Visualizing Directional Rab7 and TrkA Cotrafficking in Axons by pTIRF Microscopy
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 wordsPseudo-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