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.