Three-dimensional distribution of TrkA neurotrophin receptors in neurite varicosities of differentiated PC12 cells treated with NGF determined by immunoelectron tomography

Abstract

Nerve growth factor (NGF) initiates the activation of TrkA tyrosine kinase receptors and numerous subsequent signaling cascades. However, the dynamics of the process including the translocation of TrkA is still unclear. In this study, the effect of NGF or membrane depolarization on the endocytic process and TrkA localization in the neuronal cell line PC12 was analyzed by live-cell imaging and immunoelectron tomography using an ultra-high voltage electron microscope (UHVEM). Both NGF re-stimulation and high potassium-induced depolarization enhanced the endocytic uptake of the fluorescent indicator into acidic organelles within varicosities as well as cell bodies. However, the transition of uptake differed completely. NGF also significantly increased the number of TrkA-containing varicosities. Immunoelectron tomography in whole-mounted cells showed that NGF induced the recruitment of TrkA to the surface membrane of neurite varicosities as well as the multivesicular bodies (MVBs) and lysosomal complexes inside the varicosities. Three-dimensional analysis revealed that invagination pits and intralumenal vesicles of MVBs contained TrkA immunoreactivity. In addition, TrkA immunoreactivity was scattered in the lysosomal matrices after NGF treatment. These results suggest that the neurite varicosities are intensely active in intracellular membrane trafficking, and play an important role in the degradation and accumulation of the NGF receptor, TrkA, after ligand stimulation.