FIGURE 8.
CAR and CTB co-transport and sorting after axonal retrograde transport. A–C, FKCAV/CAR were co-transported with CTB in axonal carriers. A, hippocampal neurons cultured in MCs were incubated with Cy5-FKCAV and Alexa 488-CTB in the axonal compartment for 60 min at 37 °C and then imaged in the microgrooves. Still frames of a movie show Cy5-FKCAV and Alexa 488-CTB in the same axonal carriers undergoing retrograde transport. Arrows show moving compartments positive for both ligands (co-transport of 75% ± 9%; n = 3, 94 structures were counted; results are expressed as mean ± S.E.). B, kymographs show similar profiles of transported Cy5-FKCAV and Alexa 488-CTB. C, CTB and CAR were sorted differently after axonal transport. Hippocampal neurons cultured in MCs were incubated with Cy5-FKCAV and Alexa 488-CTB in the axonal compartment for 3 h at 37 °C, and imaged in the cell body compartment. Dashed line and n identify the nucleus. Insets show minimal colocalization between FKCAV and CTB. D, CAR was targeted to lysosomes after axonal transport. Hippocampal neurons cultured in MCs were incubated with Cy5-FKCAV in the axonal compartment for 3 h at 37 °C, fixed, stained for the lysosomal marker LAMP1, and imaged at the cell body compartment. Numerous FKCAV-positive structures contain LAMP1 (88% ± 3%; n = three independent experiments, 140 structures were counted. Results are expressed as mean ± S.E.). Dashed line and n identify the nucleus. Scale bars, 5 μm.