Microtubule and kinesin/dynein-dependent, bi-directional transport of autolysosomes in neurites of PC12 cells.

Abstract

Autophagy, a major degradative pathway of the lysosomal system, has been implicated in various neurodegenerative diseases. During autophagic process, organelles and proteins are encapsulated in double-membrane vacuoles called autophagosomes, which finally fuse with lysosomes to form autolysosomes where incorporated materials are degraded. Despite extensive investigations in identifying the molecular components that participate in autophagy, little is known about routes and dynamics of autophagosomes/autolysosomes in the neurites of live cells. Hence, in the present study, we aim to investigate the biophysical characteristics of neuritic transport of autolysosomes in PC12 cells. Our study demonstrated that monomeric red fluorescence protein-light chain 3 (mRFP-LC3)-labeled autolysosomes were motile and moved along PC12 neurites in both anterograde and retrograde directions with a bias towards the nucleus during starvation. By using image processing, quantitative analysis was made to show the dynamic biophysical characteristics of these vesicles. The average velocity of anterograde and retrograde transport was 0.33±0.04μm/s and 0.39±0.05μm/s, respectively. Disruption of microtubules by nocodazole completely abolished their movements, suggesting the neuritic transport of autolysosomes depends on microtubules. The directional transport of autolysosomes was also affected by blockage of motor protein activity. Altogether, our study documents many aspects of the highly dynamic movement of autolysosome in PC12 neurites. Autolysosomes transported in a bi-directional manner along microtubules by dynein and kinesin motor proteins. These findings provide valuable insight into understanding the mechanism and control of autophagy in neurites under physiological and pathological conditions.