Single-Molecule Imaging of NGF Axonal Transport in Microfluidic Devices

Abstract

Nerve growth factor (NGF) plays a critical role in neuron survival and functioning. NGF binds and activates membrane receptors TrkA, endocytosed NGF-TrkA complex then recruits various motor proteins to transport along cytoskeletal tracks towards cell body, where downstream signaling processes are regulated. Our recent study revealed that the majority of endosomes contain a single NGF molecule, which makes single-molecule imaging an essential tool for NGF studies. However single-molecule imaging in live cells is often limited by the intrinsic background fluorescence. Here, we efficiently reduced the background by employing a microfluidic culture platform, which can guide the growth of neurons and allow separately controlled microenvironment for cell bodies or axon termini. Single-molecule imaging of Qdot-labeled NGF in live axons shows exclusive retrograde transport of cargoes toward cell body with a stop-and-go pattern. Measurements at various temperatures show that the rate of NGF retrograde transport decreased exponentially over the range of 36-14 degree Celsius. A 10-degree decrease in temperature resulted in a threefold decrease in the rate of NGF retrograde transport. Our successful measurements of NGF transport suggest that the microfluidic device can serve as a unique platform for single-molecule imaging of molecular processes in live neurons.