HCA, HCE and HCT share axonal retrograde carriers.

Motor neurons were incubated with 15 nM HCA and 40 nM AlexaFluor555-TeNT HC (HCT) for 30 min at 37°C, either under resting (5 mM KCl) or stimulating conditions (60 mM KCl; data not shown), and then imaged. (A, top) Individual frames from a confocal time series are shown. Cell bodies are located out of view on the right. Arrows point to double-positive carriers for HCA and HCT moving towards the soma. Scale bar, 10 µm. (A, bottom) Kymographs of motor neuron axons treated as described above. The soma is out of view on the right. HCT-positive carriers are frequent and fast, whilst several HCA-positive organelles are stationary or oscillate. Carriers containing only HCA or HCT are also present. (B) Speed profile of HCA (in green), HCE- (in red) and HCT (in black) carriers. Note the overlap between the three curves, which display a speed peak around 0.8–1 µm/s. (C) Motor neurons were incubated with 15 nM HCA and 7.5 nM HCE for 30 min at 37°C under stimulating conditions (60 mM KCl), and then imaged. (C, top) Individual frames from a confocal time series are shown. Cell bodies are located out of view on the right. Arrowheads point to a carrier moving towards the soma containing both HCA and HCE. Asterisks indicate an example of stationary double-positive carrier. Scale bar, 10 µm. (C, bottom) Kymographs of motor neuron axons correspond to the stills above. Note the high number of double-positive carriers, either moving towards the soma or oscillatory. This analysis was performed using three (HCA, HCT) or two (HCE) independent primary motor neuron cultures, and replicated at least twice. At least ten (HCA, HCT) or five (HCE) movies per conditions were used to assemble the speed distribution curves shown in (B).