Lysosome Mobility Probed with Live Cell Imaging and Single Particle Tracking

Abstract

Lysosomes are the major degradative compartments of eukaryotic cells and their mobility plays an essential role in intracellular trafficking of the degraded cargo. There has been a growing interest in understanding the role of lysosomal membrane proteins in intracellular trafficking. The lysosome-associated membrane proteins LAMP1 and LAMP2 are key lysosomal membrane proteins. They have a large luminal domain and an 11 amino acid long cytosolic domain. Heavy glycosylation of LAMP1 and LAMP2 protects the proteins and the lysosomal membrane from degradation by proteases and lipases, respectively. Previous work has suggested that LAMP1 and LAMP2 may also play a role in lysosomal transport. We report the use of live cell fluorescence microscopy and single particle tracking to study the mobility of lysosomes following degradation of LAMP1 and LAMP2. LAMP1 and LAMP2 were degraded using endoglycosidase H (Endo H), a glycosidase which cleaves the glycans on the luminal domain of LAMPs. Fluorescence microscopy revealed that Endo H treatment caused the lysosomes to become enlarged and cluster in the perinuclear region. Particle tracking revealed that although both enlarged and punctate lysosomes had similar speeds, the mobility of the enlarged lysosomes was significantly lower than that of punctate lysosomes. Control experiments with sucrose-swollen vesicles show that the increase in size of the lysosomes is not responsible for the change in the mobility. Collectively, our results suggest that degradation of luminal domains of LAMPs inhibits the mobility of lysosomes. We believe that LAMPs may have a role in the interaction of dynein and kinesin motor proteins with lysosomes. Further work is underway to better understand the interaction of LAMPs with motor proteins and microtubules in regulating the mobility of lysosomes.