Methodology for Detecting and Tracking Brain-Derived Neurotrophic Factor Complexes in Neurons Using Single Quantum Dots
We describe a methodological workflow for detecting and tracking single brain-derived neurotrophic factor (BDNF) receptor complexes in primary cultures of neurons. This methodology includes the preparation of BDNF-conjugated quantum dot fluorescent probes for sensitive detection of single BDNF complexes in fixed neurons as well as single-particle tracking (SPT) in live neurons. These methods are valuable for high-resolution localization and quantitation of low-abundance proteins in single neurons and for obtaining nanoscale-resolution, dynamic information of BDNF trafficking for long time durations (min) in live neurons.
KeywordsBDNF Dynamics Growth factor Intracellular trafficking Quantum dot Single-particle tracking TrkB
This work was supported by NIH-NINDS R01 NS071116 01 and W81XWH-07-2-0107 to T.Q.V.
This movie shows tracking of several quantum dot-labeled brain-derived neurotrophic factors (QD-BDNFs) bound to TrkB receptors moving along distal axons to the cell body. Most of the selected trajectories exhibit the active/pause transport dynamics that characterizes the primary motion observed along axons. Some trajectories are apparently stationary or confined and are shown for comparison to the active behavior. The first half of the movie shows dynamic tracking from a 2D perspective where colors distinguish different particle trajectories, and we also show a 3D flyover of a static rendering of the trajectories in x, y, and time dimensions. Of particular interest is the nature of the pauses in the active transport trajectories which are well resolved at our high spatiotemporal tracking resolutions. (MP4 14001 kb)
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