Nerve growth factor (NGF) signaling begins at the nerve terminal, where it binds and activates membrane receptors and subsequently carries the cell-survival signal to the cell body through the axon. A recent study revealed that the majority of endosomes contain a single NGF molecule, which makes single molecule imaging an essential tool for NGF studies. Despite being an increasingly popular technique, single molecule imaging in live cells is often limited by background fluorescence. Here, we employed a microfluidic culture platform to achieve background reduction for single molecule imaging in live neurons. Microfluidic devices guide the growth of neurons and allow separately-controlled microenvironment for cell bodies or axon termini. Designs of microfluidic devices were optimized and a three-compartment device successfully achieved direct observation of axonal transport of single NGF when quantum dot labeled NGF (Qdot-NGF) was applied only to the distal-axon compartment while imaging was carried out exclusively in the cell-body compartment. Qdot-NGF was shown to move exclusively toward the cell body with a characteristic stop-and-go pattern of movements. Measurements at various temperatures show that the rate of NGF retrograde transport decreased exponentially over the range of 36–14°C. A 10°C 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 neurons.
Summary
Microtubules are essential cytoskeletal tracks for cargo transportation in axons and also serve as the primary structural scaffold of neurons. Structural assembly, stability, and dynamics of axonal microtubules are of great interest for understanding neuronal functions and pathologies. However, microtubules are so densely packed in axons that their separations are well below the diffraction limit of light, which precludes using optical microscopy for live-cell studies. Here, we present a single-molecule imaging method capable of resolving individual microtubules in live axons. In our method, unlabeled microtubules are revealed by following individual axonal cargos that travel along them. We resolved more than six microtubules in a 1 μm diameter axon by real-time tracking of endosomes containing quantum dots. Our live-cell study also provided direct evidence that endosomes switch between microtubules while traveling along axons, which has been proposed to be the primary means for axonal cargos to effectively navigate through the crowded axoplasmic environment.
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