Understanding Pikfyve Control Of Lysome Dynamics

Abstract

Lysosomes are organelles that receive external cargo through phagocytosis and endocytosis, and internal cargo through autophagy, followed by degradation in the acidic and hydrolase rich lumen and redistribution of substrates for maintaining cellular integrity. Lysosomes undergo homotypic or heterotypic repeated fusion and fission or kiss and run cycles with other organelles to exchange and receive cargo, as well as maintain lysosome number and size. Lysosome membranes display the phosphoinositide lipid phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P₂) synthesized by the lipid kinase PIKfyve. PtdIns(3,5)P₂ act as a signalling lipid on lysosomes to regulate maturation of endosomes, phagosomes and autophagosomes maturation by fusing with lysosomes, and recycling from the lysosomal lumen, lysosome ion channel activity, and lysosome-associated actin turnover. Of these defects, the most dramatic phenotype of PtdIns(3,5)P₂ depletion from PIKfyve inhibition is the appearance of enlarged lysosomes. Our work demonstrates that PtdIns(3,5)P₂ is an important regulator of lysosome size and number by governing the balance between lysosome fusion and fission and/or kiss and run. Depletion of PtdIns(3,5)P₂ arrests lysosome fission disrupting the balance between the continuous fusion and fission cycle, leading to lysosome coalescence and causing lysosome enlargement and reduction in their numbers. Microtubules, cytoskeletal tracks for lysosome positioning, and associated motor protein complexes, kinesin-1 and dynein, regulate lysosome coalescence during PIKfyve inhibition. Our experimental observations revealed ROS as a novel regulator of lysosome fusion and fission. Specifically, ROS arrested lysosome enlargement from acute PIKfyve inhibition and accelerated lysosome fragmentation during PIKfyve re-activation. However, depending on the ROS produced and/or site of ROS synthesis, lysosome dynamics are affected distinctly. H₂O₂ impaired lysosome mobility to arrest coalescence. However, superoxide generated from mitochondrial ETC complex 1, or thioredoxin reductase, or glutathione inhibition through rotenone, or CDNB, or MCB respectively depolymerised microtubules without affecting mobility. Instead, superoxide generation through pharmacological manipulations promoted actin clearance from lysosomes, which otherwise accumulate on lysosomes to hinder fission upon PIKfyve inhibition, to promote fission. Indeed, actin depolymerisation arrested lysosome enlargement during acute PIKfyve inhibition and accelerated lysosome fragmentation during PIKfyve re-activation, further indicative of ROS stimulating lysosome fission through actin clearance.