Bianxiao Cui, a postdoc in Steve Chu‘s lab here at Stanford, gave a job talk in the Chemistry Department last week. The title was approximately “Single-Molecule Analysis of NGF in Live Neurons (Using Quantum Dots).” Anyway, that gets the point of the talk. It was pretty cool. Here are my notes:
Summary: Nerve-growth factor (NGF) is transported down the axon from the distal end near a target to the nerve cell body, and this tells the axon to grow. To image this transport in live cells, they labeled NGF with quantum dots (QDs) using a biotin/streptavidin linkage and tracked the labeled molecules using “pseudo TIRF”—somewhere between TIR illumination and columnated epi so that the excitation extended a little more into the sample than TIR, but kept background low. They used special compartmented sample containers (and later, microfluidic cells) in order to separate the cell body from the axon (because the labeled NGF nonspecifically labels the entire cell body and makes it too difficult to image).
Cui showed some beautiful (false-color) movies of individual QD-NGF-endosome complexes being actively transported down microtubules toward the cell body. Also found were instances where endosomes moved backward (toward distal axon) and endosomes allegedly passed over each other (although it was unclear to me how they proved that the two endosomes were on the same microtubule and if they actually passed over, because each diffraction-limited spot is indistinguishable). She made some claims about only one NGF per endosome (i.e. the endosome does not wait for more passengers), but that part was a little fuzzy to me (although it was probably the point of the talk). Also, they found that labeled NGF co-localized with signaling molecules in the cell body. Cool.
By fitting the point-spread function of the QDs, they were able to localize each endosome to individual microtubules (imaging below the diffraction limit). This was very cool, and Cui showed tracks of endosomes switching microtubules and then changing speed or direction. Finally, she even did a mouse study, but this seemed tacked on. The real results were the imaging and single-particle tracking.