Abstract
Lysosomes are terminal, degradative organelles of the endosomal pathway that undergo repeated fusion-fission cycles with themselves, endosomes, phagosomes, and autophagosomes. Lysosome number and size depends on balanced fusion and fission rates. Thus, conditions that favour fusion over fission can reduce lysosome numbers while enlarging their size. Conversely, favouring fission over fusion may cause lysosome fragmentation and increase their numbers. PIKfyve is a phosphoinositide kinase that generates phosphatidylinositol-3,5-bisphosphate to modulate lysosomal functions. PIKfyve inhibition causes an increase in lysosome size and reduction in lysosome number, consistent with lysosome coalescence. This is thought to proceed through reduced lysosome reformation and/or fission after fusion with endosomes or other lysosomes. Previously, we observed that photo-damage during live-cell imaging prevented lysosome coalescence during PIKfyve inhibition. Thus, we postulated that lysosome fusion and/or fission dynamics are affected by reactive oxygen species (ROS). Here, we show that ROS generated by various independent mechanisms all impaired lysosome coalescence during PIKfyve inhibition and promoted lysosome fragmentation during PIKfyve re-activation. However, depending on the ROS species or mode of production, lysosome dynamics were affected distinctly. H2O2 impaired lysosome motility and reduced lysosome fusion with phagosomes, suggesting that H2O2 reduces lysosome fusogenecity. In comparison, inhibitors of oxidative phosphorylation, thiol groups, glutathione, or thioredoxin, did not impair lysosome motility but instead promoted clearance of actin puncta on lysosomes formed during PIKfyve inhibition. Additionally, actin depolymerizing agents prevented lysosome coalescence during PIKfyve inhibition. Thus, we discovered that ROS can generally prevent lysosome coalescence during PIKfyve inhibition using distinct mechanisms depending on the type of ROS.
Highlights
Lysosomes are typically defined as terminal organelles with an acidic and degradative lumen that digest macromolecules received through endocytosis, phagocytosis and autophagy [1,2,3]
Stimulation of reactive oxygen species (ROS) suppresses lysosome enlargement during acute PIKfyve inhibition We previously observed that extended laser excitation by spinning disc confocal fluorescence microscopy arrested lysosome enlargement during acute PIKfyve suppression [30]
We speculated that this arrest may be due to ROS production caused by light energy [41]; RAW cells that were more frequently exposed to laser light displayed higher levels of nitroblue tetrazolium (NBT) staining, a ROS indicator (S1A Fig)
Summary
Lysosomes are typically defined as terminal organelles with an acidic and degradative lumen that digest macromolecules received through endocytosis, phagocytosis and autophagy [1,2,3].
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