3.4. Axonal transport imaging in freely orientated axons
In addition to the above work for fluorescence imaging, we further undertook axonal transport imaging in freely orientated axons. Unlike the microchannel-culturing method,[10,27] coverslip-culturing method provided a freely orientated growth pattern for natural morphogenesis owing to no space limitation in the culture environment, thus obtaining multifurcated axons with mCherry located at lysosomes. Hence, four axonal transport models were observed. The first one is the forward or backward transport model (Figure 4A and Video S1) in a ring-like axon, most of the particles moved forward while others moved backwards. One classic interpretation comes from the tug-of-war model[28] that mainly contained two kinds of motor proteins, dyneins and kinesins, which determine the direction by integrating the force from a mole ratio between these two proteins, retrograde or anterograde.[29] The second model is the stop-and-go model in which particles previously moved then suddenly came to rest together at a certain moment (Video S2). In Figure 4B, particles were moving before 215 s, and keeping still for the rest of the time. On the one hand, we attributed this static state to the discontinuity of ATP supply because axonal transport is dependent on the supply of ATP to drive the motor proteins.[30] On the other hand, axonal transport-related regulatory proteins in axons may also contribute to stop or start the transport of particles in the axons.[31]