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.

Acknowledgement:ARPE-1 (RPE) cells stably expressing clathrin heavy chain-eGFP were a kind gift from Dr.Costin Antonescu. We would also like to thank Mr. Janusan Baskararajah for technical assistance. AbstractLysosomes 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.Bissig, C., Croisé, P., Heiligenstein, X., Hurbain, I., Lenk, G. M., Kaufman, E., Sannerud, R., Annaert, W., Meisler, M. H., Weisman, L. S., et al. (2019). PIKfyve complex regulates early melanosome homeostasis required for physiological amyloid formation. . Dense core lysosomes can fuse with late endosomes and are re-formed from the resultant hybrid organelles. . (2001). Rab-interacting lysosomal protein (RILP): the Rab7 effector required for transport to lysosomes. EMBO J. 20, 683-93.Cao, Q., Zhong, X. Z., Zou, Y., Murrell-Lagnado, R., Zhu, M. X. and Dong, X.-P. (2015). Calcium release through P2X4 activates calmodulin to promote endolysosomal membrane fusion.