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]