Lysosomes are terminal, degradative organelles of the endosomal pathway that undergo repeated fusion-fission cycles with themselves and other organelles like endosomes, phagosomes, and autophagosomes. Lysosome number, size and degradative flux depends on the equilibrium between fusion and fission rates. Thus, conditions that favour fusion over fission will reduce lysosome numbers while enlarging remaining lysosomes. Conversely, conditions that favour fission over fusion will cause lysosome fragmentation and increase their numbers. PIKfyve is a phosphoinositide kinase that generates phosphatidylinositol-3,5-bisphosphate to modulate several lysosomal functions. PIKfyve inhibition causes a dramatic 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 acute 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 four independent mechanisms all arrested lysosome coalescence during PIKfyve inhibition and accelerated lysosome fragmentation during PIKfyve re-activation. However, depending on the ROS species and/or mode of production, lysosome dynamics were affected distinctly. H2O2 impaired lysosome motility and reduced lysosome fusion with phagosomes, suggesting that H2O2 prevents lysosome coalescence in PIKfyve-impaired cells by reducing lysosome fusogenecity. In comparison, inhibitors of oxidative phosphorylation, glutathione, and thioredoxin that produce superoxide, 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.

Reactive oxygen species rescue lysosome coalescence during PIKfyve inhibition