Imaging Diversity in Slow Axonal Transport.

The polarized morphology of neurons necessitates the delivery of proteins synthesized in the soma along the length of the axon to distal synapses; critical for sustaining communication between neurons. This constitutive and dynamic process of protein transport along axons termed "axonal transport" was initially characterized by classic pulse-chase radiolabeling studies which identified two major rate components: a fast component and a slow component. Early radiolabeling studies indicated "cohesive co-transport" of slow transport cargos. However, this approach could not be used to visualize or provide mechanistic insights on this highly dynamic process. The advent of fluorescent and photoactivatable imaging probes have now enabled real-time imaging of axonal transport. Conventional fluorescent probes have helped visualize and characterize the molecular mechanisms of transport of vesicular proteins. These proteins typically move in the fast component of axonal transport and appear as "punctate structures" along axons. However, a large majority of transported proteins that move in the slow component of transport, typically show a "uniform diffusive glow" along axons when tagged to conventional fluorescent probes. This makes it challenging to unequivocally track them in real time. Our lab has used photoactivatable fluorescent probes to tag three individual cytosolic proteins moving in the slow component of axonal transport, and identified three distinct modes of transport along axons. Our data from these experiments argue against the prevailing hypothesis based on classic radiolabeling studies, which suggested that all slow-transport proteins may move along the axon as one large macromolecular protein complex. Although other labs have started using photoactivation to study axonal transport of cytosolic proteins, this technique remains largely under-utilized. Here, we describe the detailed protocols to image and analyze axonal transport of three typical slow-component cargoes along axons of cultured hippocampal neurons.