Single-molecule imaging of NGF axonal transport in microfluidic devices

Abstract

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.

A new method for quantifying mitochondrial axonal transport

“…1A and 1B; also see Li et al, 2014). Several improvements were made based on published designs (Cui et al, 2007; Zhang et al, 2010), including reducing the overall size of the microfluidic device and decreasing the space between the microgrooves. This improved design made it possible to assemble the microfluidic chamber onto a standard 18-mm circular coverslip and to fit the entire device in a single well of conventional 12-well cell culture dishes.…”

A close look at axonal transport: Cargos slow down when crossing stationary organelles

“…Media and reagents were obtained from Invitrogen (Carlsbad, CA).DRG cells were transiently transfected using Nucleofector method (Lonza, Switzerland) before plated in microfluidic devices. The devices used in this study featured two individual chambers connected by a set of micron-size channels [12]. This design allowed the separation of cell bodies and axonal terminals, thus making it straight forward to differentiate the direction of axonal transport, i.e.…”

“…Fluorescence imaging of cargo transport was carried out using a homebuilt pseudo total internal reflection fluorescence (pseudo-TIRF) microscope as previously described [12]. Briefly, the incident angle of the excitation laser was adjusted to be slightly smaller than the critical angle so that the laser beam could penetrate approximately 1–3µm deep into the sample (a depth comparable to the diameter of DRG axons).…”

Synapse-to-nucleus signaling

“…Many studies have focused on the binding of trophic factors such as nerve growth factor (NGF) to TrkA tyrosine receptors at axon terminals, a process that regulates neuronal survival and axonal outgrowth [15]. The NGF-bound activated TrkA receptor is endocytosed into a signaling endosome and then trafficked to the cell body via microtubules [14,16–19]. Once the endosome arrives in the soma, the active complex interacts with a range of kinases or second messenger molecules including Ras-MAPK, PI3K, PLCγ and ERK5, which in turn activate transcription factors such as CREB to trigger gene expression in the nucleus [18,20,21].…”