3.2. Colocalization of mCherry proteins, axonal lysosomes and
tubulin
To figure out how mCherry protein granules formed and moved, we first
analyzed the colocalization between the mCherry signal and lysosome
signal in the axon. Notably, the fluorescence of mCherry highly merged
with the lysosomal signal (Figure 2), indicating the mCherry protein
granules were formed by wrapping the mCherry in the lysosome. This
result agrees with previous founding that the over-expressed GFP-like
proteins were accumulated in the lysosome of
cells.[20] It is known that lysosomes are
responsible for endocytic and autophagic components degradation and
transportation, thus maintaining cellular homeostasis in
neurons.[21] Thereby, the formation of lysosomes
with fluorescent mCherry may attribute to the strategy of removing extra
proteins via axonal transport to maintain the homeostasis of substances
in axons.[22] In axons, the microtubule serves as
a basic cytoskeleton for neuronal polarity, axon growth, branching and
axonal
transport.[23] To further confirm whether mCherry
protein granules can be loaded on microtubules, we analyzed the
colocalization between the mCherry and microtubule. It was found that
the mCherry protein granules were intermittently distributed on the
microtubule skeleton of the axon (Figure 2C). These results suggested
the over-expressed mCherry were able to be gradually wrapped in the
lysosome and loaded on microtubules for axonal transport imaging. And
the sparse labelling strategy can be used as a convenient model for the
study of lysosomal transport in axons of freely orientated neurons.