Figure 5. Lysosomal transport velocity analysis by comparing TrackMate and KymographClear algorithm. (A) A diagram of the motion of particles a and b. Scale bar: 20 μm. (B) A typical screenshot of the TrackMate algorithm during analyzing the velocity of axonal transport. The green line represents the trajectory of target particle a. Scale bar: 20 μm. (C) The result of the KymographClear algorithm about particle a. Red color notes forward-moving while green color notes backward moving, and blue color notes static. Scale bar: 5 μm. (D) The velocity-time diagram based on TrackMate and KymographClear algorithms, respectively.
In addition, in terms of the complexity of application operation, the KymographClear algorithm can only extract the motion velocity of one target particle at a time, while the TrackMate algorithm can analyze the motion condition of multiple target particles at the same time, which makes the operation more convenient and efficient. To sum up, compared with the KymographClear algorithm, the merits of the TrackMate algorithm can promote more concise and accurate analyses of lysosomal transport and a better understanding of the axonal transport mechanisms.
3.6. Factors involved in the velocity of axonal transport in freely orientated axons
After confirming that the TrackMate algorithm can be used as a concise and efficient tool for axonal transport analysis, we applied it to single-particle velocity analysis nearby axon branch point and multiple particle velocity analysis for axonal transport interpretation. Judging from the four stages in which moving particles passing by axonal branch (Figure 6A), the first stage is in slow motion (~0.106 μm/s), the velocity of the second stage is accelerated (~0.217 μm/s) and it seems to suddenly slow down while encountering with another particle. The velocity of the third stage is the slowest (0.024 μm/s) when it reaches the branching point. After passing by the axonal branch, the fourth stage velocity remains ~0.067 μm/s (Figure 6B). All these velocities illustrate that transportation of cytoplasmic protein mCherry wrapped in the lysosome belongs to a slow transport.[35] Then, we made a statistical analysis of the particle velocity before and after the branching point (Figure S3A), and it is obvious that the particle velocity before the branching point is higher than that after the branching point. The different microtubule arrangements nearby the branching point may bring about a different integrated force exerted on lysosomes during transportation.[23] These results suggested the velocity of axonal transport is partly dependent on the structure of axons. To further explore whether the size of particles affects the axonal transport velocity, we analyzed the velocity of 17 target particles in Figure 6C (Table S1) and confirmed the negative correlation between particle sizes and velocities, when the particle size ranged from 0.644 μm to 0.721 μm, the velocity could decrease from 0.439 μm/s to 0.067 μm/s (Figure 6D).
Moreover, at the molecular level, we provide a possible reason why larger particles move slower and most of the movement is in the same direction. First, the lysosomal transport homeostasis is generally maintained by a mole ratio of dynein to kinesin motors in the tug-of-war model.[7,29] Without considering the fusion between lysosomes, once particles get bigger, more sparse ligand proteins anchor in the lysosome membrane, and fewer chance particles have to be recognized by dynein/kinesin motors. Hence, the mole ratio of dynein to kinesin motors becomes unbalanced, and the bidirectional lysosomal transport tends to be unidirectional. It is also possible that lysosomes are in the one-way transport stage due to their developmental state.[15,36,37] Second, we simplify the tug-of-war model to one-way transportation for a concise explanation of moving velocities.[29] We have inferred those particles will have fewer chances to be recognized by kinesin motors if they get bigger, therefore the force from kinesin motor decreases. On the other hand, the increased load caused by larger particles further hinders the motion of kinesin protein, which makes the movement speed slower than smaller particles.
But this movement correlation doesn’t exist once we expanded the applicability to axons of different neurons, we found the velocity and diameter of particles in new statistics were irregular (Figure S3B, Video S1-S5, and Table S2). Maybe in different cells, the subtle microenvironment of axonal transport, such as microtubule density and arrangement,[23] motor protein quantity, regulatory protein concentration etc., makes a uniform and reasonable judgement intractable.[38]