White MK, Khalili K (2013) Compartmentalized neuronal cultures. Methods Mol Biol 1078:147–152CrossRefPubMedCentralPubMedGoogle Scholar
  14. 14.
    Park JW, Vahidi B, Taylor AM, Rhee SW, Jeon NL (2006) Microfluidic culture platform for neuroscience research. Nat Protoc 1(4):2128–2136CrossRefPubMedGoogle Scholar
  15. 15.
    Zhang K, Osakada Y, Vrljic M, Chen L, Mudrakola HV, Cui B (2010) Single-molecule imaging of NGF axonal transport in microfluidic devices. Lab Chip 10:2566–2573CrossRefPubMedCentralPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of BiochemistryUniversity of Illinois at Urbana-ChampaignUrbanaUSA
  2. 2.Department of ChemistryStanford UniversityStanfordUSA

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