Visualizing directional rab7 and trka cotrafficking in axons by ptirf microscopy

Kai Zhang, Praveen D. Chowdary, Bianxiao Cui

Research output: Contribution to journalArticlepeer-review

Abstract

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 cotraffi cking 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 refl ection fl uorescence (pTIRF) microscopy, with microfl uidic neuron cultures that enables the simultaneous tracking of fl uorescently labeled Rab7- and TrkA-containing endosomes in axons.

Original languageEnglish (US)
Pages (from-to)319-329
Number of pages11
JournalMethods in Molecular Biology
Volume1298
DOIs
StatePublished - 2015

Keywords

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

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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