As a result, neurons rely on active axonal transport for all the materials needed at synaptic terminals, including neurotransmitter receptors, synaptic proteins, ion channels, lipids, and mitochondria 3. Such long axons preclude effective diffusion of soma-produced proteins for cell growth and maintenance given there are minimal or no ribosomes in axons to synthesize proteins. Neurons are highly polarized cells with long axons extending out 1000 times the length of their cell bodies 1, 2. 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. A 10☌ decrease in temperature resulted in a threefold decrease in the rate of NGF retrograde transport. Measurements at various temperatures show that the rate of NGF retrograde transport decreased exponentially over the range of 36–14☌.
Qdot-NGF was shown to move exclusively toward the cell body with a characteristic stop-and-go pattern of movements. 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. Microfluidic devices guide the growth of neurons and allow separately-controlled microenvironment for cell bodies or axon termini. Here, we employed a microfluidic culture platform to achieve background reduction for single molecule imaging in live neurons. Despite being an increasingly popular technique, single molecule imaging in live cells is often limited by background fluorescence. 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.
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.
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