Figure 5.

Simultaneous visualization of transport kinetics of α-synuclein and synaptophysin in the same axon. Cultured hippocampal neurons were double-transfected with mRFP::α-SYN and SYP::GFP and visualized by simultaneous dual-color video microscopy. A, Selected frames from a time-lapse sequence shows a single mRFP::α-SYN particle (red) transported anterogradely (arrowhead). In the same axon, several SYP::GFP particles (green) are seen, including those being transported (marked with asterisks, also see the corresponding kymograph). (For corresponding video, see supplemental Video 5, available at www.jneurosci.org as supplemental material.) B–D, Kymographs from dual-cam experiments wherein each three-frame panel shows the original mRFP::α-SYN kymograph (left), SYP::GFP kymograph (middle), and a pseudocolor overlay of anterograde tracings (mRFP::α-SYN in red and SYP::GFP in green) from the two kymographs (right). The kymograph in B is from the time-lapse shown above. Note the different transport frequencies and cotransport (yellow diagonal lines in merged kymographs) of mRFP::α-SYN and SYP:GFP. Retrograde mRFP::α-SYN particles were often cotransported with SYP::GFP; the asterisk in D marks one such particle. Scale bars for the kymographs are at the bottom left of the kymograph(s). Scale bar, 5 μm.